draft...this study was an exploratory, open label, randomized, 13-week, 2-arm trial conducted at the...

Draft

A 13-week, randomized, pilot trial of a low glycemic load

diet and lifestyle modification program combining low glycemic load protein shakes and targeted nutraceuticals improved weight loss and cardio-metabolic risk factors over

a low glycemic load diet alone in subjects with cardiovascular risk factors

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0704.R2

Manuscript Type: Article

Date Submitted by the Author: 04-Jun-2017

Complete List of Authors: Dahlberg, Clinton ; Nature\'s Sunshine Products Inc, Hughes Center for Research and Innovation Ou, Joseph ; Nature\'s Sunshine Products Inc, Hughes Center for Research and Innovation Babish, John; Bionexus, Ltd., Executive Lamb, Joseph ; Hypertension Institute of Nashville, St. Thomas Medical Group, 4230 Harding Road Eliason, Sarah; Nature\'s Sunshine Products Inc, Hughes Center for Research and Innovation Brabazon, Holly ; Nature\'s Sunshine Products Inc, Hughes Center for Research and Innovation

Gao, Wei; Nature\'s Sunshine Products Inc, Hughes Center for Research and Innovation Kaadige, Mohan ; Nature\'s Sunshine Products Inc, Hughes Center for Research and Innovation Tripp, Matthew; Nature\'s Sunshine Products Inc, Hughes Center for Research and Innovation

Is the invited manuscript for consideration in a Special

Issue?: N/A

Keyword: Cardio-metabolic syndrome, Obesity, Nitric oxide, Low glycemic load diet, Bergamot polyphenols

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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A 13-week, randomized, pilot trial of a low glycemic load diet and lifestyle modification

program combining low glycemic load protein shakes and targeted nutraceuticals

improved weight loss and cardio-metabolic risk factors over a low glycemic load diet alone

in subjects with cardiovascular risk factors

Clinton J. Dahlberg, Joseph J. Ou, John G. Babish, Joseph J. Lamb, Sarah Eliason, Holly

Brabazon, Wei Gao, Mohan R. Kaadige, and Matthew L. Tripp

Clinton J. Dahlberg, Joseph J. Ou, Sarah Eliason, Holly Brabazon, Wei Gao, Mohan Kaadige,

and Matthew L. Tripp: Hughes Center for Research and Innovation, Nature’s Sunshine, 2500

Executive Parkway, Lehi, UT 84043, USA

John G. Babish: Bio Nexus Ltd., 53 Brown Road, Suite B, Ithaca, NY 14850

Joseph L. Lamb, Hypertension Institute of Nashville, St. Thomas Medical Group, 4230 Harding

Road, Nashville, TN 37205

Corresponding author: John G. Babish, Bio Nexus, Ltd., 53 Brown Road, Suite B, Ithaca, NY

14850, USA; Phone (607) 227-4948; Fax (607) 266-9481; email: [email protected].

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Abstract: An open label, randomized, exploratory study comprising 44 healthy, overweight

subjects with cardio-metabolic syndrome (CMS) risk factors was conducted to assess the safety,

tolerability and efficacy of a proprietary lifestyle modification program without (DIET) and with

(PROG) targeted nutraceutical supplementation including soy, pea and whey proteins,

phytosterols, antioxidants, probiotics, fish oil and berberine over 13-weeks. Key metrics were

recorded at baseline, weeks 9 and 13. For DIET and PROG compliance was 85% and 86%,

respectively, with no adverse events related to the diet or supplements. Twelve subjects

discontinued prior to week 9 for reasons unrelated to the study. PROG subjects experienced

greater decreases (P<0.05) than DIET in body weight, fat mass, total cholesterol, LDL

cholesterol, TG, cholesterol/HDL, TG/HDL, ApoB/ApoA, and hs-CRP. The Framingham 10-

year cardiovascular disease risk score decreased by 40% (P<0.01) in the PROG arm versus no

change for the DIET arm. As a pilot study, it was not possible to state whether the observed

effects were a result of nutraceutical supplementation alone or the result of additive or

synergistic interactions among diet, lifestyle modifications and nutraceutical supplementation.

Moreover, individuals with CMS risk factors following a lifestyle modification program received

additional health benefits from targeted nutraceutical supplementation. NCT03097965

Keywords: Cardio-metabolic syndrome; Obesity; Low glycemic load diet, Nitric oxide,

Bergamot polyphenols

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Introduction

A recent analysis has shown that the number of overweight and obese people worldwide

is now over 2.1 billion, up from 857 million in 1980, representing a 28% increase among adults

and a 47% increase among children.(Wise 2014) Cardio-metabolic syndrome (CMS)1, also

known as metabolic syndrome, or syndrome X, is a combination of metabolic disorders or risk

factors including central obesity, diabetes mellitus, systemic arterial hypertension, and

hyperlipidemia.(Kelli et al. 2015) According to the NCEP ATP III definition, CMS is present if

three or more of the following five criteria are met: waist circumference over 40 inches (men) or

35 inches (women), blood pressure (BP) over 130/85 mmHg, fasting triglyceride (TG) level over

150 mg/dL, fasting high-density lipoprotein (HDL) cholesterol level less than 40 mg/dL (men) or

50 mg/dL (women) and fasting blood glucose over 100 mg/dL.(Huang 2009)

While new pharmacological approaches to CMS and type 2 diabetes such as alogliptin, a

dipeptidyl peptidase 4 inhibitor, and canagliflozain, a sodium-glucose co-transporter 2 inhibitor,

are continually under development(Said et al. 2014; Sarnoski-Brocavich and Hilas 2013), the

pharmaceutical approach is not suitable or economically sustainable for a pandemic rooted

primarily in poor dietary choices and inactivity. Lifestyle modification including diet and

exercise are recommended as first-line intervention for treating insulin resistance and CMS by

1 Abbreviations: ACC/AHA: American College of Cardiology/American Heart Association;

ANOVA: analysis of variance; Apo A: Apolipoprotein A1; Apo B: Apolipoprotein B; BMI:

body mass index; BP: blood pressure; CFU: colony forming units; CMS: cardio-metabolic

syndrome; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; HbA1c: glycated

hemoglobin; HDL: high density lipoprotein; HIV: human immunodeficiency virus HOMA-IR:

homeostatic model assessment insulin resistance; hs-CRP: high sensitivity C-reactive protein;

LDL: low density lipoprotein; MPO: myeloperoxidase; MUFA: monounsaturated fatty acids;

NCEP ATP III: National Cholesterol Education Program Adult Treatment Panel III; ORAC –

oxygen radical absorbance capacity; OxLDL: oxidized low density lipoprotein; PAI-1:

plasminogen activator Inhibitor-1: PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids;

TG: triglycerides.

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the National Cholesterol Education Program and American Heart Association.(Grundy 2016;

National Cholesterol Education Program Expert Panel on Detection and Treatment of High

Blood Cholesterol in 2002)

An important aspect to consider in dietary recommendations for CMS is the incorporation

of diverse, targeted, biologically active nutraceuticals to address the multiple, dysfunctional

metabolic pathways associated with the etiology and pathogenesis of CMS. Noting that the

addition of soy protein, phytosterols and selective kinase response modulators to a modified,

Mediterranean-style, low glycemic load diet could favorably reduce plasma lipids and CMS risk

factors(Lerman et al. 2010), we developed a program consisting of a modified Mediterranean-

style, low glycemic load diet and a broader range of targeted nutraceuticals to address metabolic

criteria associated with obesity and CMS.

The objectives of this pilot study were to discern the safety, tolerability and efficacy of a

low glycemic-load, modified Mediterranean-style diet and aerobic exercise plan coupled with

low-glycemic, protein snack shakes and targeted nutraceutical supplementation on CMS risk

factors in generally healthy, overweight people. We hypothesized that all subjects would benefit

from the low glycemic-load diet and aerobic exercise plan, but that participants receiving the

low-glycemic protein shake and targeted nutraceutical supplementation would have lipoprotein

profiles and glycemic control variables associated with a decreased risk for cardiovascular

disease.

Methods

Subjects

Potential subjects for this study were recruited in Lehi, Utah and surrounding cities.

Radio advertisements, distribution emails, flyer, and word-of-mouth were used in recruiting.

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Subjects selected were healthy men and women between 27 and 64 years old, overweight or

obese with BMI >25 kg/m2 and ≤ 55 kg/m2, and exhibited at least two of the following

additional cardiometabolic risk factors: visceral adiposity (waist circumference ≥ 35 - 61 inches

for women and ≥ 40 inches for men), elevated LDL cholesterol ≥ 130 mg/dL, elevated TG

defined as TG ≥ 150 mg/dL, HDL: 29 - 81 mg/dL for women and 26 – 49 mg/dL for men,

elevated blood glucose ≥ 100 mg/dL, HbA1C 4.8 – 7.0%, and/or Homeostatic Model

Assessment of Insulin Resistance (HOMA-IR) score 0.5 – 5.8.

Exclusion criteria were pregnancy; lactation; recent changes in prescription medications,

over-the-counter medications, medical foods, and nutritional supplements; recent or regular use

of narcotics, investigational drugs, corticosteroids, anticoagulants, neuroactive medications, or

medication or supplements relevant to hyperglycemia or hyperlipidemia; allergy or intolerance to

study products; serious, unstable medical conditions; known infection with HIV, tuberculosis, or

hepatitis; cardiovascular disease; diabetes mellitus; autoimmune diseases; malignancy;

psychiatric disease; substance abuse; and abnormal laboratory findings.

Clinical protocols were approved by the Aspire Group Independent Review Board of

Santee, California. This study was conducted based on good clinical practice guidelines.

Informed written consent was obtained from each participant before enrollment in the study. The

study was registered on ClinicalTrials.gov (https://clinicaltrials.gov/) and assigned the NCT

identifier number NCT03097965.

Study design

This study was an exploratory, open label, randomized, 13-week, 2-arm trial conducted at

the Hughes Center for Research and Innovation in Lehi, UT from August 10, 2015 through

November 12, 2015. Eighty-four subjects were evaluated for participation (Fig. 1). Forty subjects

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were excluded for not meeting entrance criteria or declined to participate. Forty-four subjects

were randomized (using Excel® 2010; Microsoft, Redmond, WA) in blocks of four by sex and

age (ages 18 to 42, and ages 43-69 inclusive) to ensure approximately equal distribution in arm

assignment by gender and age.

Diet and lifestyle modifications for all participants

All subjects were required to make lifestyle changes during the study including following

a low glycemic load food plan, exercising per standardized recommendations and participating in

a cognitive behavioral program by attending 11 didactic/experiential group visits. Additionally,

all subjects received written instruction regarding the lifestyle change program and verbal

instruction in the lifestyle change program at Week 1 and continued instruction, counseling and

feedback at follow-up Group Weeks 2-9 and 11-13 and also at Week 10 (an individual visit).

Participants in both arms received written instruction regarding expected visit attendance and

prohibited therapies and behaviors during the study. Table 1 contrasts the elements of the study

for the DIET and PROG groups.

Diet

Both DIET and PROG arms were provided with information on a specific food plan to be

followed through the 13 weeks of the study. Our food plan offered a diverse variety of protein-

rich and phytonutrient-dense foods while maintaining a low glycemic load focus. Further, it

provided a relatively simple way to choose quality food to promote achieving health goals by

counting servings of allowed food, rather than calorie counting (Table 2). The diet plan ensured

that the required number of servings each day would contain a balanced blend of protein,

carbohydrates and quality fats from foods rich in vitamins, minerals and phytonutrients. All

participants were counseled to eat 5 times daily (3 meals and 2 snacks). The PROG arm

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consumed the low glycemic load shakes twice per day as the 2 snacks. DIET recommended

snacks approximated the caloric contribution of the low-glycemic load protein shakes. PROG

and DIET protocol differences are indicated in shaded areas in Table 2 (A representative listing

of 5 days of DIET and PROG plans is provided in Supplemental Table 1).

While our dietary program shared some features of a Mediterranean-style food plan, it

differed in several significant regards. Specifically, the standard Mediterranean diet is not

generally low glycemic due to a strong focus on grains. Our previous experience (Lerman et al.

2008) and Riccardi et al.(Riccardi et al. 2003) suggested that the standard Mediterranean diet

would not be optimally beneficial to individuals with pre-existing insulin resistance due to its

high carbohydrate content. Thus, our modified, low glycemic load Mediterranean diet excluded

grains and both natural and synthetic sweeteners except for limited amounts of stevia (Table 2).

Moreover, in this study we reduced total carbohydrates an additional 34% and increased protein

by 54% from our former modification of the Mediterranean diet, with similar daily energy intake

(Lerman et al. 2008). Viscous fiber was increased nearly 3-fold and MUFA were increased 21%.

Daily cholesterol consumption was also reduced by approximately 30%. Finally, our diet plan

excluded all alcohol and limited coffee and all teas to one cup daily, while the Mediterranean

diet allows for the consumption of alcoholic and caffeinated beverages.

The macronutrient compositions of the DIET and PROG arms are described in Table 3.

Estimates of the relative soy, pea and whey protein consumption during the study were made

from returned product canisters. Soy protein shakes were most popular and represented 50% of

the consumed snakes followed by pea protein at 30% and whey protein at 20%.

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Physical activity

In addition to diet, all subjects received instruction and were required to follow a lifestyle

change program consisting of specific exercise recommendations and cognitive behavioral

changes. The exercise requirements included (1) using a pedometer, (2) keeping a log of daily

steps aiming for at least 5,000 steps per day (about 2.5 miles), with at least 30 minutes of

moderate intensity focusing on aerobic movement exercising, and (3) adding resistance training

and flexibility exercises at least twice a week. Instruction on monitoring heart rates and activity

levels was also provided. All subjects received written instruction regarding the lifestyle change

program, expected visit attendance and prohibited therapies and behaviors during study

participation. .

Cognitive behavioral program

Our cognitive behavioral program required attendance at 11 didactic/experiential group

visits. During these visits, study staff presented educational and experiential content to support

healthy lifestyle change. Short seminars on mindfulness and visualization techniques designed

for stress reduction, relaxation and mind-body connectivity were presented weekly by one of the

authors (JL). All subjects provided a self-assessment of food-plan compliance for review at each

visit.

Low glycemic load protein shake formulations

The low glycemic load protein shake formulations used in these studies were designed to

provide 40 g protein, 6 to 10 g fat, 10 to 18 g carbohydrate equivalent to 346 to 368 calories per

day. As protein type was not an independent variable within the study we allowed subjects

within the study to choose between commercially available soy, whey or pea proteins distributed

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by Nature’s Sunshine Products (Spanish Fork, UT), with scoop size normalized to deliver 20 g of

protein. Supplemental Table 2 details the nutritional content available within each of the low

glycemic load protein shakes studied, as normalized to the delivered 20 g of protein per serving.

PROG nutraceutical supplementation overview

Subjects in the PROG arm additionally received (1) a bulk jar containing 2 grams of

phytosterol per serving taken 2 times daily added to the protein replacement low-glycemic

shakes, (2) CardioxLDL®

antioxidant formulation: 2 capsules per day with dinner, (3) Probiotic

Eleven®

probiotic capsules: 1 capsule, 2 times daily with meals, (4) Super Omega-3 fish oil

capsules: 1 capsule, 2 times daily with meals, (5) BerberineIR®

: 333 mg per capsule, 3 times

daily with meals, and (6) Super Supplemental® a high potency vitamin mineral supplement: 2

tablets, 2 times daily with meals. Subjects in the control DIET arm received none of the

nutraceutical supplements (Table 2; Supplemental Tables 2 and 3 provide a complete description

of all protein shakes and nutraceutical supplementation, respectively).

Compliance monitoring

All subjects completed a Food Plan Questionnaire that was scored at weekly meetings to

provide feedback on dietary compliance. Subjects in the PROG arm were instructed to return all

unused beverage powder and tablets, and the percentage of the amount consumed was calculated

to indicate compliance.

Clinical measurements

Blood Pressure (BP) was measured using the Welch Allyn 4200 Spot Vital Signs Monitor

(Welch Allyn, Skaneateles, NY). The measurement was taken from a bare, left arm in a quiet

room. Waist circumference was measured at the midpoint between the lowest rib and the top of

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the hipbone (iliac crest) with a flexible plastic tape. The subject was requested to remove any

outdoor clothes before measurements. BP and waist circumference were measured at baseline

and upon completion of 9 and 13 weeks.

Body composition was measured weekly using the FitScan (BC-557) Body Composition

Monitor (TheCompetitiveEdge.com, Seattle, WA). Besides the body weight, the monitor also

calculated the percentage of body fat, total body water, muscle mass, daily calorie intake,

metabolic age, bone mass and visceral fat.

Laboratory analyses

Following an overnight fast, blood samples were collected from subjects at baseline, 9

weeks, and 13 weeks. Analysis of serum and whole blood samples was conducted in batches and

were conducted by Quest Diagnostics for (CBC, CMP, HbA1c and insulin). Analysis of oxLDL,

MPO, hs-CRP and Atherotech VAP+ lipid panel of serum and whole blood samples was

conducted in batches on freshly frozen blood by Cleveland Heart Lab. Retained serum and

plasma specimens were maintained and stored at -80°C.

Model curve fitting for weight loss responders

Current treatment guidelines recommend that obese individuals lose 5% to 10% of their

starting weights to minimize the risk factors for cardiovascular disease and reduce the risk for

developing type 2 diabetes or hypertension.(Wharton 2016) Clinical weight loss in individuals

typically stabilizes at 6 months and validated models indicate that weight loss generally plateaus

between 1 and 2 years.(Thomas et al. 2014) To estimate this plateau and 95% confidence interval

for responders in our study, we modeled the cumulative number of responders observed in each

arm during the 13 weeks using nonlinear regression (GraphPad Software, San Diego, CA).

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CMS diagnosis and Framingham 10-year cardiovascular risk

The proportion of subjects fitting the diagnostic criteria of CMS initially and at

termination was determined using the modified NCEP ATP III criteria previously

published.(Huang 2009) According to the NCEP ATP III definition, CMS is present if three or

more of the following five criteria are met: waist circumference over 40 inches (men) or 35

inches (women), blood pressure over 130/85 mmHg, fasting triglyceride (TG) level over 150

mg/dL, fasting high-density lipoprotein (HDL) cholesterol level less than 40 mg/dL (men) or 50

mg/dL (women) and fasting blood sugar over 100 mg/dl. The NCEP ATP III definition is one of

the most widely used criteria of metabolic syndrome.

Estimated risk of heart disease or stroke over the next 10 years was assessed using the

ASCVD algorithm published in the 2013 ACC/AHA Guideline on the Assessment of

Cardiovascular Risk.(Goff et al. 2014) Briefly, the algorithm incorporates the variables of age,

gender, race, total cholesterol (mg/dL), HDL cholesterol (mg/dL), and systolic BP (mm Hg) as

well as background on treatment for hypertension, diabetes and smoking status.

Chemicals and reagents

Sodium Fluorescein and 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) were

purchased from Sigma-Aldrich (St. Louis, MO). 6-hydroxy-2,5,7,8-tetramethylchroman-2-

carboxylic acid (Trolox) was purchased from ACROS (Geel, Belgium). Potassium phosphate

buffer was purchased from Alfa-Aesar (Haverhill, MA). Black-sided, optical bottom, 96-well

plates were purchased from Nunc (part #265301). All other chemicals and reagents, unless

specified were purchased from Sigma-Aldrich.

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Ex vivo inhibition of myeloperoxidase chlorination activity

The ability of subjects’ plasma to inhibit MPO chlorination activity was assessed using

the Cayman Chemicals MPO kit. Plasma samples were filtered through 10 kDa cutoff VivaSpin

500 columns (Sartorius, Bohemia, NY). Filtered plasma was then serially diluted ten times

starting with a 5% concentration and mixed with MPO, hydrogen peroxide and chlorination

substrate in a final reaction volume of 0.1 ml per well in a 96-well microplate. Commercial

sodium heparin plasma (BioreclamationIVT, Hocksville, NY) was used at a concentration of

0.25% as a reference, standard plasma. The microplate was incubated on a plate shaker at room

temperature for 20 minutes and the fluorescence was measured on a Cytation 5 plate reader

(BioTek Instruments, Winooski, VT) with excitation and emission wavelengths of 490 nm and

520 nm, respectively.

Assay of salivary nitrite

Berkeley Test®

Salivary Nitric Oxide Strips, which detect salivary NO2 as a biomarker

for NO, have been shown to be useful as a reliable indicator of physiological NO

levels.(Clodfelter et al. 2015) Nitric Oxide Test Strips (Berkeley Test, Berkeley, CA) were used

in this study to measure the appearance of the NO biomarker NO2 in saliva. Subjects recorded

morning, lunch and dinner strip scores one hour after meals on the day of the weekly clinical

visit.

Assay of plasma nitrate

Nitrate/Nitrite (NOx) fluorometric assays were performed according to the

manufacturer's instructions (Cayman Chemicals, Item No. 780051, Ann Arbor, MI). Plasma was

filtered with 10 kDa MWCO VivaSpin 500 columns (Sartorius Stedim, VS0102). The microplate,

with 10 µL of plasma per well, was incubated with nitrate reductase and cofactors at room

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temperature for 45 minutes before adding the 2,3-diaminonaphthalene (DAN) reagent. After a

10-minute incubation, 2.8 M sodium hydroxide was added and fluorescence was measured on a

Cytation 5 plate reader (BioTek Instruments) with excitation and emission wavelengths of 375

and 417 nm, respectively.

Plasma oxygen radical absorbance capacity (ORAC)

Plasma ORAC assays were performed according to Huang et al.(Huang et al. 2002) with

no modifications. Briefly, plasma samples were diluted with buffer and assayed with fluorescein

(Sigma, St. Louis, MO) and AAPH (Sigma, St. Louis, MO). Fluorescence readings were taken

on a Cytation5 (BioTek Instruments, Winooski, VT) every two minutes for 70 minutes with an

excitation and emission of 485 nm and 528 nm, respectively. Trolox (ACROS, Geel, Belgium)

was used to generate a standard curve. The mean intra- and inter-day coefficient of variation was

≤ 15%, and the limit of detection and limit of quantitation were 5.00 and 6.25 µM, respectively.

Values were reported as µmole Trolox equivalents/L for each subject.

Statistical methods

Sample size was determined based on the results of an earlier study in which a decrease

of 10% in LDL cholesterol was observed in 11 subjects with moderate dyslipidemia.(Babish et al.

2016) Setting the probability of a type I error at 5% and type II error at 20%, a group size of 17

subjects per arm was estimated for lipid variables. We therefor recruited more than 34 subjects to

account for possible attrition.

A modified intent-to-treat analysis was conducted with one subject not completing all

three clinical visits, missing the 13-week final. The 9-week results for this subject in the DIET

arm were carried forward to the final clinical values.

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Differences between macronutrient components in the DIET and PROG arms were

analyzed using the Student t-test with n=5 representative diet-days. Clinical response variables

were generally not normally distributed and percent change from baseline within arms was

assessed using the non-parametric Wilcoxon Rank Sign Test. The Mann-Whitney U-Test was

used to assess differences between arms for resolution of CMS and percent reduction in 10-year

Framingham risk scores. Significance between DIET and PROG was determined using a 2-way

ANOVA of the log (week/baseline) with time and treatment as factors. Spearman's rank

correlation coefficient r was used to evaluate the relationship between plasma NO3 and salivary

NO2. Confidence intervals for proportions were computed using the Wilson Score Interval as

described by Brown et al.(Brown et al. 2001) All other calculations were performed using

GraphPad Software (San Diego, CA). The probability of a type I error was set at the nominal 5%

level using a two-tailed test for all variables.

Results

Subject engagement

Of the 44 subjects selected for the study, 23 participants were randomly assigned to the

control diet only arm (DIET) with 16 completing. One subject in the DIET arm left the study

after the week 9 clinical visit (Fig. 1). Therefore, 17 subjects were used in data analysis via last

visit forward starting from the second clinical visit of week 9. Twenty-one subjects were

randomized to the treatment program arm (PROG) with 15 completing. By week 9, 6 subjects in

the DIET and 6 subjects in the PROG arm had discontinued. Through phone calls it was

determined that all subjects had discontinued for personal reasons unrelated to the study. All

subject in the study were Caucasian.

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Safety and tolerability

Baseline cardio-metabolic profiles of the DIET and PROG arms were well balanced

among the major variables with the only statistically significant difference (P<0.01) being an 8%

greater ORAC value in the PROG arm (Table 4). Complete blood counts and metabolic profiles

covering baseline, week 9 and week 13 were normal (data not shown).

No serious or severe adverse events were reported during the study. Five of 17 subjects in

the DIET arm reported adverse events (severity: 4 mild and 1 moderate) and 4 of 15 subjects in

the PROG arm reported adverse events (severity: 4 mild and 1 moderate). Muscles aches and

constipation were reportedly equally by each group and treated symptomatically with

magnesium supplementation. One subject in the DIET arm suffered a knee injury during exercise

that resolved with rest and conservative treatment. Two subjects (one with a prior diagnosis of

acquired hypothyroidism) in the DIET arm were noted on non-study measurements to have mild

TSH elevations. Subjects had not been screened for hypothyroidism prior to study enrollment.

One subject in the PROG arm experienced a brief flare of acne symptoms and one subject in the

PROG arm experienced headaches likely related to a pre-existent dental infection.

Supplementation in the PROG arm was not associated with an increase in adverse events

reported by study participants.

As determined from weekly personal interviews, reviews of dietary records and returned

study products, compliance and tolerability for both DIET and PROG arms were estimated at

85% and 86%, respectively, over the study period.

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Weight, body fat mass and waist circumference

PROG subjects experienced greater decreases (P<0.05) in BMI and body weight 11.9%

vs 7.8% and fat mass 21.5% vs 13.7% (P<0.01). Waist circumference was reduced from baseline

in both arms (P<0.01), but did not differ between arms, respectively -8.4% and -11.5% for DIET

and PROG (Table 5). Weight and fat mass loss were similar between arms for the first 7 and 3

weeks, respectively (Fig. 2). Subsequent to these times, the PROG arm continued losing weight

and fat mass at rates relatively similar to their initial rates, while the DIET arm exhibited

plateauing with a decreased rate of loss in both variables. For weight loss and BMI at termination,

the overall rate of change from baseline was 33% greater (P<0.05; 95% CI = 17 to 50%) in the

PROG arm vs the DIET arm. Nonlinear, maximum likelihood estimates of the maximum number

of expected responders, subjects achieving a weight loss of 5% or greater during the 13 weeks of

the study, were 93% (89 – 97%) in the PROG arm and 66% (59 – 73%) in the DIET arm (Fig. 3).

Serum lipids and apolipoproteins

While a majority of serum lipids were reduced in both arms, PROG subjects experienced

greater decreases relative to DIET subjects in total cholesterol 17.5% vs 7.7% (P<0.01), LDL

cholesterol 18.6% vs 10.3% (P<0.01), TG 50.8% vs 30.6% (P<0.05), cholesterol/HDL 15.9% vs

4.3% (P<0.01), TG/HDL 47.0% vs 26.0% (P<0.05), ApoB 19.7% vs 9.2% (P<0.01).

Cholesterol/HDL, LDL/HDL and ApoB/ApoA were reduced from baseline only in the PROG

arm 15.9% (P<0.01), 15.8% (P<0.01) and 9.6% (P<0.01). Neither HDL nor lipoprotein(a) were

changed from baseline by either treatment.

Glucose, insulin, HbA1c and HOMA-IR score

In general, effects on glucose-related variables were similar for both arms (Table 5). No

effect of either DIET or PROG was seen on plasma glucose or HbA1c. Fasting insulin and

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HOMA-IR scores were both reduced from baseline by DIET and PROG, respectively, 31.9%

(P<0.01) vs 29.7% (P<0.05) and 29.6% (P<0.01) vs 31.3% (P<0.01).

hs-CRP, myeloperoxidase and plasma ORAC

The decrease in hs-CRP was approximately 5-tmes greater in the PROG arm vs the DIET

arm (P<0.05) with a 26% median reduction of hs-CRP from baseline in the PROG and -5.2%

change (P>0.05) in the DIET group (Table 5). Individual subject responses for BMI, TG, LDL

cholesterol and hs-CRP are presented in Fig. 4 for DIET (N=17) and PROG (N=15) arms at

baseline, 9, and 13 weeks.

No effect of either DIET or PROG was noted on MPO abundancy or inhibition by

plasma. Additionally, plasma ORAC changes from baseline were not affected by either treatment

(Data not shown).

Blood pressure and nitric oxide biomarkers

Subjects in the PROG group experienced decreases from baseline in both systolic

(P<0.01) and diastolic (P<0.01) BP, 11.2% and 12.1%, respectively, while the DIET arm

exhibited a 7.4% decrease (P<0.05) in diastolic BP and no change in systolic BP.

The NO biomarkers salivary NO2 and plasma NO3 were increased relative to baseline in

both the DIET and PROG arms during the study (Fig. 5 A and B, respectively). The PROG arm,

however, displayed a 3-fold increase (P<0.05) in AUC for salivary NO2 relative to DIET alone.

Plasma NO3 at 44 µM (31 – 64) was also greater in the PROG arm (P<0.05) at week 13 than in

the DIET arm, 25 µM (17 – 36). For both arms, the plasma NO3 concentration was significantly

correlated to salivary NO2 for the morning, afternoon and evening readings (Table 6).

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Reduction of cardio-metabolic syndrome and 10-year Framingham risk

Both arms exhibited similar rates of resolution of CMS. Initially both groups had 53% of

participants meeting criteria for CMS, 9/17 vs 8/15, respectively for DIET and PROG. These

numbers were reduced at week 13 to 2/17 (12%; 95% CI = 3.3% to 24%) in the DIET arm and

3/15 (20%; 95% CI = 7.1% to 45%) at termination in the PROG group.

While the DIET arm failed to reduce the Framingham 10-year risk of a cardiovascular

event (-16.7%, P >0.05), the PROG arm, exhibited a reduction of 40.0% (P <0.05) from base line

and was greater (P<0.05) than the overall decrease exhibited by the DIET group (Fig. 6).

Discussion

Obesity, whether central or total body, is a primary target for reduction in CMS with the

goal to reduce total body weight by 7 to 19% in the first 7 months to 1 year.(Kelli et al. 2015)

Both DIET and PROG groups in this study achieved this goal with weight losses of 7.8 and

11.9%, respectively. One aspect of the success of both treatments was the emphasis on lowering

carbohydrate consumption, as low carbohydrate diets tend to exhibit faster weight loss.(Johnston

et al. 2014)

In addition to weight loss the reduction of serum lipids is considered an essential element

for dietary management of cardio-metabolic risk. Combining foods and food components such as

plant sterols, viscous fiber, soy protein and almonds into a dietary portfolio has demonstrated

clinical efficacy in lowering serum lipids, coronary artery disease risk, hsCRP and 10-year

Framingham risk scores.(Jenkins et al. 2011; Jenkins et al. 2003a; Jenkins et al. 2003b) Using a

portfolio diet providing ~20 g viscous fiber, ~16.2 g soy protein, ~2.9 g phytosterols and

almonds, these researchers reported reducing LDL levels 35% or the same extent as reported in

large, randomized controlled trials of the statin drugs in 4 weeks.(Jenkins et al. 2003a)

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When tested concurrently against lovastatin, a portfolio diet containing 9.8, 21.4, and 1.0

g/day of viscous fiber, soy protein and phytosterols, respectively, per day for 4 weeks decreased

LDL cholesterol levels similarly to a 20 mg/day dose of lovastatin.(Jenkins et al. 2003b) In these

studies, the portfolio diets exhibited a 35% and 29% reduction in LDL cholesterol, respectively,

compared to the 19% reduction seen in the PROG arm of this study. The latter trial also reported

a decrease in hsCRP nearly twice that of the PROG group. The PROG, however, affected

substantially greater TG decrease (8 to 6-fold), systolic BP lowering (2.3-fold), and 10-year

cardiovascular risk reduction (1.3 to 1.5-fold) than the portfolio diets.

In a more recent study of the effect of intensity of dietary advice on serum lipids with a

portfolio diet containing 8.2, 15, and 0.8 g/day of viscous fiber, soy protein and phytosterols,

respectively, per day for 24 weeks(Jenkins et al. 2011), the current PROG group outperformed

both the Routine and Intensive Dietary Portfolio arms in all primary outcome variables. From

these studies it appears that the approximate 2-fold greater content of viscous fiber and soy

protein in the earlier portfolio diets relative to the PROG arm resulted in greater decreases in

serum lipids. When levels of these dietary components were reduced to below those of the

PROG group in the more recent portfolio diet, the differences in reduction of LDL cholesterol

and hsCRP between the portfolio diet and the PROG arm disappeared.

Other differences between the portfolio diets and the PROG arm likely contributed to

the consistently greater weight loss, reductions in TG, systolic BP, and 10-year cardiovascular

risk seen in this study. The portfolio diets were metabolically controlled to ensure that weight

loss did not occur. Supplementation of the PROG arm with omega-3 fatty acids supports the

finding of decreased serum TG. The magnitude of the TG effect in the PROG subjects, however,

is unlikely due to the omega-3 supplementation alone, which would be expected to be 27% or

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roughly half of the observed decrease.(Skulas-Ray et al. 2011) The additional TG-lowering

effect may have come from the 4 g phytosterols and bergamot antioxidant blend (CardioxLDL)

that also function to reduce LDL cholesterol.(Babish et al. 2016; Gylling et al. 2014) Increased

NOx by the PROG group would likely explain the decreases in systolic BP that are not usually

observed in portfolio diets. Taken together, it is likely that the combined effectiveness of

decreasing serum cholesterol and systolic BP were the primary divers in the enhance reduction in

10-year cardiovascular risk observed in this study versus portfolio diets.

The PROG group in this study also compared favorably against well-known diet plans

employing low glycemic load, meal-replacing shakes. Over 6 months, the Medifast 5&1, Atkins

and Slim Fast diets failed to achieve the 7% weight loss goal with losses of 6.7%, 6.2% and 4.9%,

respectively.(Shikany et al. 2013; Truby et al. 2006) While the 7.0% and 7.3% weight losses

reported, respectively, by the Rosemary Conley and Weight Watchers diets did achieve greater

than 7% weight loss goal, however, these results were less than the 11.9% reported here for the

PROG group.(Truby et al. 2006) Moreover, the PROG nutraceutical supplementation of low

glycemic load, high protein snack formulations also proved superior to low glycemic load snack

or meal replacement programs coupled with caloric restriction and exercise - PROG’s 11.9%

weight loss versus -6.5% for the Herbalife Program and -8% for the Shaklee Program.(Lee et al.

2009; Truby et al. 2006) Our pilot study is the first to demonstrate that low-glycemic load, high

protein shake/snacks coupled with targeted nutraceutical supplementation may reduce BMI and

fat mass as well as lipid biomarkers and CMS risk beyond the more traditional caloric restriction,

aerobic exercise plans with or without high protein low glycemic load shake formulations.

Waist circumference has been reported positively correlated with insulin and the

inflammatory markers interleukin 6 and tumor necrosis factor alpha and negatively correlated

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with plasma adiponectin.(Ackermann et al. 2011) Moreover, among the various components of

CMS, waist circumference appears to exert the greatest influence upon the presence and intensity

of the micro-inflammatory response, specifically erythrocyte sedimentation rate, plasma

fibrinogen, white blood cell count and most significantly hs-CRP.(Rogowski et al. 2010) Both

the DIET and PROG arms exhibited reductions (P<0.01) in waist circumference, 8.4 vs 11.5%,

respectively, indicating that lifestyle modifications common to both affected general systemic

markers of inflammation, but only the PROG arm reduced hs-CRP, a major contributor to

cardiovascular disease risk, 25.6% (P<0.01) vs 5.2% (P>0.05), respectively.(Cook et al. 2012)

This discordant response of the PROG and DIET arms could likely be independent of weight

loss and a result of an interaction of diet, exercise and nutraceutical supplementation, specifically

the probiotics, berberine and the bergamot/antioxidant formulation CardioxLDL™.(Asemi et al.

2013)

Decreases in blood pressure mirrored the overall increases in salivary NO2 and plasma

NO3 seen for both arms during the study with higher salivary NO2 and plasma NO3 levels in the

PROG arm consistent with the greater BP reduction in that group. The question arises what

PROG components could be responsible for these increases? Nitrates from green leafy

vegetables form a significant component of our diet. Mediterranean diets in particular supply

approximately 10-times the 40 to 100 mg NO3/day estimated in standard western diets.(Hord et

al. 2009) Research over the last decade suggests that the NO3-NO2-NO pathway may be used to

supplement circulating NO concentrations, with both anti-obesity and anti-diabetic effects, as

well as improving vascular function.(McNally et al. 2016)

NO has a short half-life approximately 0.05–1.18 ms in human blood, limiting its

functioning to only local signaling changes.(Rassaf et al. 2002) The half-lives of both NO2 and

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NO3 are relatively longer about 110 sec and 8 hr, respectively, in human blood.(Kelm 1999)

Therefore, NO2 and NO3 are more stable and can circulate in the blood, where they may be

reduced via the NO3-NO2-NO pathway back to NO, and function in an endocrine manner and

serve as biomarkers for NO.

The source of the increase NO biomarkers NO2 and NO3 in the PROG arm may be due to

the supplemental berberine, bergamot polyphenols, probiotics or a combination of the three.

Because berberine acts topically in the gastrointestinal tract and is poorly absorbed, berberine

might modulate gut microbiota without systemic anti-infective activity.(Han et al. 2011) In a rat

study, berberine supplementation showed enrichment of short-chain, fatty acid-producing

bacteria and reduction of microbial diversity.(Zhang et al. 2015) Additionally, berberine can

directly stimulate gastric NO production in mice through increased eNOS expression;(Pan et al.

2005)and has been shown to upregulate the number and functioning of circulating endothelial

progenitor cells through increased NO production in healthy subjects.(Xu et al. 2009)

Dietary polyphenols also show an interesting connection with NO biology. At acidic

gastric pH, dietary polyphenols not only promote NO2 reduction to NO but also produce higher

nitrogen oxides with signaling functions through post-translational modifications. These

modified endogenous molecules, such as nitrated proteins and lipids, can in turn alter important

physiological functions. Thus, local and systemic effects of NO such as modulation of vascular

tone, mucus production in the gut and protection against ischemia-reperfusion injury can be

triggered by dietary polyphenols.(Rocha et al. 2014) In a 1-year study of 200 subjects at high

cardiovascular risk, it was shown that the addition of polyphenols as extra virgin olive oil or nuts

to a Mediterranean-style diet decreased systolic and diastolic BP in supplemented diets relative

to a control, low-fat diet. Moreover, plasma NO and total polyphenol excretion correlated with

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the changes in BP, adding to the growing evidence that polyphenols might protect the

cardiovascular system by improving the endothelial function and enhancing endothelial synthesis

of NO.(Medina-Remon et al. 2015)

While probiotic bacteria can elicit a number of beneficial effects in the gut, mechanisms

for these health-promoting effects are not well characterized. It is suggested from in vitro studies

that bacterial NO3 reduction to ammonia, as well as the related NO formation in the gut, could be

an important aspect of the overall mammalian NO3/NO2/NO pathway and illustrates yet another

way in which the microbiome links diet and health.(Tiso and Schechter 2015) In rats, dietary

supplementation with lactobacilli and NO3 resulted in a 3-8 fold NO increase in the small

intestine and caecum, but not in colon, indicating that commensal bacteria can be a significant

source of NO in the gut in addition to the mucosal NO production.(Sobko et al. 2006)

From our study, we hypothesize that modulation of NO production through direct

stimulation or modification of gut microbiota by the nutraceuticals berberine, polyphenols in the

anti-oxidant formulation and probiotics, may underlie the increased salivary NO2 and plasma

NO3 seen in the PROG arm.

Categorical changes, such as change in diagnosis for CMS, or multicomponent risk

analyses, like the 10-Year Framingham Risk Score, are helpful outputs for understanding the

overall effects of interventions affecting multiple variables. Consider the categorical diagnosis of

CMS. Both arms exhibited similar rates of resolution of CMS. Initially both arms had 53% of

participants meeting criteria for CMS, 9/17 vs 8/15, respectively for DIET and PROG. These

numbers were reduced at week 13 to 2/17 for the DIET arm and 3/15 for the PROG group. The

similarity in resolution of CMS was due primarily to the dramatic decreases seen in TG in both

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arms (-31% for DIET and -51% for PROG) and removing this singular criterion from most

subjects.

While the DIET arm failed to reduce the Framingham 10-year risk of a cardiovascular

event (-16.7%, P >0.05), the PROG arm, exhibited a reduction of 40.0% (P <0.05) from baseline

and was greater (P<0.05) than the overall decrease exhibited by the DIET group (Fig. 6).

Continuous variables contributing to the estimation of risk in the algorithm for the Framingham

model are depicted in Fig. 7. As can be seen, the quantitatively greater decreases in the combined

factors of cholesterol, systolic and diastolic BP were primary drivers in the reduction of

cardiovascular disease risk by the PROG group, while no decrease was seen in the DIET group.

Moreover, this reduction in risk is likely under estimated as it fails to include the contribution of

the reduction of hs-CRP observed only in the PROG group as well. Further, as discussed above,

the changes in BP are strongly correlated with nutraceutical supplementation and likely

independent of weight loss.

As a pilot study, there were a few inherent limitations. Specifically, the study had a

relatively small number of participants, but was adequately powered even for the large variability

seen within arms, as demonstrated by the statistical significance of the results. Participants were

predominantly white and generalizability of this clinical trial to more diverse, higher-risk,

populations is unknown. Due to the complex nature of the study, it was not possible to state

whether the observed effects were a result of nutraceutical supplementation alone or the result of

additive or synergistic interactions among diet, lifestyle modifications and nutraceutical

supplementation. Finally, there was no follow-up after the study to assess the sustainability of the

changes noted during the study, this is as by design we were testing the safety and efficacy of a

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13-week intervention for rapid weight loss and cardiovascular benefits. Many of these limitations

are currently being addressed with a larger, observational study.

Conclusions

Lifestyle modifications including dietary alteration and daily aerobic exercise have

demonstrated success in correcting obesity-associated CMS risk factors and variations of this

regimen form the basis of recommendations provided by national organizations. The results of

this study, however, demonstrate that supplementation of a low glycemic load diet and exercise

plan with low glycemic load, high-protein shake formulations and targeted nutraceuticals may

produce significantly greater improvements in multiple CMS risk factors than diet and lifestyle

modification alone. Combined targeted nutraceutical supplementation with a low glycemic load

diet may represent a novel, nutritional approach for CMS risk factor reduction in generally

healthy, overweight persons.

Contributions

All authors materially contributed to the research or article preparation. The subsequent

listing identifies the authors with their responsibilities in the development of this manuscript: (1)

protocol design: MLT, JJL, JJO, CJD, JGB; (2) recruiting, screening participants, informed

consent: MLT, SE, JJO, JJL, CJD, JGB, WG, MRK; (3) weekly meeting presentations and

monitoring: SE, JJO, JJL, CLD, MRK, WG; (4) subject compliance: JJO; (5) development and

testing of NO2 scoring strips: JGB, CJD, MLT, MRK, SE, WG, HB; (6) ORAC, MPO, plasma

nitrate/nitrite assays: HB; (7) data analysis, interpretation and preparation of the manuscript: CJD,

JGB, MLT.

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Financial disclosures

This study was funded by Nature’s Sunshine Products, Inc. that developed and

manufactured the commercial foods used in this study and are sold under the InForm Program®.

All authors have approved the final manuscript. Drs. Joseph J. Lamb and John G. Babish served

as paid consultants to Nature’s Sunshine Products and all other authors are or were employees of

Nature’s Sunshine Products.

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production in healthy subjects. Cardiology 112(4): 279-286. doi: 10.1159/000157336.

Zhang, X., Zhao, Y., Xu, J., Xue, Z., Zhang, M., Pang, X., Zhang, X., and Zhao, L. 2015.

Modulation of gut microbiota by berberine and metformin during the treatment of high-

fat diet-induced obesity in rats. Scientific reports 5: 14405. doi: 10.1038/srep14405.

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34

Figure Captions

Fig. 1. Flow chart of study design and participant enrollment.

Fig. 2. Percent change from baseline for body weight [A] and fat mass [B] by week for DIET

(n=17) and PROG (n=15) arms.

Fig. 3. Time course for achieving a 5% loss of body weight for DIET (n=17) and PROG (n=15)

arms. Solid lines are nonlinear regression estimates and dotted lines represent the 95%

confidence intervals. Best-fit estimates of the maximum number of 5% responders were 93%

(95% CI = 89-97) in the PROG arm and 66% (95% CI = 59-73).

Fig. 4. Individual responses in BMI, triglycerides, LDL cholesterol, and hs-CRP in the DIET

(n=17) and PROG (n=15) arms at baseline, 9 and 13 weeks.

Fig. 5. Percent change from baseline of evening salivary NO2 strip scores over 13 weeks [A] and

plasma NO3 concentrations (mean±95% CI) at baseline, week 9 and week 13. *P<0.05 vs

baseline; # P<0.05 between DIET (n=17) and PROG (n=15).

Fig. 6. Reduction of 10-year cardiovascular event risk (medians with interquartile range) at the

completion of 13 weeks for the DIET (n=17) and PROG (n=15) arms. **Significantly changed

from baseline (P<0.01). The PROG arm was more effective (P<0.01) in the reduction of 10-year

risk of a cardiovascular event than the DIET arm, respectively 40.0% vs 16.7% (P<0.01). The

Wilcoxon Rank Sign Test was used for paired observations within arms and the Mann-Whitney

U-Test was used to assess differences between arms.

Fig. 7. Medians with interquartile ranges for percent changes from baseline for continuous

cardiometabolic variables used in the estimation of 10-year Framingham risk for DIET (n=17)

and PROG (n=15) arms. *P<0.05 and **P<0.01 significantly changed from baseline. The

Wilcoxon Rank Sign Test was used for paired observations within arms and the Mann-Whitney

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35

U-Test was used to assess differences between arms.*P<0.05 and **P<0.01 significantly

changed from baseline. The Wilcoxon Rank Sign Test was used for paired observations within

arms and the Mann-Whitney U-Test was used to assess differences between arms.

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Table 1. Study design contrasting control and treatment arms

Event Details DIET PROG

Recruiting Screening Recruit generally healthy overweight and obese adults. Record

physical measurements including height, weight and waist

circumference. Perform clinical assessment including medical

history questionnaire and limited physical examination. Phlebotomy

for determining complete metabolic profile, HbA1c, insulin and

serum pregnancy test for women of child-bearing potential.

Week 0

Baseline

exams

Measurements included: Physical measurements (height, weight and

waist circumference), vital signs (including blood pressure and heart

rate), body impedance analysis, clinical assessment (completion of

medical history questionnaire, and visit with study clinician), urine

pregnancy test for women of child bearing potential, fasting

phlebotomy for CBC, CMP, insulin, HbA1c, oxidized LDLc,

myeloperoxidase, high sensitivity C-reactive protein†

Randomize to open-label, 2 arm study

Weeks

1-12

Food Plans

Low-glycemic food plan

Physical activity


Low-glycemic food plan

Physical activity


-------------------------------------

Low-glycemic load, high protein

shake

• 40 g protein

Nutraceutical Supplementation

• 4 g phytosterols

• 2 capsules antioxidant

formulation

• 12 billion CFU probiotic

• 2 g fish oil capsules

• 1g berberine

• 4 high potency vitamin

mineral tablets

Weeks

1-12

Counseling

and group

meetings

All subjects met collectively with the study clinician in small groups

and had Body Impedance Analysis and weight collected.

Weeks 9

and 12

Repeated

physical

exams

Review questionnaires, assess for signs and symptoms of adverse

events, review compliance to the study products and answer any

participant questions. Measurements of baseline variables repeated.

Note: † Additional variables were measured, but are not discussed within this report.

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Table 2. Representative diet and supplementation for DIET and PROG groups

DIET PROG

Breakfast Eggs (2 large) Eggs (2 large)

Peppers (0.5 cup) Peppers (0.5 cup)

Olive oil (1 tsp) Olive oil (1 tsp)

Super Supplemental® (2 tablets); Super Omega 3

EPA® (1 capsule containing 380 mg EPA, 190 mg

DHA); Probiotic Eleven® (1 capsule); Berberine

IR® (1 capsule 333 mg)

Snack Mixed greens (2.5 oz.) Protein Shake (Soy, Pea, or Whey)

Olive oil (1 tsp) Phytosterols (1 scoop ~2 g)

Almonds (16)

Olives (6)

Lunch Salmon, chicken or turkey breast (1 palm size) Salmon, chicken or turkey breast (1 palm size)

Broccoli (2 cups) Broccoli (2 cups)


Berberine IR® (1capsule 333 mg)

Snack Chicken leg (1) Protein Supplement Shake (Soy, Pea, or Whey)

Apple (1, small) Phytosterols (1 scoop~ 2 g)

Dinner Salmon, chicken or turkey breast (1 palm size) Salmon, chicken or turkey breast (1 palm size)

Bok choy (1 cup) Bok choy (1 cup)

Carrots (0.5 cup) Carrots (1/2 cup)

Cucumber (0.5 cup) Cucumber (1/2 cup)

Mixed greens (2.5 oz.) Mixed greens (2.5 oz.)


Avocado (1/8) Avocado (1/8)

Super Supplemental® (2 tablets); Super Omega 3

EPA® (1 capsule containing 380 mg EPA, 190 mg

DHA); Probiotic Eleven® (1); Berberine IR

® (1

capsule 333 mg); CardioxLDL® (2 capsules)

Beverages Water and herbal teas: unlimited; optional: coffee or tea (black, green, white): 1 cup caffeinated or

decaffeinated

Condiments/Sweeteners All fresh/dry herbs: dill, oregano, basil, lavender, tarragon, etc. All spices: cinnamon, chili powder,

pepper, ginger, etc. Mustards, horseradish, lemon/lime juice, salsa, vinegars (all types) (unsweetened),

soy sauce, fish sauce (unsweetened). Stevia.

†Vegetables could be eaten raw or steamed and dressed with one of their fats as primary cooking

method. *Shaded areas denote differences between arms.

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Table 3. Representative macronutrient composition of the study diets over 5 days

Daily Intake (g/day)

P-Value*

Daily Intake (% kcal)

P-Value* Composition DIET PROG DIET PROG

Calories (kcal) 1332 (1197-1466) 1409 (1316-1502) NS - -

Protein 115 (98.4-133) 131 (119-143) NS 34.7 (31.4-38.0) 37.4 (33.7-41.1) NS

Soy Protein Shake†† - 20 5.7 (5.3-6.1) -

Pea Protein Shake - 12 3.4 (3.2-3.21) -

Whey Protein Shake - 8.0 2.3 (2.1-2.4) -

Available Carbohydrates 69 (56-82) 100 (89.5-110) <0.001 20.7 (17.9-23.4) 28.4 (26.3-30.4) <0.005

Sugars 25 (21-28) 41 (38-45) <0.001 7.5 (6.1-8.8) 12 (10-13) <0.002

Fiber

Total 21 (18-25) 33 (30-36) <0.001 16.0 (14.1-18.0)b 23.4 (21.4-25.5)

b <0.001

Viscous 5.1 (4.3-5.9 12 (11 -13) <0.001 3.8 (3.4-4.3)b 8.2 (7.5-9.0)

b <0.001

Total Fat 68 (60-76) 58 (51-65) NS 46 (44-48) 37 (34-40) <0.005

SFA 15 (10-20) 12 (7.4-16) NS 10 (7.0-13) 7.6 (5.0-10) NS

MUFA 26 (21-31) 21 (16-26) NS 18 (13-23) 18 (13-23) NS

PUFA 10 (6.5-14) 8.0 (5.3-11) NS 6.9 (4.5-9.4) 5.2 (3,3-7.2) NS

Trans fatty acids 0.11 (0.00-0.24) 0.043 (0.004-0.081) NS 0.072 (0.0-0.160) 0.026 (0.004-0.049) NS

Dietary cholesterol 336 (189-483)a 251 (164-337)

a NS 266 (119-413)

b 178 (119-238)

b NS

Alcohol - - - -

†Data are expressed as mean grams per day for a 5-day period; parenthetic values are 95% confidence intervals of the estimate, n=5).

††Percentoftotalshakesconsumedwasestimatedfromproductreturns.

*ComputedusingStudent’st-testwithP<0.05consideredstatisticallysignificant;NS=notsignificantlydifferentbetweenarms.(a) mg/day; (b) mg/1000 kcal.

SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

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Table 4. Baseline median cardiometabolic profiles of DIET and PROG subjects

Variable DIET Arm†

(n=17)

PROG Arm†

(n=15) P††

Gender, Male/Female 5/12 5/10 -

Age (yrs) 47 (33-64) 46 (27-59) NS

Weight (lb) 224 (163-390) 226 (171-292) NS

BMI (kg/m2) 36.3 (26.0-53.1) 34.6 (30.4-42.4) NS

Body Fat Mass (%) 46.9 (23.8 – 53.6) 44.3 (26.8 – 53.6) NS

Waist circumference (in) 45.0 (35.5-61.0) 44.5 (35.0-52.0) NS

Systolic BP (mm Hg) 129 (102-172) 133 (115-160) NS

Diastolic BP (mm Hg) 85 (73-99) 83 (78-99) NS

Salivary Nitrite (Score units) 5.5 (3.0-7.0) 5.0 (3.0-8.0) NS

Plasma Nitrate (µM) 17.0 (11.4-25.3) 24.5 (17.1-35.0) NS

Total Cholesterol (mg/dL) 215 (186-277) 220 (147-340) NS

LDL Cholesterol (mg/dL) 142 (107-195) 149 (71-239) NS

TG (mg/dL) 162 (77-284) 184 (96-332) NS

HDL Cholesterol (mg/dL) 50 (32-71) 46 (27-83) NS

Non-HDL Cholesterol (mg/dL) 168 (130-229) 176 (101-259) NS

oxLDL (U/L) 47 (32-63) 43 (23-79) NS

Cholesterol/HDL 4.42 (2.87-8.06) 5.09 (3.11-7.67) NS

LDL/HDL 2.92 (1.55-5.91) 3.13 (1.51-5.18) NS

TG/HDL 3.02 (1.27-7.63) 3.47 (1.06-9.24) NS

oxLDL/HDL 1.00 (0.59-1.78) 1.02 (0.36-1.61) NS

Lipoprotein(a) (mg/dL) 7 (3-23) 6 (1-17) NS

ApoA1 (mg/dL) 144 (122-180) 143 (114-206) NS

ApoB (mg/dL) 112 (84-142) 113 (68-161) NS

ApoB/ApoA Ratio 0.79 (0.47-1.16) 0.80 (0.46-1.17) NS

Glucose (mg/dL) 93 (71-121) 90 (79-99) NS

Fasting Insulin (µIU/mL) 7.5 (2.5-18) 9.0 (2.9-27) NS

HbA1C (%) 5.8 (5.1-7.0) 5.7 (4.8-6.2) NS

HOMA-IR 1.78 (0.50-4.24) 1.97 (0.615-5.79) NS

hs-CRP (mg/L) 2.6 (0.80-42) 3.6 (0.40-9.8) NS

Myeloperoxidase (pmol/L) 298 (209-742) 330 (211-472) NS

Myeloperoxidase (% Inhibition) 45 (25-76) 56 (33-83) NS

Plasma ORAC (µmole Trolox/mL) 8.9 (7.1-10) 9.6 (8.5-11) <0.01

Note: BMI: Body Mass Index; BP = Blood Pressure; LDL: Low Density Lipoprotein; HDL:

High Density Lipoprotein; OxLDL: Oxidized Low Density Lipoprotein; Apo A: Apolipoprotein

A1; Apo B: Apolipoprotein B; HbA1c: Glycated Hemoglobin; HOMA-IR: Homeostatic Model

Assessment Insulin Resistance computed using the formula HOMA-IR = [(Glucosemg/dL) x

(Insulin µU/mL)]/405; hs-CRP: high sensitivity C-reactive protein; ORAC – Oxygen Radical

Absorbance Capacity.

†Tabulated values are medians with parenthetic minimum and maximum values, respectively.

†† P-value computed from Mann-Whitney U-Test; NS = P>0.05.

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Table 5. DIET and PROG effects on cardio-metabolic risk variables

DIET Arm PROG Arm PROG vs DIET

Variable Median %Change(a)

(Min - Max) Median %Change(a)

(Min - Max) P-Value(b)

BMI (kg/m2) -7.8** (-19.6 to -0.9) -11.9** (-16.3 to -4.4) <0.05

Weight (lbs) -7.8** (-19.6 to -0.9) -11.9** (-16.3 to -4.4) <0.05

Fat Mass (lbs) -13.7** (-26.6 to -2.8) -21.5** (-35.4 to -1.0) <0.01

Waist circumference (in) -8.4** (-17.0 to -4.9) -11.5** (-16.9 to -3.0) NS

Systolic BP (mm Hg) -5.0 (-24.4 to 12.7) -11.2** (-23.8 to 7.1) NS

Diastolic BP (mm Hg) -7.4* (-18.7 to 8.2) -12.1** (-20.5 to 2.6) NS

Total Cholesterol (mg/dL) -7.7** (-32.2 to 20.4) -17.5** (-36.6 to -8.1) <0.01

LDL Cholesterol (mg/dL) -10.3* (-37.0 to 37.2) -18.6** (-40.2 to 8.4) <0.01

TG (mg/dL) -30.6** (-52.0 to 16.2) -50.8** (-64.9 to 72.3) <0.05

HDL Cholesterol (mg/dL) -5.7 (-26.1 to 12.5) -2.6 (-16.4 to 9.4) NS

Non-HDL Cholesterol (mg/dL) -8.0** (-38.5 to 24.0) -22.7** (-44.0 to -10.7) <0.01

oxLDL (U/L) -15.9* (-41.3 to 37.2) -11.5** (-36.7 - 6.7) NS

Cholesterol/HDL -4.3 (-39.7 to 26.4) -15.9** (-34.0 to -4.4) <0.01

LDL/HDL -0.9 (-44.0 to 46.7) -15.8** (-42.4 to 8.4) <0.01

TG/HDL -26.1** (-57.4 to 28.4) -47** (-67.5 to 88.6) <0.05

oxLDL/HDL -12.5 (-45.4 to 35.7) -6.2* (-42.2 to 19.6) NS

Lipoprotein(a) (mg/dL) -8.7 (-58.3 to 40.0) 33.3 (-33.3 to 200) <0.01

ApoA (mg/dL) -5.1** (-19.0 to 3.1) -6.8** (-13.8 to -1.3) NS

ApoB (mg/dL) -9.2** (-33.8 to 12.4) -19.7** (-35.4 to -6.0) <0.01

ApoB/ApoA 0.0 (-31.9 to 34.0) -9.7** (-34.7 to 4.1) <0.01

Glucose (mg/dL) -2.0 (-37.2 to 15.5) -2.0 (-16.2 to 11.7) NS

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Fasting Insulin (µIU) -31.9** (-71.9 to 30.6) -29.7* (-75.6 to 57.4) NS

HbA1c (%) 0.0 (-14.3 to 8.6) -1.8 (-11.3 to 4.2) NS

HOMA-IR -29.6** (-82.4 to 29.5) -31.3* (-79.4 to 69.4) NS

hs-CRP -5.2 (-63.0 to 438.5) -25.6** (-55.4 to 66.6) <0.05

Note: BMI: Body Mass Index; BP: blood pressure; LDL: Low Density Lipoprotein; HDL: High density lipoprotein; TG: triglycerides;

High Density Lipoprotein; OxLDL: Oxidized Low Density Lipoprotein; Apo A: Apolipoprotein A1; Apo B: Apolipoprotein B

HbA1c: Glycated Hemoglobin; HOMA-IR: Homeostatic Model Assessment Insulin Resistance computed using the formula HOMA-

IR = [(Glucosemg/dL) x (Insulin µU/mL)]/405; hs-CRP: high sensitivity C-reactive protein.

(a) Significance of Median % change between baseline and week13 within arms was computed using the Wilcoxon Rank Sign Test as

described in Methods with * = P<0.05 and ** = P<0.01

(b) P-values between arms were computed using a 2-way ANOVA of the log (week/baseline) as described in Methods.

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Table 6. Correlation between salivary nitrite and plasma nitrate

Salivary Nitrite Scoring(a) Spearman r

(b) 95% Confidence Interval P

Morning 0.2489 0.02118 - 0.4520 <0.05

Lunch 0.3147 0.09089 - 0.5082 <0.01

Dinner 0.2833 0.05659 - 0.4822 <0.05

Note: (a) Scoring of salivary NO2 performed using Berkeley Test® Nitric Oxide Test Strips and

plasma NO3 was determined as described in Methods.

(b) Spearman ranking correlation coefficient r and 95% confidence intervals were computed

using GraphPad statistical software (N= 96 pairs of data).

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161x127mm (300 x 300 DPI)

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170x190mm (300 x 300 DPI)

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153x152mm (300 x 300 DPI)

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203x152mm (300 x 300 DPI)

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133x171mm (300 x 300 DPI)

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158x101mm (300 x 300 DPI)

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127x66mm (300 x 300 DPI)

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Supplemental Table 1. Five-day representative diets† and targeted nutraceutical

supplementation* followed in DIET and PROG groups

Day 1

DIET PROG

Breakfast

Egg (1 large) Egg (1 large)

Part-skim ricotta cheese (3 oz.) Part-skim ricotta cheese (3 oz.)

Spinach (2.5 oz.) Spinach (2.5 oz.)

Plum tomatoes (1/4 cup) Plum tomatoes (1/4 cup)

Mozzarella cheese (1 oz.) Mozzarella cheese (1 oz.)

Scallion (1/4 cup) Scallion (1/4 cup)


Coconut milk (1 Tbsp.) Coconut milk (1 Tbsp.)

(prepared as baked spinach & cheese omelet) (prepared as baked spinach & cheese omelet)

Super Supplemental® (2 tablets); Super

Omega 3 EPA® (1 capsule); Probiotic

Eleven® (1 capsule); Berberine IR

® (1

capsule)

Snack

Vegetarian soy sausage (1/2 palm size) Protein Shake (Soy, Pea, or Whey)

Phytosterols (1 scoop)

Beets (1/4 cup) Beets (1/4 cup)

Lunch

Turkey breast, sliced (1 palm size) Turkey breast, sliced (1 palm size)

Red bell peppers (1/2 cup) Red bell peppers (1/2 cup)

Cucumber (1/2 cup) Cucumber (1/2 cup)



Olives (8) Olives (8)

(prepared as wraps) (prepared as wraps)

Berberine IR® (1capsule)

Snack

Parmesan cheese crisps (equivalent to 6 Tbsp.

fresh grated)

Protein Shake (Soy, Pea, or Whey)


Orange (1 small) Orange (1 small)

Dinner

Diced chicken breast (1 palm size) Diced chicken breast (1 palm size)

Mushrooms (1 cup) Mushrooms (1 cup)

Parmesan cheese (2 Tbsp.) Parmesan cheese (2 Tbsp.)


(prepared as spinach and chicken stuffed

mushrooms)

(prepared as spinach and chicken stuffed

mushrooms)

Broccoli (1 cup) (seasoned, steamed or

baked)

Broccoli (1 cup) (seasoned, steamed or

baked)


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® (1

capsule); CardioxLDL® (2 capsules)

Beverages

Water and Herbal teas: unlimited; coffee or tea (black, green, white): 1 cup caffeinated or

decaffeinated

Condiments/Sweeteners

All fresh/dry herbs: dill, oregano, basil, lavender, tarragon, etc. All spices: cinnamon, chili

powder, pepper, ginger, etc. Mustards, horseradish, lemon/lime juice, salsa, vinegars (all

types) (unsweetened), soy sauce, fish sauce (unsweetened). Stevia.

†Vegetables could be eaten raw or steamed and dressed with one of their fats as primary cooking

method. *Shaded areas denote differences between arms.

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Day 2

DIET PROG

Breakfast

Eggs (2 large) Protein Shake (Soy, Pea, or Whey)


Peppers (0.5 cup) Peppers (0.5 cup)





® (1

capsule)

Snack

Almonds (16) Almonds (16)



Lunch

Salmon, chicken or turkey breast (1 palm

size)

Salmon, chicken or turkey breast (1 palm

size)




Snack

Sliced turkey breast (1/2 palm) Protein Shake (Soy, Pea, or Whey)


Apple (1, small) Apple (1, small)

Dinner

Salmon (1 palm size) Salmon (1 palm size)

Bok choy (1 cup) Bok choy (1 cup)

Carrots (1/2 cup) Carrots (1/2 cup)





(vegetables, greens, oils prepared as salad) (vegetables, greens, oils prepared as salad)




®

(1capsule); CardioxLDL® (2 capsules)

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Day 3

DIET PROG

Breakfast

Egg (1 large) Egg (1 large)

Diced chicken breast (1/2 palm size) Diced chicken breast (1/2 palm size)


Onion (1/4 cup) Onion (1/4 cup)

Mushrooms (1/4 cup) Mushrooms (1/4 cup)


(prepared as omelet) (prepared as omelet)




® (1

capsule)

Snack

Almonds (16) Protein Shake (Soy, Pea, or Whey)


Baby Carrots (1/2 cup) Baby Carrots (1/2 cup)

Olives (8) Olives (8)

Lunch

Tuna (4 oz.) Tuna (4 oz.)

Celery (1/2 cup) Celery (1/2 cup)



Snack

Mozzarella cheese stick (2 oz.) Protein Shake (Soy, Pea, or Whey)


Blueberries (1/4 cup) Blueberries (1/4 cup)

Dinner

Halibut (1 palm size) Halibut (1 palm size)

Broccoli (1 cup) Broccoli (1 cup)







(broccoli steamed; other vegetables, greens,

oils prepared as salad)

(broccoli steamed; other vegetables, greens,

oils prepared as salad)




® (1


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Day 4

DIET PROG

Breakfast

Tofu (1 palm size) Protein Shake (Soy, Pea, or Whey)


Spinach (1 oz.) Spinach (1 oz) (blend with shake)

Olive oil (1 tsp) Coconut milk (1 TBS) (blend with shake)

(scramble tofu with sautéed spinach and olive

oil)

(blend spinach and coconut with protein

shake)




® (1

capsule)

Snack

Vegetarian Soy Sausage (1/2 palm size) Vegetarian Soy Sausage (1/2 palm size)

Baby Carrots (1/2 cup) Baby Carrots (1/2 cup)

Hummus (1/2 cup chickpeas, 1 tsp olive oil) Hummus (1/2 cup chickpeas, 1 tsp olive oil)

Lunch




Butter Lettuce (2 oz.) Butter Lettuce (2 oz.)

Mayonnaise, unsweetened (1 tsp) Mayonnaise, unsweetened (1 tsp)

(prepared as chicken salad lettuce wraps) (prepared as chicken salad lettuce wraps)


Snack

Peanut butter (unsweetened) (2 tablespoon) Protein Shake (Soy, Pea, or Whey)



Dinner

Turkey breast (1 palm size) Turkey breast (1 palm size)

Tomato (1/2 cup) Broccoli (1 cup)




Radishes (1/4 cup) Radishes (1/4 cup)

Mixed greens (2 oz) Mixed greens (2 oz.)



(vegetables, greens, oils prepared as salad) (vegetables, greens, oils prepared as salad)




® (1


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Day 5

DIET PROG

Breakfast

Vegetarian soy sausage (1 palm size) Protein Shake (Soy, Pea, or Whey)


Banana (1/2 small) Banana (1/2 small) (blended in shake)




® (1

capsule)

Snack

Cottage cheese (6 oz.) Cottage cheese (6 oz.)

Baby carrots (1/2 cup) Baby carrots (1/2 cup)

Mixed greens (2.5 oz) Mixed greens (2.5 oz)

Lunch



Walnuts (5 halves) Walnuts (5 halves)

Green Onions (1/4 cup) Green Onions (1/4 cup)

Mayonnaise, unsweetened (2 tsp) Mayonnaise, unsweetened (2 tsp)

(prepared as broccoli salad) (prepared as broccoli salad)


Snack

Mixed nuts (1/4 cup) Protein Shake (Soy, Pea, or Whey)



Mixed greens (2.5 oz) Mixed greens (2.5 oz)

Dinner

Chicken breast (1 palm size) Chicken breast (1 palm size)

Peppers (1 cup) Peppers (1 cup)

Onions (1/4 cup) Onions (1/4 cup)

Tomato (1/4 cup) Tomato (1/4 cup)



(fajita skillet, tomato and avocado on the

side)

(fajita skillet, tomato and avocado on the

side)




® (1


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Supplemental Table 2. PROG low glycemic load, high protein shakes (HPS) to be taken twice

per day†

Ingredient HPS1 HPS2 HPS3 % Daily Value

Serving Size (g) 46 45 45 *

Calories 184 180 173 *

Fat (g) 5 3 5 *

Saturated Fat (g) 2 1 1 *

Trans Fat (g) 0 0 0 *

Cholesterol (mg) 32 0 0 *

Sodium (mg) 104 150 307 *

Potassium (mg) 368 95 187 *

Carbohydrate (g) 14 16 16 *

Dietary Fiber (g) 5 3 3 *

Sugars (g) 5 9 8 *

Protein (g) 20 20 20 *

Phytosterols (mg) 2000 2000 2000 *

Vitamin A (IU) 2400 3750 2333 47 - 75 Vitamin C (mg) 29 45 28 47 - 75 Calcium (mg) 960 20 333 2 - 96 Vitamin D (IU) 0.0 300.0 186.7 0 - 75 Vitamin E (IU) 14.4 0.0 14.0 0 - 48 Thiamin (B1) (mg) 0.7 1.1 0.7 47 - 75 Riboflavin (B2) (mg) 0.8 1.3 0.8 47 - 75 Niacin (mg) 9.6 15.0 9.3 47 - 75 Vitamin B6 (mg) 1.0 1.5 0.9 47 - 75 Folate (mcg) 416.0 300.0 186.7 47 - 104 Vitamin B12 (mcg) 2.9 4.5 2.8 47 - 75 Biotin (mcg) 144.0 225.0 140.0 47 - 75 Pantothenic Acid (mg) 4.8 7.5 4.7 47 - 75 Phosphorus (mg) 480.0 0.0 200.0 0 - 48 Iodine (mcg) 0.0 112.5 70.0 0 - 75 Magnesium (mg) 288.0 0.0 133.3 0 - 72 Zinc (mg) 0.0 11.3 7.0 0 - 75 Selenium (mcg) 0.0 52.5 32.7 0 - 75 Copper (mg) 0.0 1.5 0.9 0 - 75 Manganese (mg) 0.0 1.5 0.9 0 - 75 Chromium (mcg) 211.2 90.0 56.0 47 - 176 Molybdenum (mcg) 0.0 56.3 0.0 0 - 75

†Values in table represent single serving amounts and should be multiplied by two to determine

total daily supplementation (e.g. calories per day = 368, 360 and 348, respectively)

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Draft

Supplemental Table 3. Daily PROG nutraceutical supplementation.

Supplement Description

Phytosterols The phytosterol supplement in this study was added to the

low-glycemic, protein shake snack replacement immediately

before preparation and administered at 4 g/day.

Antioxidant formulation CardioxLDL™ - A bergamot fruit extract antioxidant

formulation used in the present study contained 250 mg of

bergamot fruit, standardized to approximately 30%

bergamot flavonoids, and 110 mg of a blend of nine other

phytoextracts: apple fruit extract, blueberry fruit, capsicum

fruit), grape seed extract, grape skin extract, green tea leaf

extract, mangosteen pericarp extract, olive leaf extract, and

turmeric root and rhizome extract per capsule. In the study,

subjects were instructed to take 2 capsules with dinner per

day. Clinically, this formulation has been shown to be well-

tolerated and effective for ameliorating dyslipidemia in

subjects with moderate cardiometabolic risk factors,

particularly in the individuals with HbA1c >5.4%.(Babish et

al. 2016) Samples of all authenticated raw materials in F105

and the commercial product are retained for seven years past

the manufacture date at Nature’s Sunshine facilities.

Probiotics Probiotic Eleven® manufactured by Natures’ Sunshine

Products was the probiotic supplement. Each capsule

containined Bifidobacterium bifidum; B. infantis; B. longum;

Lactobacillus rhamnosus; L. acidophilus; L. bulgaricus; L.

brevis; L. plantarum; L. salivarius; L. casei; Streptococcus

thermophiles, and inulin; fructooligosaccharide; prebiotic

fibers, and magnesium stearate.

Omega-3 fatty acids Super Omega-3 EPA softgels contained approximately

1,000 mg fish oil (380 mg EPA, 190 mg DHA) per softgel.

During the study 1 softgel was taken 2 times daily for a total

of 2g of fish oil (760 mg EPA, 280 mg DHA) as the targeted

daily dose.

Berberine As Berberine IR™ manufactured by Nature’s Sunshine

Products and contained 333 mg berberine HCl powder per

capsule as well as cellulose, magnesium stearate, and silicon

dioxide. Within the study 1 capsule was taken 3 times daily

with meals for a total of 1,000 mg of Berberine as the

targeted daily dose.

High-potency vitamin and

mineral formulation

Super Supplemental®, manufactured by Nature’s Sunshine

Products, is a high-potency multiple vitamin and mineral

supplement with added phytonutrients including ten herbs,

twelve fruit powders and three vegetable powders. Super

Supplemental was directed be administered as 2 tablets

twice daily with meal.

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draft...this study was an exploratory, open label, randomized, 13-week, 2-arm trial conducted at the...

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