Develop a Novel Botanical Formula Preventing Diabetes for China Market Juntao Kan Nutrilite Health Institute Amway (China) R&D Center 1

Background High Prevalence of Diabestes in China IDF Diabetes ATLAS, sixth edition, 2013

Background High Prevalence of Pre-diabestes in China with Rapid Increase 2010 2013 15.5% 50.1% N ENGL J MED 2010,362(12):1090-1101 JAMA 2013,310(9):948-958.

Background Prevent Pre-diabetes for Developing Diabetes The Lancet 2009,373(9682):2215-2221.

CFDA approved products Market Analysis Develop Product for China Market CFDA approved products

Ingredients Screening Cinnamon, Fenugreek seed, Mulberry leaf, Bitter melon American ginseng Dendrobium officinale Literature Astragalus mongholicus, Tree peony bark Tuckahoe Atractylodes macrocephala TCM Philosophy PPARα binding assay PPARγ binding assay Glucose uptake assay α-amylase activity α-glucosidase activity FTL Hits 6 4 24 Screening by functional tests & supplier chain evaluation 11 ingredients 17 samples 13 suppliers American ginseng extract Fenugreek seed extract Mulberry leaf extract

Ingredients Screening PPARγ Binding Assay

Ingredients Screening Activity of Carbohydrate Digestive Enzymes The effect of fenugreek seed (FS) and mulberry leaf (ML) on carbohydrate digestive enzymes. The effect of fenugreek seed on α-amylase (A) and mulberry leaf on α-glucosidase (B) activity were determined in a cell-free, enzyme-substrate assay model. Data were fitted with log (inhibitor) vs. response, variable slope (four parameters) equation in Prism Graph Pad 6.00. Data are presented as mean ± SD of triplicates.

Ingredients Screening Glucose Uptake Assay The effect of fenugreek seed (FS), mulberry leaf (ML) and American ginseng (AG) extracts of insulin sensitivity in differentiated primary human subcutaneous adipocytes. After 20 h of pre-treatment with extracts or botanical test formula, glucose uptake was initiated with the addition of a cocktail containing 2-DOG and 3H-2-DOG with 1 nM insulin and incubate for 2 hours. Change in insulin sensitivity was determined by setting 1 nM insulin as 0% and 100 nM as 100%, and calculating the percent change in the delta between 1 nM and 100 nM insulin. Data are presented as mean ± SD of triplicates. *P < 0.05 vs. 1:1:1 ratio group.

Functional Evaluation Animal Study (High Fat Diet + Alloxan) The effect of the botanical test formula on fasting glycemia status. Fasting (3 h) serum glucose (A), insulin (B) and HOMA-IR (C) in a chronic HFD fed and an acute alloxan induced model of insulin resistance and T2DM treated with the botanical test formula comprised of fenugreek seed, mulberry leaf and American ginseng for 33 d. Naïve control (n=10); model control (n=10); 1X (n=10); 2X (n=9); 4X (n=7). Data are presented as mean ± SD. ###P < 0.001, ##P < 0.01 and #P < 0.05 vs. naïve control group, ***P < 0.001 and **P < 0.01 vs. model control group.

Functional Evaluation Animal Study (High Fat Diet + Alloxan) The effect of the botanical test formula on oral glucose tolerance. Serum glucose levels (A) and AUC (B) in a chronic HFD fed and an acute alloxan induced model of insulin resistance and T2DM treated with the botanical test formula comprised of fenugreek seed, mulberry leaf and American ginseng for 33 d. Animals were fasted 3 h followed by an administration of an oral glucose load (2.5 g/kg) and blood was collected at 0, and 0.5 and 2 h post challenge. Naïve control (n=10); model control (n=10); 1X (n=10); 2X (n=9); 4X (n=7). Data are presented as mean ± SD. ###P < 0.001 vs. naïve control group, ***P < 0.001, **P < 0.01 and *P < 0.05 vs. model control group.

Functional Evaluation Animal Study (High Fat Diet + Alloxan) The effect of the botanical test formula on fasting lipids. Fasting (3 h) triglycerides (A) and total cholesterol (B) levels in a chronic HFD fed and an acute alloxan induced model of insulin resistance and T2DM treated with a test formula comprised of fenugreek seed, mulberry leaf and American ginseng for 33 d. Naïve control (n=10); model control (n=10); 1X (n=10); 2X (n=9); 4X (n=7). Data are presented as mean ± SD. ###P = 0.0015 vs. naïve control group, #P < 0.05 vs. naïve control group.

Mechanism Research Clustering Analyses of Signal Molecules

Mechanism Research Western Blot Analysis for INS/PDK1/GLUT4 Pathway M N F M N F M N F P-IRS1 IRS1 GAPDH GAPDH M N F M N F M N F PDK1 GLUT4 The effect of the test formula (1X dose) on adipose tissue expression of IRS, PDK1 and Glut4. Western blot of proteins in epididymal adipose tissue in a chronic HFD fed and an acute alloxan induced model of insulin resistance and T2DM treated with the botanical test formula comprised of fenugreek seed, mulberry leaf and American ginseng for 33 d. Model control (M); Naïve control (N); Test Formula (1X) (F). Nat Rev Mol Cell Biol 2012,13:383-396

Before (baseline value) Clinical Trial Subjects: 7 T2DM patients Dose: 1 tablet before each meal, 3 times per day Duration: 12 weeks The value of blood parameters and the decline percentage after treatment Blood parameters Before (baseline value) After treatment Decline percentage FPG 12.16±3.93 10.59±3.90 0.1294 2hPG 17.14±3.91 15.155±3.16 0.1166 HbA1c 9.34±1.87 8.83±1.95 0.0550 Cho 5.32±0.60 4.67±1.03 0.1230 LDL 3.57±0.73 2.97±1.08 0.1699 The ratio of the change of blood parameters after treatment for each subject Subjects FPG 2h PG TG Cho HDL-C LDL-C HbA1c(%) #1 0.38 0.17 0.19 0.35 0.20 0.43 0.26 #2 -0.43 -0.25 0.11 0.18 -0.06 0.39 -0.02 #3 0.04 0.08 0.07 0.00 #4 -0.17 0.06 -0.13 0.13 -0.05 #5 0.31 0.01 -0.11 0.09 #6 0.16 -0.04 #7 0.27 0.02

Raw Material Development American ginseng extract Total Ginsenosides ≥ 5.50% Ginsenosides (Rd,Re,Rg1) ≥ 2.0% Ginsenoside (Re) ≤1.5% Import Commercialized Fenugreek seed extract 4-Hydroxyisoleucine ≥ 4.5% Ion exchange and crystallization Import Commercialized Mulberry leaf extract 1-DNJ ≥ 1.12% Resin technology Back up supplier: NGH & Waggot Granulation Patent：1. Mulberry leaf extract (IminoNorm)：CN 102018766 B; 2. Fenugreek seed extract：ZL 200310110779.4; 3. Mixture of Mulberry leaf extract and Fenugreek seed extract： ZL 200510020691.2

Raw Material Development Characteristic of the American Ginseng Constitutes: Over 50 ginsenosides, Rg1, Rc, Re, Rb1, Rb2, Rd…… belonging to 3 types. Specific constitute, 24( R )-pseudoginsenoside F11 (p-F11) Content of ginsenosides fibrous root > main root positive related with cultivated year Panaxan Potential Adulterants Materials Panax ginseng C. A. Mey. leaves Leaves of Panax quinquefolium L. (Fam. Araliaceae). Panax notoginseng (Burk.) F.H.Chen Panax ginseng Panax quinquefolium Panax notoginseng specific constitute Rf p-F11 R1

Raw Material Development Raw Material & Suppliers’ Sample Analysis Content of ginsenosides of Panax quinquefolium L. Content of ginsenosides of P. quinquefolium L. extract Raw material analysis by HPLC, ChP method Fibrous root > main root; Feedstock standard ChP: Rg1+Re+Rb1 > 20 mg/g Hongjiu supplier : 19.2 mg/g (Rg1+Re+Rb1) 绿之健 supplier : 39.7 mg/g (Rg1+Re+Rb1) Lihua : 11.1 mg/g (Rg1+Re+Rb1) Extract ＞210 mg/g , chromotographied

Raw Material Development Extraction Process Evaluation Samples from different process, analysis by HPLC Rg1+Re+Rb1 All 6 ginsenosides Ext. rate Ext. w./100 g Lab sample supplier Feedstocka 44.00 61.3 Process Ab 138.16 192.31 91.4% 29.2 g Hongjiu Pro. b 86.60 19.2 119.70 31.8 52.1% 26.7 g 绿之健 Pro. b 57.82 39.7 77.90 53.90 49.5% 38.9 g Process A: 70% EtOH, 12 V, reflux, 3h, 2 times Hongjiu Pro.: 70% EtOH, 6/5 V, reflux, 1.5h, 2 times 绿之健 Pro.: Water, 8V, 95 ºC, 2h, 2 times A reasonable ratio of the American Ginseng extracts should be determined; Appropriate extraction process can yield a reasonable content of ginsenosides of extracts;

Formulation Development Tooling Design Tooling Formula table Ingredients Qty.(%) Fenugreek seed extract (Granulated) 24.1 Mulberry leaf extract (Granulated) 32.9 American ginseng extract (Granulated) 20.6 MCC 18.4 Silicon Dioxide 0.98 Cellulose Gum 1.9 Magnesium stearate 1.0 Total (mg) 510 Serving Size 3 tablets/Day 510 mg/Tablet

Formulation Development Stability Test Items Unit Initial Target RT 4°C 37°C 1st Mon 2nd Mon 3rd Mon 1-Deoxynojirimycin g/100g 0.32 0.44 0.41 0.38 0.35 0.40 0.49 0.37 4-Hydroxyisoleucine 0.93 1.00 1.13 1.11 0.99 1.14 1.08 1.01 1.09 Total Ginsengsides 0.87 0.98 1.12 Items Units Target Release Min. 1-Deoxynojirimycin g/100g 0.32 0.35 4-Hydroxyisoleucine 0.93 1.02 Total Ginsengsides 0.87 0.95

Summary Multi-Targets  Three ingredients synergistically reduce blood glucose with multi-targets. Multi-Targets  Three paper have been published to support product registration in China: Plos One, 2016, 11(4):e0152728. Chin J Mod Appl Phar, 2016, 33(5):551 BMC Complement Alternat Med, 2017, 17(1):352. American ginseng  Three patents support the extraction process and function of two ingredients, fenugreek seed extract and mulberry leaf extract: CN 102018766 B; ZL 200310110779.4; ZL 200510020691.2  Fenugreek seed extract is an unique ingredient of healthy food in China market .  The quality of a precious ingredient, American ginseng extract, is guaranteed by scientific validation.

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