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

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

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

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

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

6. 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

7. Ingredients Screening
PPARγ Binding Assay

8. 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 Data are presented as mean ± SD of triplicates.

9. 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.

10. 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 < and **P < 0.01 vs. model control group.

11. 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 < vs. naïve control group, ***P < 0.001, **P < 0.01 and *P < 0.05 vs. model control group.

12. 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 = vs. naïve control group, #P < 0.05 vs. naïve control group.

13. Mechanism Research
Clustering Analyses of Signal Molecules

14. 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:

15. 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

16. 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 B;
2. Fenugreek seed extract：ZL ;
3. Mixture of Mulberry leaf extract and Fenugreek seed extract： ZL

17. 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

18. 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

19. 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;

20. 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

21. 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

22. 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):e
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 B; ZL ; ZL 
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.

23. Thanks for Your Attention !23