Probiotics

:

= Cell density = 10 11- 1012 CFU/ml Volume = 6 Cm diameter x 1.5 m Total bacterial number = 1014 CFU/ colon Total bacterial weight = 1.0 ~ 1.5 kg/colon Major products = short chain fatty acids, ammonia, methane, H2, amine, etc. Host : control of medium Who is harmful ? Who is beneficial ? : 1.0-1.5kg 80%

(, Bacteroides) ( ) ( 10-15%) : : ,

=

Microscopy of human fecesElectron microscopy of fecal materials

Birth Infants Maturity Aged 111086 4C. perfringensLactobacillusE. coli, StreptococcusBifidobacteriumBacteroides, EubacteriumLog counts / g feces(Mitsuoka)

/Probiotic BacteroidesBifidobacteriumEubacteriumStreptococcusLactobacillusE. coliC. perfringensStaphylococcusProteus Log CFU/g feces81162 : 1

Actions of the intestinal bacteria on human healthHarmful/pathogenicBeneficialBacteroidesBifidobacteriumEubacteriumStreptococcusLactobacillusE. coliC. perfringensStaphylococcusProteusInfection DirrheaPutrefactionCarcinogen productionInhibition of pathogensDigestionReduction ofputrefactionLog CFU/g feces81162

1970 71990 42007 2

Metabolism of xenobioticsxenobiotics

* Deconjugation and reduction nitroreductase b-glucuronidase b-glucosidase bile acid 7-a dehydroxydsae : secondary bile acid* Fecapentaene* IronDMH colon cancer model : based on elctrophilic DNA binding property and needs activation byb-glucuronidaseSuggested colon cancer causing agents

Primary bile aicdSecondary bile acidBile acidDecrease of detergent propertyTumor promotionEubacteriumClostridiumBacteroidesBifidobacteriumFusobacteriumClostridiumLactobacillusStreptococcus

Metabolism of bile acid by intestinal bacteria

Amino acidAmmoniaProteinBacteroidesE. coliClostridiumBacteroidesFusobacteriumClostridiumStreptococcusProduction of ammonia and amines from proteins by intestinal bacteria amine Decarboxylases arginine agmatine lysine cadaverine ornithine putrescine histidine histamineDeaminaseUreaseTryptophanase

Primary bile acidSecondary bile acidBile acidDecrease in fat solubilization Tumor promotorEubacteriumClostridiumIntestinal bacteriaProduction of tumor promotor from bile acids by intestinal bacteriaHOC NH CH2 CH2 SO3HOHOHOHDeoxycholic acidHOOHOHOCH

Human gut microbes associated with obesity (Ley etc., Nature 444, 1022-3, 2006)

Significance of intestinal microflora to the human healthRole of intestinal microbiota in the develoment of atherosclerosis (Nature medicine 2013, Robert et al.)

- Metaanalysis : no association between dietary saturated fat and atherosclerosis

Meat consumption production of trimethylamine-N-oxide from L-carnitne, choline, phosphatidyl choline by intestinal microbiota atherosclerosis

Calories: 10-15% of the daily caloreis come from the low MW fatty acids produced by intestinal microflora

***

(Turnbaugh , Nature 444, 1027-1031, 2006)

TG (Backhed , PNAS 104, 979-984, 2007)

AMPK Fiaf (Backhed , PNAS 104, 979-984, 2007)

LPL TG

(Ley , PNAS 102, 11070-11075, 2005)

LPS (Cani , Diabetes 56, 1761-1772, 2007)LPS CD14/TLR4 Cf: Bacteroides LPS

Bifidobacterium (Kalliomaki , Am. J. Clin. Nutr. 87, 534-538, 2008)

B2,B6,B12 ? : Bifidobacterium Lactobacillus . (2001. WHO & FAO )

LactococcusLactobacillusBifidobacterium ProbioticsCf) Potential pathogens : Enterococcus Weak probiotics : Streptococcus thermophilus, Lactobacillus bulgaricus

: Lactobacillus : 1 /g : Bifidobacterium : 100 /gL. BulgaricusSt. thermophilus10 /g Leu. mesenteroidesL. plantarum10 /g

WHO & FAO Probiotics Expert Committee (2002) Potential pathogensEnterococcus : Recommended not to be used.L. rhamnosus, Bacillus subtilis, Saccharomyces boulardii : infection- associated cases

Korean FDA: Recommended Enterococcus not to be used.

Health Canada (2003): Prohibited for use in natural health products Enterococcus sp. Bacillus cereus Bifidobacterium dentium Parascardovia denticolens Scardovia inopinta Bacillus clausii CNCM MA23/3V & CNCM MA66/4M Lactobacillus plantarum CNCM MA40/5B-p Pediococcus acidilactici CNCM MA28/6BRecommended not-to-be-used or prohibited lactic acid bacteria

J Clin Gastroenterol. 2006;40(3):276Beneficial in the alleviation of the following -Acute diarrhea -Antibiotic-associated diarrhea -Pouchitis -Immune modulation (TNF-,IL-12,IL-10)

J Allergy Clin Immunol 2008;121, 116-121 Meta analysis: 10 double-blind randomized controlled clinical trials which pooled data from the 6 prevention studies (n = 1581) and 4 treatment trials (n = 299) - Beneficial in the improvement of the atopic dermatitis

Aliment Pharmacol Ther. 2003;17:895-904, Neurogastroenterol Motil. 2005;17:687-696, Am J Gastroenterol. 2006;101(7):1581-90 - Beneficial in the alleviation of irritable bowel syndrome

Clinical effects of some probioticsppt12

Association with endocarditis: Endocarditis by Lactobacillus rhamnosus due to yogurt ingestion? Scand J Infect Dis 2001; 33:710-714. Association with liver abscess: Liver abscess due to a Lactobacillus rhamnosus strain indistinguishable from L. rhamnosus strain GG. Clin Infect Dis 1999;28:1159-1160.

Increase in death from pancreatitis : Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial. Lancet 2008;371:651-659.

Increase in atopy: Probiotic (L. acidophilus LAVRI-A1) supplementation for the first 6 months of life fails to reduce the risk of atopic dermatitis and increases the risk of allergen sensitization in high-risk children: A randomized controlled trial. J Allergy Clin Immunol 2007; 119:184-191No effect of Lactobacillus GG on atopic dermatitis and increased rate of recurrent episodes of wheezing bronchitis Pediatrics 2008; 121, e850-856 Clin. Exp. Allergy 2006; 36, 899-906

.Safety issues related to the probiotics in human studies

Desired characteristics of the probiotics- Beneficial and safe stain of human origin- Selection of the strain with strong ability in various nutrient utilization- High adhesion to the human intestinal cells

The administered probiotic bacteria after meal can survive in the stomach since stomach pH reach 5.0 after food intake

Evaluation of the effect of probiotics on the composition of intestinal microflora through human study

Selection of the probiotic strain and intestinal colonization of the probiotics

Viable cell counts of lactic bacteria in commercial probiotic productsRef : Fundamental Food Microbiology, CRC Press, 2001, p220

Product typecfu/gBenefitClaimedActualHealth Products (Dry)H1Not specified1.5 103 NoH21 1081.6 103No H34 1087.3 102No H4Not specified 2.5 106No

Pharmaceutical Products (Dry)P1Not specified4.8 106MaybeP2Not specified1.3 108YesP35 1093.2 109YesP42 107 2.2 103No

B. bifidum BGN4 : reported in 20 peer-reviewed journals since 1999, full genome sequenced

B. lactis AD011: good growth, full genome sequenced

B. longum BORI: anti-rotavirus, genome sequenced

L. acidophilus AD031

L. casei IBS041

Some of the commercialized probiotic strains developed in our lab

Probiotic Bifidobacterium Screening

Mean numbers of adhering bifidobacteria per 50 Caco-2 cell

Sheet1

SugarB.bifidum BGN-4B.bifidum ATCC 15696

ArabinoseNN

CellobioseNN

FructoseP3P3

GalactoseP3P3

GluconateNN

GlucoseP2P3

LactoseP3P3

MaltoseNN

MannitolNN

MannoseNN

MelezitoseNN

RaffinoseNN

RhamnoseNN

RiboseNN

SalicinNN

SorbitolNN

SucroseNN

TrehaloseNN

XyloseNN

Sheet2

Bifidobacterium strainsAdherent bacteria

BGN-4500

E2-18168

E-15132

JS-984

RD-5467

SI61

SH-247

RD-6042

B. bifidum ATCC 295235

CN230

SJ-3226

KJ24

23

B. animalis ATCC 255222

B. adolescentis ATCC 1570319

SH-517

B. infantis ATCC 1569715

B.longum ATCC 1570711

M-610

MS-110

Sheet3

Bifidobacterium strainsCell surface hydrophobicity

BGN-493

KJ90

90

B. adolescentis ATCC 1570390

B. animalis ATCC 255286

M-685

RD-6069.6

SI66.3

CN-221

E2-18~

E-15~

JS-9~

RD-54~

SH-2~

SJ-32~

MS-1~

SH-5~

B. bifidum ATCC 2952~

B. infantis ATCC 15697~

B.longum ATCC 15707~

30 bifidobacteria , bifidobacteria BGN4 Bifidobacterium sp. BGN4 30 bifidobacteria strain strain BGN4 Electron scanning morphology of BGN4

BGN4 whole cells cell extract ; HT-29 colon cancer cell

Cell line specificity ; BGN4

BB-pol : glucose(31.5%), ribose(23.8%), galactose(11%),rhamnose(3.9%), chiro-inositol(26.3%)

Microarray Analysis of BB-pol on Human 10K oligonucleotide chip - 154 genes in HT-29 and 254 genes in HCT-116 were expressed more than 2-fold in BB-pol treatment- Expression of 62 genes were increased and expression of 13 genes were decreased, respectively, in both cell lines - 24 genes were the genes known to be involved in signaling pathways such as ppp5c, cdc42bpb, ptprf, map3k3, prkdc, prkar1a, ptpra, psphl, and prkcq genes belonging to the PTK or PTPase group. - The expression of tumor suppressors such as TGFBR2 and BIN1 were up-regulated

The effect of Bifidobacterium spp RD65 and BGN4 on number and size of aberrant crypt foci 1) All the numbers are expressed as Mean SE Anti-tumor effect of selected Bifidobacterium strains

GroupACFSizeControlBGN4 102.66 23.1133.00 16.97+++++++

GMPBIFIDO

BGN4

Chart1

9.089.621 ()4.488.558.77

9.58.667.224.877.567.48

8.9710.17.455.097.798.09

10.049.927.974.727.948.38

9.089.877.475.37.68.35

bifidus

bacteroides

Lactobacillus

Clostridium

E.coli

total aerobes

Log of CFU/g

Sheet1

11%22%33%44%55%

0.9180.5360.3660.74881.48148148151.4060.7960.3350.94567.21194879091.1860.7240.3340.79667.11635750421.6390.8020.3371.17471.62904209882.1370.5420.3361.93190.3603182031

1.480.8740.3580.96465.13513513511.5030.7470.3281.08472.1224218231.1850.6630.3320.85472.06751054851.1460.7950.3420.69360.47120418851.9430.8260.3341.45174.6783324756

0.9290.580.3560.70575.88805166851.1880.8340.3290.68357.49158249161.5770.860.3451.06267.34305643631.890.6950.3491.54481.69312169311.1140.5910.3610.88479.3536804309

1.0390.7010.3620.767.37247353221.3650.7680.3340.93168.20512820510.9210.6940.3360.56361.12920738331.3940.720.3391.01372.6685796271.0680.7150.3310.68464.0449438202

1.0510.730.3620.68364.98572787821.0250.7730.340.59257.7560975611.0650.7440.3330.65461.40845070420.9070.6330.3310.60566.70341786110.9090.6840.3330.55861.3861386139

0.490.530.3380.29860.81632653061.7061.0520.3360.9958.0304806565xx00.6030.5720.3310.36260.03316749590.7270.6210.3340.4460.522696011

2.4220.6480.3432.11787.4071015691.1230.570.340.89379.51914514691.3870.7210.3461.01272.96322999280.9020.5460.330.68676.05321507761.3930.5980.3311.12680.8327351041

1.8740.6020.3321.60485.59231590181.1520.4980.3471.00186.89236111111.4310.5810.3371.18782.94898672260.90.4810.3320.75183.44444444441.1430.5820.3430.90479.0901137358

1.970.780.3261.51676.95431472081.4310.7850.3470.99369.3920335431.3220.8930.3330.76257.63993948560.6920.5150.3390.51674.56647398840.7740.6270.3420.48963.1782945736

1.0710.7260.3350.6863.49206349212.1131.0130.3511.45168.67013724561.1430.6530.3390.82972.52843394581.6730.6380.3361.37181.94859533771.0020.6220.3480.72872.6546906188

1.5320.7330.3311.1373.75979112271.2480.7060.3350.87770.27243589740.9920.6760.3380.65465.92741935481.1860.7740.330.74262.5632377741.3640.6990.3471.01274.1935483871

1.2840.660.3460.9775.54517133961.5790.6950.3351.21977.20075997471.5140.7030.3431.15476.22192866581.110.7850.3260.65158.64864864861.0620.550.3370.84979.9435028249

1.6950.7010.3441.33878.93805309730.8220.5030.3340.65379.44038929441.130.5120.3430.96185.04424778761.0410.5160.3280.85381.94044188281.5320.7590.3341.10772.2584856397

Mean73.643692882370.169609364770.194897377671.720276162973.2690369568

SD8.63891228340.32983824710.16670790520.00696603480.23163961299.05501152260.20774509070.10421045140.004956630108.46891625730.39000473370.11786035850.006461920.34233970619.2050016730.42384871210.08425016170.00868316970.41161663778.9423927969

controlintake

Mean72.44803316470.9449427638

SD8.5934661788.9807197096

12345

mean73.6436970.1696170.194971.7202873.26904

control: bifidus

intake: bifidus

Sheet2

pH12345

7.766.726.747.35.83

6.368.298.696.976.17

6.656.836.915.888

7.438.76.79

6.716.877.966.296.71

6.686.716.58

6.155.96.337.425.96

5.726.736.68

7.858.426.797.76.87

6.466.36.275.736.74

7.486.677.148.096.58

6.96.185.847.385.64

6.926.595.475.216.56

mean6.976.76416666676.80636363646.92769230776.5358333333

SD0.5789818650.8272348060.90806687780.98631598930.6151638116

pHcontrolintake

mean6.76382352946.8492

SD0.71503343730.8983360544

Log of CFU1 ()2()3()4()5()

bifidus9.089.58.9710.049.08

bacteroides9.628.6610.19.929.87

Lactobacillus7.227.457.977.47

Clostridium4.484.875.094.725.3

E.coli8.557.567.797.947.6

total aerobes8.777.488.098.388.35

Sheet2

9.089.6214.488.558.77

9.58.667.224.877.567.48

8.9710.17.455.097.798.09

10.049.927.974.727.948.38

9.089.877.475.37.68.35

bifidus

bacteriodes

LBS(lactobacillus)

NN(Clostridium)

DHL(E.coli)

TS( )

Log of CFU/g

Sheet3

bifidus

bacteroides

Lactobacillus

Clostridium

E.coli

total aerobes

Log of CFU/g

95%

BIFIDUSname11 log22 log33 log44 log55 log

A2.0E+099.301.0E+1010.001.3E+1010.112.3E+1010.361.2E+088.08Control mean9.041 2

B6.0E+088.782.2E+1010.341.1E+1010.045.0E+099.701.3E+099.11SD0.69644134140.0479366163

C2.3E+099.363.0E+099.482.7E+077.431.1E+1010.041.2E+077.082 3

D2.0E+099.303.0E+099.481.1E+099.041.7E+1010.233.2E+099.510.0613155172

E2.0E+088.301.0E+099.001.7E+099.232.0E+1010.305.0E+088.70intake mean9.773 4

F9.0E+088.954.0E+088.609.0E+099.951.2E+1010.082.0E+099.30SD0.55979183770.0002334557

H8.0E+088.902.0E+099.301.5E+088.184.0E+1010.606.0E+088.7845

I8.0E+088.906.0E+099.781.1E+099.042.0E+099.305.0E+088.700.0009492926

J1.7E+1010.233.0E+1010.482.0E+088.301.7E+1010.235.0E+099.70

K1.5E+099.186.0E+099.784.5E+088.651.2E+1010.083.5E+099.54

L3.0E+088.485.0E+088.703.0E+088.481.5E+099.183.8E+099.58

M1.3E+099.112.1E+099.322.6E+099.412.0E+1010.308.0E+099.90

N1.6E+099.202.0E+099.306.0E+088.781.5E+1010.181.2E+1010.08

mean9.089.508.9715038461538.4610.043117846153.859.08

SD0.470.570.790.410.82

BACTEROIDES BL name10.0020.3030.4840.6050.70

A9.0E+099.952.0E+1010.302.0E+1010.304.9E+1010.69Control mean9.601 2

B5.0E+099.707.0E+099.851.4E+1010.155.0E+099.701.0E+1010.00SD1.71595547490.7957837163

C7.4E+099.873.0E+088.487.0E+099.855.0E+099.705.0E+099.702 3

D0.006.0E+088.781.5E+1010.185.0E+099.701.0E+099.000.0025039388

E1.3E+099.111.0E+088.001.8E+1010.261.7E+1010.231.0E+1111.00intake mean9.293 4

F2.8E+099.451.0E+099.001.4E+1010.151.7E+1010.23SD1.23554132360.2292461375

H2.1E+099.323.0E+088.482.0E+1010.301.0E+1010.007.0E+099.8545

I5.0E+088.701.0E+088.001.0E+099.003.0E+099.48

J1.0E+1010.009.0E+099.954.0E+1010.606.0E+099.782.0E+099.30

K1.2E+1010.087.0E+088.851.5E+1010.181.7E+1010.231.0E+1010.00

L7.5E+099.881.0E+088.005.0E+099.705.0E+099.703.0E+1010.48

M6.7E+099.834.0E+044.601.8E+1010.267.0E+099.851.1E+1010.04

N4.0E+099.602.0E+1010.302.5E+1010.405.0E+099.702.0E+099.30

mean9.628.6610.1011615384615.389.9217800000000.009.87

SD0.411.490.400.3430065853647.540.59

LBSname10.0020.3030.4840.6050.70

A0.002.9E+088.465.2E+088.722.5E+1010.405.0E+066.70Control mean7.46

B0.002.4E+088.384.0E+077.603.0E+099.48SD1.2588913939

C0.004.9E+088.697.0E+055.853.2E+088.501.0E+077.00

D0.004.0E+055.603.0E+055.481.0E+077.002.0E+077.30

E0.007.0E+055.851.7E+066.235.4E+088.731.6E+088.20intake mean7.69

F0.005.0E+055.701.9E+088.285.5E+055.741.0E+077.00SD1.4148745064

H0.002.4E+088.383.9E+088.591.6E+099.213.4E+088.53

I0.009.0E+077.955.5E+077.744.6E+055.66

J0.008.5E+055.933.0E+055.482.0E+055.30

K0.002.1E+088.323.2E+088.516.0E+077.78

L0.001.2E+066.081.0E+077.00

M0.002.2E+099.343.3E+088.521.6E+099.20

N0.007.0E+077.855.5E+077.741.7E+088.231.0E+088.00

mean0.007.227.452184680769.237.97408896923.087.47

SD1.431.331.391.24

NNname10.0020.3030.4840.6050.70

A2.5E+033.403.0E+044.483.3E+055.52XControl mean4.93

B4.0E+044.60too many to countx3.0E+044.48SD0.8604835141

C2.0E+044.305.0E+044.706.0E+044.782.0E+055.303.5E+055.54

D2.0E+055.305.0E+033.706.5E+044.816.0E+044.783.5E+044.54

E5.0E+044.701.3E+077.115.5E+044.746.0E+044.786.5E+033.81intake mean4.79

F5.0E+033.706.5E+066.811.3E+055.117.0E+033.855.0E+066.70SD1.0818010215

H3.0E+044.482.8E+055.454.5E+044.653.2E+055.513.0E+044.48

I8.0E+033.901.1E+033.044.0E+044.606.0E+044.781.1E+055.04

J1.5E+055.187.0E+033.853.3E+055.522.0E+044.309.5E+033.98

K1.5E+055.181.3E+066.116.0E+055.788.0E+055.907.5E+066.88

L1.5E+055.181.2E+044.083.5E+055.545.0E+022.706.5E+044.81

M7.0E+044.854.6E+044.661.0E+066.001.0E+066.006.3E+055.80

N3.0E+033.482.5E+044.401.0E+044.002.0E+044.305.0E+066.70

mean4.481635084.624.87231923.085.09198269.234.721441230.775.30

SD0.681.260.590.921.11

BL()name10.0020.3030.4840.6050.70

A1.2E+1010.080.000.000.000.00

B5.5E+099.740.000.000.000.00

C1.1E+1010.040.000.000.000.00

D0.000.000.000.000.00

E1.6E+1010.200.000.000.000.00

F7.0E+099.850.000.000.000.00

H2.5E+1010.400.000.000.000.00

I5.0E+099.700.000.000.000.00

J2.0E+1010.300.000.000.000.00

K2.0E+1010.300.000.000.000.00

L1.2E+1010.080.000.000.000.00

M8.5E+099.930.000.000.000.00

N7.0E+099.850.000.000.000.00

mean10.040.000.000.000.000.000.000.000.00

DHLname10.0020.3030.4840.6050.70

A2.0E+099.309.2E+099.964.9E+099.698.0E+077.90Control mean7.97

B3.4E+088.531.1E+077.045.3E+077.724.0E+077.609.4E+066.97SD0.8492713456

C2.4E+088.381.1E+066.043.0E+066.480.004.9E+066.69

D1.6E+088.201.3E+077.111.5E+077.181.4E+088.151.7E+066.23

E3.5E+088.543.4E+066.536.0E+077.783.0E+088.48intake mean7.44

F2.2E+088.349.9E+067.001.0E+077.001.4E+088.15SD1.7724742134

H5.1E+088.713.6E+077.565.3E+077.725.0E+077.708.0E+077.90

I9.6E+088.983.0E+077.481.6E+077.206.0E+077.782.8E+066.45

J5.6E+088.753.6E+066.568.0E+077.903.0E+077.485.2E+088.72

K2.4E+088.383.2E+088.511.8E+088.265.3E+088.721.3E+088.11

L2.7E+088.435.0E+088.705.7E+077.766.0E+077.784.7E+077.67

M7.9E+088.903.7E+099.571.3E+099.115.7E+077.763.7E+077.57

N3.0E+077.481.8E+077.261.5E+077.187.0E+077.858.0E+077.90

mean8.55404675000.007.56915158333.337.79500583333.337.94119400000.007.60

SD0.461.030.962.300.78

TS()name10.0020.3030.4840.6050.70

A2.0E+099.303.0E+099.481.4E+088.15Control mean8.41

B4.6E+088.661.3E+066.113.7E+088.572.9E+088.461.1E+088.04SD0.6365416962

C3.6E+088.563.8E+066.585.7E+066.765.0E+088.701.7E+077.23

D4.0E+088.604.0E+066.601.2E+077.086.0E+077.789.3E+077.97

E2.8E+099.451.1E+077.041.3E+099.118.0E+077.902.9E+088.46intake mean7.95

F7.0E+077.852.3E+088.364.5E+077.659.0E+077.953.9E+088.59SD0.9052736915

H1.1E+099.043.8E+077.582.2E+088.348.0E+077.905.0E+088.70

I9.9E+089.001.0E+088.005.0E+077.701.4E+088.159.7E+077.99

J7.4E+088.875.6E+066.751.9E+088.281.0E+088.001.3E+099.11

K4.3E+088.635.8E+077.763.4E+088.538.3E+088.922.2E+088.34

L6.0E+088.782.5E+088.401.4E+088.151.7E+088.231.9E+088.28

M9.8E+088.993.2E+099.511.9E+099.283.2E+099.512.4E+099.38

N1.7E+088.231.3E+077.114.0E+077.609.0E+077.952.2E+088.34

mean8.77301130769.237.48354823076.928.09664615384.628.38459000000.008.35

SD0.430.970.760.600.54

TS()name10.0020.3030.4840.6050.70

A2.6E+077.410.000.000.000.00

Bx0.000.003.5E+077.540.000.00

Cx0.000.002.3E+066.360.000.00

Dx0.000.008.0E+055.900.000.00

E3.6E+099.560.000.000.000.00

F1.0E+077.000.002.1E+088.320.000.00

H1.8E+088.260.000.000.000.00

I1.0E+077.000.000.000.000.00

J2.0E+077.300.000.000.000.00

K1.5E+088.180.000.000.000.00

L1.4E+088.150.000.000.000.00

M1.6E+088.200.000.000.000.00

N7.0E+077.850.000.000.000.00

0.000.000.000.000.00

0.000.000.000.000.00

TS(name10.0020.3030.4840.6050.70

A2.0E+099.300.008.8E+099.940.000.00

B4.6E+088.661.3E+066.112.1E+088.320.000.00

C3.6E+088.563.8E+066.582.3E+066.360.000.00

D4.0E+088.604.0E+066.607.8E+066.890.000.00

E2.3E+088.361.1E+077.040.000.000.00

F7.0E+077.852.3E+088.360.000.000.00

H1.0E+099.003.8E+077.580.000.000.00

I9.9E+089.001.0E+088.000.000.000.00

J7.1E+088.855.6E+066.750.000.000.00

K2.8E+088.455.8E+077.760.000.000.00

L4.6E+088.662.5E+088.400.000.000.00

M8.2E+088.913.2E+099.510.000.000.00

N0.001.3E+077.110.000.000.00

0.01% .

.

* Damage of host epihelial and immune cells* Proliferation or death of bacteria* Immunostimulation or immunosuppression : effector cells and cytokines mitogenic activity* Outcome : defense from pathogenic bacteria and tumor inflammation

Factors and implicationsof bacterial immunomodualtion

Immunomodulation of Bifidobacterium

Animal model * I. P. or S. C. injection of Bifidobacterium * Inhibition of transplated tumor * Activation of macrophage * Induction of cytokines (IL-6, TNF-a, IL-1b, IL-10)

Cell culture model : mice peritoneum or cell line * Proliferation of B cell * Activation of macrophage * Phagocytic activity * Cytotoxic activity against tumor cell line

Human : oral feeding * Increase of phagocytic activity of blood monocytes and granulocytes

: . , . .

: VS

Dendritic cell Figure from Kapsenberg. Nature Review immunol 2003 Innate immunityAdaptive immunityInnate immunityAdaptive immunityBacterial components

(Ref: Haller, 2005)

(Ref: Haller, 2005)

Too cleanenvironmentTh2dominantAllergyMicrobial challenge Benign challenge ProbioticsSevere Infection Enhanced Treg (Tr1, Th3) activity Normal Th1/Th2/Th17 balanceAllergy aggravation by Th1/Th17-associated inflammationNo AllergyAllergy prevention by probioticsModified from Ji GE. Forum Nut. (61, 117-128, 2009)

Effect of B. bifidum BGN-4 in peanut antigen-induced allergy mice

Effect of the integrity of the administered B. bifidum BGN-4 on the prevention of allergic symptoms

Effect of timing of the administration of B. bifidum BGN-4 on the prevention of allergic symptoms

BloodIgE IgG1 Histamine Eotaxin IgG2a

Clinical experiment Effect of probiotics on the occurrence of atopy-3~6 month -4~8 w Birth 3 month 6 month 9 month 12 month Probiotics motherbabyBrest-feedingweaningRestriction on eggs and nuts

volunteerExam: SASSADexamexamexamfecesfecesBlood:CAP-FEIADesign; Double-blind, Randomized, Placebo-controlled, Clinical trial Probiotics; B. bifidum BGN4, B. lactis AD011, L. acidophilus AD031 each with 1.6 x 109 CFU /stick. Once daily.

Participants characteristics

ProbioticsPlacebop valuen (%)n (%)study completed numbers 3335Mother age (Years)*30.24 0.4929.70 0.510.446Indirect smoking (father)7 (21.2)13 (37.1)0.15Older sibling4 (12.1)6 (17.1)0.379Allergic disease Mother24 (72.7)28 (80.0)0.48 Father18 (54.5)13 (37.1)0.15 Older sibling4 (12.1)4 (11.4)0.929Family history AD21 (63.6)25 (71.4)0.492 Asthma, AR, and FA16 (48.5)13 (37.1)0.345DP levels of parents (KU/L)*8.29 2.0112.48 3.790.324DF levles of parents (KU/L)*13.29 3.7416.96 4.390.526Boys15 (45.5)15 (42.9)0.829Caesarean delivery5 (15.2)11 (31.4)0.114Gestation age (weeks)*39.66 0.2539.47 0.190.556Birth weights of infants (Kg)*3.33 0.073.25 0.060.406

: immune cell (peyers patches, spleen cells): intestinal cell (without pp cells): Probiotics (whole cell, cell wall, cell extract)Co-culture designImmune cells and intestinal cells were co-cultured with probiotic BifidobacteriumThe unit of IFN-r is pg/ml.The unit of BGN4 is ug/ml. wh: whole cellscw: cell wall fractionex: cell extract fraction Co-culture

Chart2

201.624261.952

191.972380.195

225.755271.605

206.451232.995

228.169232.995

211.277216.103

158.188259.539

182.319274.018

230.582232.995

148.536237.821

191.972305.388

237.821237.821

PP

SP

BGN4 (ug/ml)

IFN-r (pg/ml)

IFN-r production in co-culture

Sheet1

2002.12.17-19

mouse C3H/Hej : intestinal cell + Peyer's patch or Spleen cell co-culture

BGN4 fraction pattern ( il-6, IFN-r )

IL-6

BGN4PPSP

wh185.271159.095

wh102.219199.084

wh5014.523417.481

cw123.751146.791

cw100426.709

cw502.219629.726

ex142.207128.335

ex1023.751254.452

ex508.371568.205

ex50 noIC694.322488.229

only IC32.979152.943

NOIMMUN00

IFN-r

BGN4PPSP

wh1201.624261.952

wh10191.972380.195

wh50225.755271.605

cw1206.451232.995

cw10228.169232.995

cw50211.277216.103

ex1158.188259.539

ex10182.319274.018

ex50230.582232.995

ex50 noIC148.536237.821

only IC191.972305.388

NOIMMUN237.821237.821

Sheet1

PP

SP

BGN4 (ug/ml)

IL-6 (pg/ml)

IL-6 production in co-culture

Sheet2

PP

SP

BGN4 (ug/ml)

IFN-r (pg/ml)

IFN-r production in co-culture

Sheet3

Probiotics in primary prevention of atopic disease at 1 year ( )PlaceboProbioticsFrequency of atopy at 1 year (%)14/35 (40%)6/33 (18%)p < 0.05

Cytokine production from peripheral blood mononuclear cells (PBMCs) isolated from probiotics and placebo group

Skim milkBGN4 Transplantation of CD4+CD45RBhigh T cell into scid mouse

4 weeks1 w2 w3 wExperimental periodMorphology HistologyCytokines Cell phenotypesBalb/cCD4CD45RBPurification of CD4+CD45RBhigh T cell (40% of CD4+ T cells)CD4CD45RBSCIDTransplanted into scid mouseAnalysisAnti-inflammation of B. bifidum BGN4 in the intestine (Co-works with GALT Immunology, Tokyo University)BGN4-, skim milk-containing diets

Skim milkBGN4**BGN4SkimLarge intestine & spleenHistology of large intestinePatterns of inflammatory T cells and cytokinesShowed that BGN4 had anti-inflammatory effect in the CD4+CD45RBhigh T-cell transplanted mouse model. Anti-inflammation of B. bifidum BGN4 in the intestine

Products containing mixtures of B. bifidum BGN4, B. lactis AD011, L. acidophilus AD031, L. casei IBS041 (each with 5 x 109 CFU /stick)

Cliniclal Experimental ProtocolsDouble-blindRandomizedPlacebo-controlledClinical trial 1 stick, twice daily for 8 weeks

Effect of B. bifidum BGN4 containing probiotic products on irritable bowel syndromes ( )

Demographic of participants* Bristol Stool Scale : average for a week Stool passaged per week

CharacteristicsProbiotics (N=36)Placbo(N=34)Age (years) Mean (SE)36 238 3 Range21-6922-72Females (n)1112Baseline score (SE) Pain50.3 (3.5)46.9 (3.4 ) Flatulence49.9( 3.6)49.3(4.8 ) Defecation53.5( 4.0)47.0(5.1 ) Sum 153.6(8.9 ) 143.2(9.0 ) QOL104.6(1.1 )104.7(1.4 )Stool consistency*4.24.0Defecation frequency8.17.1

PainFlatulenceITT population(N=70)Subgroup(N=58)P=0.045*P=0.006P=0.004**Effect of B. bifidum BGN4 containing probiotic products on irritable bowel syndromes

Defecation discomfortSum ofscoresITT population(N=70)Subgroup(N=58)P=0.043**P=0.013P=0.010P=0.010**Effect of B. bifidum BGN4 containing probiotic products on irritable bowel syndromes

Probiotics vs. placebo Abdominal pain 64% vs 38% (P=0.045) Defecation discomfort 55% vs 29% (P=0.043)

< Subgroup > : baseline Bristol Stool Form Scale 3 Abdominal pain 66% vs 29% (P=0.006)Defecation discomfort 57% vs 24% (P=0.013) Sum of scores 59% vs 31% (P=0.010).Effect of B. bifidum BGN4 containing probiotic products on irritable bowel syndromes

Global Metabolomics : , , Extraction and isolation of metabolite Urine samples 100 l Add 100% cold H2O 400 l Vortexing for 10 min Centrifuge at 14,000 g for 20 min to remove particulates (4C) Injection 5 l into HPLC

Chromatographic separation (Agilent 1200 series HPLC)Mobile phase A: 2 mM ammonium formate in H2O (0.1% formic acid)Mobile phase B: 100% methanol (0.1% formic acid) Flow rate : 0.4 ml/minColumn : Agilent SB-C18 1.8 m (2.1 50 mm)Gradient condition : 2% B at 0.1 min 98% B at 13 min 98% B at 15 min 2% B at 17 min Elapsed time 21 min

Q-TOF MS(Agilent Q-TOF 6530 mass spectrometer) Ion polarity mode: positive Data storage : centroid Mass range : 100-1100 m/z ESI vcap : 3500 V MS TOF Fragment : 170 V MS TOF Skimmer : 65 V OCT 1 RF Vpp : 750 V Nozzle voltage : 1000 V ESI gas temperature : 325 Drying gas : 11 l/min Nebulizer : 30 psig

Molecular Feature Extraction (MFE)Agilent Mass Hunter Qualitative Software (version B.02.02) Restrict retention time : 0.1-14 min Restrict m/z :100-800 Absolute height threshold : 100K, 50K, 10K counts Peak spacing tolerance : 0.0025 m/z, plus 5 ppm

Multivariate analysis : PCAAgilent Mass Hunter Profiler Professional (MPP) software (version B.02.02) Possible flags : present, marginal Retain entities : 80% of the values in any 1 out of 2 conditions Frequency : 100 P-value < 0.05 (t-test) Fold change (FC) Absolute > 2.0

Fold changes for endogenous metabolites from global metabolomics

Metabolite (ESI+)4,6-DihydroxyquinolinePhenylpropionylglycinePalmitic acid methyl esterN-palmitoyl glycinePalmitoleoyl ethanolamide PregnanetriolProbiotics treatment (days)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)Fold change (vs. day 0)0.630.660.960.851.151.791.332.271.122.041.121.28p-value (vs. day 0)0.020.010.720.130.170.000.020.000.280.000.010.00

Metabolite (ESI-)2-Ketobutyric acidHomogentisic acid L-KynurenineN-Acetylserotonin4-(2-Aminophenyl)-2,4-dioxobutanoic acid Cholic acidProbiotics treatment (days)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)28 (V2)56 (V3)Fold change (vs. day 0)0.910.650.720.860.170.381.081.120.320.320.480.58p-value (vs. day 0)0.640.010.050.680.000.030.610.690.040.050.070.09

Palmitic acid methyl ester (MP)

A fatty acid ester whose concentration in cells is modulated by methanol. In studies with isolated Kupffer cells, MP inhibits phagocytosis and decreases cell viability. In cells treated with lipopolysaccharide, it also decreases secretion of interleukin-10, TNF-, nitric oxide, and prostaglandin E2. This effect is thought to occur by the inhibition of NF-B.N-palmitoyl glycine

The acyl amides are a family of endogenous lipids that act as potent modulators of pain and inflammation.ESI+-> anti-inflammation-> anti-inflammationp

: Reoviridae 1943 Light Hodes .

rotavirus 47% 20% .

2001 .

1999 (Wyeth) (FDA) .

: probiotics

Screening for anitrotaviral bifidus Internal sequencingPCR-cloning

Chart1

00

2020

4020

4020

8060

100100

47

180

(mg/ml)

(%)

Sheet1

47180

000

0.12020

0.54020

14020

28060

3100100

Sheet1

00

00

00

00

00

00

47

180

(mg/ml)

(%)

Sheet2

Sheet3

BIFIDOBORI ( )[, ]

Human Milk Oligosaccharide/Structure 3 , , , Prebiotics

Bifidobactrium longum RD47

()

GRAS Cell factory Oral delivery, Tissue delivery, ,

(, 2006.5)

- ,, ,

- . . . . . . . . . . . . . .

- :

GRAS-hostCORE TECHNOLOGYBifidobacteriumGRAS promoterGRAS rep geneMCSGRAS-vectorGRAS : Generally Recognized As SafeGRAS selection markerBIFIDO

RepARepBMobAMobBiteronpKJ50pKJ36Genetic organization of two plasmids, pKJ50 and pKJ36OriTiteronOriTIdentity : 57% similarity : 70%Identity :48% similarity :61%PstIPstIBamHIBamHIMembBMembA

marker Genomic DNA from BifidobacteriumShot-gun cloning; EcoRI, BamHI, Sau3AIpBR322-xylosidase-galactosidaseamylaseAprTcr4MU - xylosideX-gal, IPTGStarch, Iodine

Genomic DNA from BifidobacteriumShot-gun cloning; Sau3AIMCSPromoter probe vectorMCSPb1Pb2Pb3Pb4Pb5Pb6Promoter vectorSequencing analysis and apply to Bifidobacterium Expression system promoter

pMG1 (3.6kb) TsR (1.3 kb)pUC19 (2.67 kb)Hind IIISph IPst IPst IpBES2-thio(7.6 kb)Kpn ISma IBamH I Xba IEcoR ITsR: StreptomycesG+C=65%CAT: StaphylocuccusG+C=30% 50

A B C D EEndostatin gene expression in Bifidobacterium-galactosidase promoter

Gene expression with Bifidobacterium vector * endostatin, * amylase * cholesterol oxidase * phytase * human IL-18 * GAD (GABA production) * BORI * Bacteriocin

Amylase expression in various Bifidobacterium hosts

Chart3

0.050.1110.0980.0570.12

-0.0110.0670.051-0.0120.088

0.1590.1260.1330.2680.161

0.3220.7280.210.9920.156

1.5792.2450.3212.9780.165

1.8361.8241.2172.8550.239

2.1521.9691.3592.3780.188

1.6721.5931.1321.8540.171

2.1571.0990.7881.3250.179

2.0340.8590.6211.0470.176

1.8760.6830.580.7350.14

Int57

BES

KJ

142T

142W

hr

OD570

DNS-broth

Sheet1

0H4H8H12H16H20H24H28H32H36H40H

Int570.05-0.0110.1590.3221.5791.8362.1521.6722.1572.0341.876

BES0.1110.0670.1260.7282.2451.8241.9691.5931.0990.8590.683

KJ0.0980.0510.1330.210.3211.2171.3591.1320.7880.6210.58

142T0.057-0.0120.2680.9922.9782.8552.3781.8541.3251.0470.735

142W0.120.0880.1610.1560.1650.2390.1880.1710.1790.1760.14

Sheet1

Int57

BES

KJ

142T

142W

hr

od570

DNS-broth

Sheet2

Sheet3

N-region : a positive charged N-terminusH-region : a hydrophobic coreC-region : a neutral or negatively charged C-terminus containing the SP cleavage site The structure of secretion proteinAll SPs display a common tripartite structure.SP : Signal PeptideGram (-) < Gram (+)Cleavage site24

660 bp750500PCRConstruction of pBESAF2Expression & secretion vector29

Medium : Modified MRS except for phosphate reagentCell extract : 9.5X concentration , SonicationSupernatant : CentrifugationPhytase Liquid Assay : Heinonen-Lahti Method94%6%Figure 1. Phytase assay measuring phosphate released by phytase

BIFIDO

Phytase activity of B. longum MG1 (appA)36

Cholesterol oxidase gene expression in Bifidobacterium16S rDNA promoter

Amylase

Bioconversion using cloned -glucosidase Ginseng extractRb1Rb2

Gene expression with Bifidobacterium vector * endostatin, * amylase * cholesterol oxidase * phytase * human IL-18 * GAD (GABA production) * BORI * bacteriocin

* b-glucosidases

Bifidobacterium 10% -glucosidase, -glucosidase, -galactosidase, -galactosidase, -xylosidase, -fucosidase, -arabinofuranosidase, -fructofuranosidase, -mannosidase

SpeciesNo. of StrainsCountryBifidobacterium bifidum BGN4Bifidobacterium lactis AD011Bifidobabcterium longum Bifidobacterium breveLactobacillus caseiLactobacillus gasseriLactobacillus helveticusLactobacillus johnsonniLactobacillus plantarumLactobacillus rhamnosusLactobacillus sakeiLactococcus lactisLeuconostoc mesenteriodes1343413111111KoreaKorea, Fr, DMNetherland, USA, JPNJPN, IRN USAFrance, USA USA, FranceUSAUSASwissNetherlandNew ZealandFranceFrance, Netherland,England, USAUSA, KoreaFrance, USA USANetherland

Full genome sequences of selected bifidobacteria(J. Bacteriol. 2009)

COG ClassBBBBLOBLDBADBLAECO**INFORMATION STORAGE AND PROCESSING (%)286 (24.96)282 (22.71)338 (25.39)271 (23.40)256 (23.17)628 (14.83)Translation, ribosomal structure and biogenesisJ130124127121126171RNA processing and modificationA111112TranscriptionK6778806252229Replication, recombination and repairL81751258374214Chromatin structure and dynamicsB000000INFO-NULL7454312CELLULAR PROCESSES AND SIGNALING (%)177 (15.45)186 (14.98)184 (13.82)175 (15.11)171 (15.48)654 (15.45)Cell cycle control, cell division, chromosome partitioningD161516141433Nuclear structureY000000Defense mechanismsV353739362648Signal transduction mechanismsT212324202288Cell wall/membrane/envelope biogenesisM5158546056207Cell motilityN1**000022CytoskeletonZ000000Extracellular structuresW000000Intracellular trafficking, secretion, and vesicular transportU71097834Posttranslational modification, protein turnover, chaperonesO4239383441118CELL-NULL44444104METABOLISM (%)401 (34.09)488 (39.29)501 (37.64)459 (39.64)420 (38.01)1377 (32.52)Energy production and conversionC4044433541266Carbohydrate transport and metabolismG7712613911594282Amino acid transport and metabolismE103123123128114273Nucleotide transport and metabolismF475252465076Coenzyme transport and metabolismH3131302921112Lipid transport and metabolismI242728242473Inorganic ion transport and metabolismP4654555249179Secondary metabolites biosynthesis, transport and catabolismQ3331134META-NULL302828292682POORLY CHARACTERIZED (%)169 (14.75)170 (13.69)182 (13.67)149 (12.87)161 (14.57)674 (15.92)General function prediction onlyR981081128794350Function unknownS7162706267324POOR-NULL000000

NULL3835383230620MULTI(example category such as LT or GATC)7581887267281Total genes (%)1146 (62.45)1242 (71.92)1331 (66.88)1158 (71.00)1105 (72.32)4234 (98.60)* threhold : Score >= 60**ECO, Escherichia coli K12 MG1655*** BBB_0945 (score, 75,3; e-value, 4e-12; Pid, 37.78) : conserved hypothetical protein is homologous with COG5492 (Bacterial surface proteins containing Ig-like domains)

COG functional classication of B. bifidum BGN4 and other human intestinal bacteria

- * -Scatter Plot MatrixB. bifidum BGN4 transcritome - correlation

- * -B. bifidum BGN4 transcritome - cluster analysis

SWOT

() (), ()

.

Gycosides are the principal bioactive components of natural phytochemicals and can be converted into more active aglycoside forms by microorganisms or their enzymes

SOFT-MORINING BIFIDOLAB FOR BETTER LIFE :

Chart8

1002000

0012040

57000

005100

0

18

Daidzin

Daidzein

Genistin

Genistein

(/)

Sheet1

1(5%)48

2(1.5%)50.5

3(0.15%)54

4(control)63

1(5%)138

2(1.5%)118

3(0.15%)122

4(control)121

1(5%)480

2(1.5%)220

3(0.15%)145

4(control)680

Daidzin100050

Daidzein200700

Genistin012005

Genistein0400100

Sheet1

0

0

0

0

group

(mg/dl)

1.

Sheet2

0

0

0

0

group

(g)

3.

Sheet3

0

0

0

0

Grpup

/60min

2. SD

0000

0000

0000

0000

0

18

Daidzin

Daidzein

Genistin

Genistein

(/)

Isoflavone aglycones of soymilk were absorbed faster than their glucosides in adults. Kano et al. J. Nutr. 136, 2291-2296 (2006) There are some other conflicting results.

Transformed ginsenosides such as Rh1, F1, and compound K were found in the serum of humans after ginseng intakes. Tawab et al. Drug Metab Dispos. 31:1065-1071 (2003)

Development and Efficacy Assessment of Fermented Ginseng

Geun Eog Ji, Seoul National University

: - (protopanaxatriol Re, Rg1) R&D . - . . . (Pharmaton, Ginsana) . $30 /.

Human large colon : Bacterial fermentor* Cell density = 10 11- 1012 CFU/ml* Volume = 6 Cm diameter x 1.5 m* Total bacterial number = 1014 cfu/ colon

GinsenosidesDegree of transformation to glycosides differ depending on the individual Bioavailable forms of ginsenosides : Rh1, F1, compound K . Tawab et al. Drug Metab Dispos. 31:1065-1071 (2003)

Positive results of ginseng in double blind randomized clinical trail

Sexual function erectile dysfunction (p.g.) de Andrade et al (2007)Sexual function erectile dysfunction (p.g.) Hong et al (2002)Sexual function erectile dysfunction (p.g.) Kim et al (2009)Endurance exercise untrained adults (p.ng.) Liang et al (2005)Acute respiratory sub healthy seniors (PS rich p.q.) McElhaney et al. (2004)Acute respiratory sub healthy seniors and adults (PS rich p.q.) McElhaney et al. (2006)Cold sub healthy adults (p.q.) Predy et al (2005)Cold antinfluenza vaccine (p.g.) Scaqlione et al. (1996)(Ab titer, NK activity)Respiratory function chronic pulmonary disease (p.g.) Gross et al (2002)Immunomodulation healthy subjects (p.g.) Scaqlione et al. (1990)Quality of life cancer (p.g.) Kim et al (2006)Cancer fatigue cancer patients (p.q.) Baton et al. (2009)Blood glucose healthy young (p.g.) Reay et al (2006)Blood glucose NIDDM patients Sotaniemi et al. (1995)Blood glucose type-2 diabetes Vuksan et al. (2008)Blood glucose healthy subjects Sievenpiper et al. (2006)Psychomotor performance exercisers Ziemba et al. (1999)Quality of life healthy young (p.g.) Ellis et al. (2002)Quality of life memory-impaired Neri et al. (1995)Cognition healthy subjects (p.g.) Kennedy et al (2002)Sleeping healthy subjects (fermented ginseng) Kitaoka et al. (2009)

Negative results of ginseng in double blind randomized clinical trailExercise performance healthy young (p.g) Allen et al (1998)Exercise recovery healthy women (p.g.) Engels et al (2001)Physical performance healthy adults (p.g.) Engels et al (2003)Blood pressure hypertension(p.q.) Stavro et al (2006)Blood sugar healthy adults (p.g.) Reay et al. (2009)Quality of life postmenopausal women (p.g.) Wiklund et al (1999) Psychological well-being healthy young adults (p.g.) Cardinal et al (2001)Psychological responses healthy adults (p.g.) Engels et al (1997)

Ginseng-related human stuies Rh1, F1, compound K . Tawab et al. Drug Metab Dispos. 31:1065-1071 (2003) , 93% Kobayashi et al. Diet and Japanese herbal medicine for recalcitrant atopic dermatitis: efficacy and safety. Drugs Exp Clin Res. 30:197-202 (2004)2. IgE . Kobayahsi . The effects of Hochu-ekki-to in patients with atopic dermatitis resistant to conventional treatment. Int J Tissue React. 26:113-117 (2004).

3. : cd63 (basophil degranulation marker ) Caruso . Effects of a new nutraceutical ingredient on allergen-induced sulphidoleukotrienes production and CD63 expression in allergic subjects. Int Immunopharmacol. 20:1781-6. 2008 Coon & Ernst. Drug Saf. 25:323-344. 2002

. Yun & Choi. Cancer Epidemiol Biomarkers Prev. 4:401-408 (1995)

Sievenpiper : Korean red ginseng rootlets decrease acute postprandial glycemia: results from sequential preparation- and dose-finding studies. J Am Coll Nutr. 25:100-107 (2006) Vuksan : Korean red ginseng (Panax ginseng) improves glucose and insulin regulation in well-controlled, type 2 diabetes: results of a randomized, double-blind, placebo-controlled study of efficacy and safety. Nutr Metab Cardiovasc Dis. 18:46-56 (2008)

Reay : The glycaemic effects of single doses of Panax ginseng in young healthy volunteers. Br J Nutr. 96:639-642 (2006)

Reay : Panax ginseng has no effect on indices of glucose regulation following acute or chronic ingestion in healthy volunteers. Br J Nutr. 101:1673-1678 (2009) Vuksan : American ginseng (Panax quinquefolius L.) attenuates postprandial glycemia in a time-dependent but not dose-dependent manner in healthy individuals. Am J Clin Nutr. 73:753-758 (2001)

Vuksan : American ginseng (Panax quinquefolius L.) reduces postprandial glycemia in nondiabetic subjects and subjects with type 2 diabetes mellitus. Arch Intern Med. 160:1009-1013 (2000)

Starvro : Long-term intake of North american ginseng has no effect on 24-hour blood pressure and renal function. Hypertension. 47:791-796 (2006)

: ( ) - Transformation of Ginseng Saponins by Food Microorganisms.

Transformation of Ginsenoside Rb1 and Rb2 by food microorganismsChi H, Ji GE, Biotechnol. Lett. 2005,765-777Rb1Rb2

Transformation of Ginsenoside Rc and Re by food microorganismsChi H, Kim DH, Ji GE. Biol. Pharm. Bull. 2005(28),2102-2105RcRe

Biotransformation of ginseng extract by crude extract of microbial enzyme

TLC of supernatant of fermented ginseng extractby various microorganism

HPLC profile of the ginsenosides Rh1 and compound K purified by prep HPLC(A) ginsenosides Rh1(B) compound K

Effect of ginsenoside Rh1 on compound 48/80-induced systemic anaphylaxis

GroupDose(mg/kg)Compound 48/80(8mg/kg)Mortality(%)40% ethanol-+100DSCG Control1000+70Rh15+8010+7050+40100+70100-0

: IgE

Chart1

334.875376.525483.2272.175167.074803606167.074803606178.3975593069178.3975593069236.679121738236.679121738117.0179865092117.0179865092

260.975117.85272.725231.878.659794897678.6597948976250.6593305664250.6593305664224.2085394597224.2085394597117.400865982117.400865982

218.9148287.275316.42590.184200390190.1842003901223.3061631483223.3061631483252.841127654252.841127654193.6212453908193.6212453908

6157.33333333332623.75281.8255728.225730.9212976329730.92129763295357.92354578715357.9235457871894.4693338324894.46933383242318.29823361882318.2982336188

27158.915060763117110.590751324821690.833734536230775.25931367017306.23455960957306.234559609510612.073106413810612.07310641388402.0974970128402.0974970125610.89048378475610.8904837847

57462.533333333331326.37541123.52554867.516472.5559007516472.5559007516572.597642091816572.597642091814819.996382225214819.99638222526675.9023856336675.902385633

62767.466666666737211.87542719.2555884.959472.93690960389472.936909603810997.595394600310997.595394600310577.92802442910577.9280244295020.7766440235020.776644023

70486.733333333337117.77550587.27559907.952561.03545140092561.03545140092791.51089376352791.510893763512968.988758155612968.988758155612210.627843365312210.6278433653

Control

Rh1 20mg/kg

Rh1 80mg/kg

FG 800mg/kg

week

IgE(ng/ml)

IgE changes in FG & Rh1 treat group

Sheet1

raw dataControlControlRh1 20mg/kgRh1 80mg/kgFG 800mg/kg(ng/ml)ControlControlRh1 20mg/kgRh1 80mg/kgFG 800mg/kg

40057.02.25841.48761.72680.876440057.0225.84148.76172.6887.64

1.67380.69040.91621.182167.3869.0491.62118.2

0.83664.9560.91623.932883.66495.691.62393.28

3.42780.95622.08562.564342.7895.62208.56256.4

40064.05.0433.0485.2664.1125.0434.6575.2663.153204.9202.3141.1213.9

2.9963.0481.3443.1532.5824.8782.3274.943108.9198.459.0140.3

2.5824.8783.8434.9435.772.5823.8430.72140064.0504.3304.8526.6411.2504.3465.7526.6315.3

5.772.9443.952.0702.9447.8922.07299.6304.8134.4315.3258.2487.8232.7494.3

40071.03.2637.2063.7848.862.6862.3153.1690.165258.2487.8384.3494.3577258.2384.372.1

8.0333.9921.4852.4284.2592.05202.157577294.43952070294.4789.2207

1.27516.375.5497.313.4940.3472.3155.874409.8348.0360.1357.0334.9376.5483.2272.2

7.5172.3235.44616.986005.4251.076154.993.4163.8123.9167.1117.0236.7178.4

40078.011.76910.3926.98020.1271.7710.7192.5382.79940071.0326.3720.6378.4886268.6231.5316.916.5

8.8506.2881.4875.7151.5724.3690.9021.972803.3399.2148.5242.8425.9205.20215.7

3.25016.7707.09612.7953.2240.2471.5236.412127.51637554.9731349.434.7231.5587.4

18.8162.6527.56127.1510.5856.5281.474751.7232.3544.61698.600542.5107.6

40085.0145.299140.764116.484189.21466.10014.21860.40414.218502.2747.3406.6889.6261.0117.9272.7231.8

69.41586.973168.444126.33353.14111.33644.71264.651328.8626.8190.1605.078.7117.4224.2250.7

70.48686.709163.52598.38465.47960.71545.442130.20440078.01176.91039.2698.02012.7177.171.9253.8279.9

69.86289.438150.76387.94218.67960.71520.056885.0628.8148.7571.5157.2436.990.2197.2

40092.0350.957272.665288.516278.786188.757221.770255.991161.513325.01677.0709.61279.5322.424.7152.3641.2

260.010220.258306.954346.862299.141126.333172.462397.8391881.6265.2756.12715.158.5652.8147.4

241.911303.749403.716279.188326.870118.887125.619372.6171067.1902.6578.11644.7218.9148.0287.3316.4

424.024248.249205.832388.173217.434313.562299.040647.9605.4287.4924.990.2193.6252.8223.3

40099.0952.674493.610658.975491.548412.993355.891582.224355.28640085.014529.914076.411648.418921.46610.01421.86040.41421.8

434.431415.278481.920478.894568.604247.542416.620760.1576941.58697.316844.412633.35314.11133.64471.26465.1

421.342500.623650.208397.630742.279265.789220.409532.2917048.68670.916352.59838.46547.96071.54544.213020.4

568.344514.369316.972611.831383.833425.688546.9666986.28943.815076.38794.21867.96071.52005.6

40106.0733.484470.020712.100461.448540.682442.229562.304432.6288876.610097.114980.412546.86157.32623.75281.85728.2

538.456456.018243.245532.896613.744364.654444.290688.7923769.22655.72343.04548.3730.92318.3894.55357.9

471.792488.742705.574420.697728.598358.531309.636515.39440092.035095.727266.528851.627878.618875.722177.025599.116151.3

672.792574.382306.897605.693323.061392.540598.58426001.022025.830695.434686.229914.112633.317246.239783.9

40116.0749.771352.574621.765453.369676.274516.485600.514615.87324191.130374.940371.627918.832687.011888.712561.937261.7

600.368281.470492.333436.533725.699247.093584.378618.52242402.424824.920583.238817.321743.431356.229904.0

480.242531.712704.970434.862712.629423.786318.432558.36531922.626123.030125.432325.227158.917110.621690.830775.3

718.782626.407353.344478.313297.347520.167603.5588460.43552.48123.85382.47306.25610.98402.110612.1

40099.095267.449361.065897.549154.841299.335589.158222.435528.6

ControlControlRh1 20mg/kgRh1 80mg/kg 800mg/kg43443.141527.848192.047889.456860.424754.241662.076015.7

40057.0108.9198.459.0140.342134.250062.365020.839763.074227.926578.922040.953229.1

40064.0154.993.4163.8123.9167.1117.0236.7178.456834.451436.931697.261183.138383.342568.854696.6

40071.0328.8626.8190.1605.078.7117.4224.2250.759419.848097.052701.949497.657462.531326.441123.554867.5

40078.0647.9605.4287.4924.990.2193.6252.8223.324804.44463.516201.08832.116472.66675.914820.016572.6

40085.03769.22655.72343.04548.3730.92318.3894.55357.940106.073348.447002.071210.046144.854068.244222.956230.443262.8

40092.08460.43552.48123.85382.47306.25610.98402.110612.153845.645601.824324.553289.661374.436465.444429.068879.2

40099.024804.44463.516201.08832.116472.66675.914820.016572.647179.248874.270557.442069.772859.835853.130963.651539.4

40106.012010.25311.425179.48148.89472.95020.810577.910997.667279.257438.230689.760569.332306.139254.059858.4

40116.012290.915882.415381.22017.52561.012210.612969.02791.560413.149729.149195.450518.462767.537211.942719.355885.0

12010.25311.425179.48148.89472.95020.810577.910997.6

40116.074977.135257.462176.545336.967627.451648.560051.461587.3

60036.828147.049233.343653.372569.924709.358437.861852.2

48024.253171.270497.043486.271262.942378.631843.255836.5

71878.262640.735334.447831.329734.752016.760355.8

63729.144804.154310.345076.970486.737117.850587.359908.0

12290.915882.415381.22017.52561.012210.612969.02791.5

(ng/ml)ControlControlRh1 20mg/kgRh1 80mg/kgFG 800mg/kg

40057.0204.9202.3141.1213.9

40064.0409.8348.0360.1357.0334.9376.5483.2272.2

40071.0502.2747.3406.6889.6261.0117.9272.7231.8

40078.01067.1902.6578.11644.7218.9148.0287.3316.4

40085.08876.610097.114980.412546.86157.32623.75281.85728.2

40092.031922.626123.030125.432325.227158.917110.621690.830775.3

40099.059419.848097.052701.949497.657462.531326.441123.554867.5

40106.060413.149729.149195.450518.462767.537211.942719.355885.0

40116.063729.144804.154310.345076.970486.737117.850587.359908.0

0

1

2

3

4

5

6

7

Sheet1

108.8672970486108.867297048659.004765909259.0047659092140.3371065209140.3371065209198.3517611887198.3517611887

154.9329828238154.9329828238163.7746291911163.7746291911123.8652628195123.865262819593.362144362793.3621443627

328.7614940956328.7614940956190.1295523233190.1295523233604.9788315856604.9788315856626.811398402626.811398402

647.9040843366647.9040843366287.3654003761287.3654003761924.8980916836924.8980916836605.4199947144605.4199947144

3769.15592009673769.15592009672342.97355369912342.97355369914548.2760370464548.2760370462655.71182297082655.7118229708

8460.40906064758460.40906064758123.76573135368123.76573135365382.38056174665382.38056174663552.39049349253552.3904934925

24804.446034044624804.446034044616200.985326696416200.98532669648832.08481404598832.08481404594463.53450156564463.5345015656

12010.182912290112010.182912290125179.358321582925179.35832158298148.80459310868148.80459310865311.39518739345311.3951873934

12290.949218937512290.949218937515381.233903039115381.23390303912017.53007474482017.530074744815882.39196161915882.391961619

Control

IgE(ng/ml)

Sheet2

167.074803606167.074803606178.3975593069178.3975593069236.679121738236.679121738117.0179865092117.0179865092

78.659794897678.6597948976250.6593305664250.6593305664224.2085394597224.2085394597117.400865982117.400865982

90.184200390190.1842003901223.3061631483223.3061631483252.841127654252.841127654193.6212453908193.6212453908

730.9212976329730.92129763295357.92354578715357.9235457871894.4693338324894.46933383242318.29823361882318.2982336188

7306.23455960957306.234559609510612.073106413810612.07310641388402.0974970128402.0974970125610.89048378475610.8904837847

16472.5559007516472.5559007516572.597642091816572.597642091814819.996382225214819.99638222526675.9023856336675.902385633

9472.93690960389472.936909603810997.595394600310997.595394600310577.92802442910577.9280244295020.7766440235020.776644023

2561.03545140092561.03545140092791.51089376352791.510893763512968.988758155612968.988758155612210.627843365312210.6278433653

Control

Rh1 20mg/kg

Rh1 80mg/kg

FG 800mg/kg

IgE(ng/ml)

Sheet3

108.8672970486108.8672970486140.3371065209140.3371065209

154.9329828238154.9329828238123.8652628195123.8652628195

328.7614940956328.7614940956604.9788315856604.9788315856

647.9040843366647.9040843366924.8980916836924.8980916836

3769.15592009673769.15592009674548.2760370464548.276037046

8460.40906064758460.40906064755382.38056174665382.3805617466

24804.446034044624804.44603404468832.08481404598832.0848140459

12010.182912290112010.18291229018148.80459310868148.8045931086

12290.949218937512290.94921893752017.53007474482017.5300747448

Control

IgE(ng/ml)

Lactobacillus brevis

167.074803606167.074803606178.3975593069178.3975593069236.679121738236.679121738117.0179865092117.0179865092

78.659794897678.6597948976250.6593305664250.6593305664224.2085394597224.2085394597117.400865982117.400865982

90.184200390190.1842003901223.3061631483223.3061631483252.841127654252.841127654193.6212453908193.6212453908

730.9212976329730.92129763295357.92354578715357.9235457871894.4693338324894.46933383242318.29823361882318.2982336188

7306.23455960957306.234559609510612.073106413810612.07310641388402.0974970128402.0974970125610.89048378475610.8904837847

16472.5559007516472.5559007516572.597642091816572.597642091814819.996382225214819.99638222526675.9023856336675.902385633

9472.93690960389472.936909603810997.595394600310997.595394600310577.92802442910577.9280244295020.7766440235020.776644023

2561.03545140092561.03545140092791.51089376352791.510893763512968.988758155612968.988758155612210.627843365312210.6278433653

Control

Rh1 20mg/kg

Rh1 80mg/kg

FG 800mg/kg

week

IgE(ng/ml)

IgE changes in FG & Rh1 treat group

HT29( )B16()MCF( )FHCCytotoxicity of various ginsenosides on tumor cells

Chart5

1.3693333333

1.4871666667

1.3263333333

1.3218333333

1.4165

1.459

1.5601666667

0.5468333333

1.483

0.1188333333

1.7166666667

1.3988333333

0.1276666667

1.1966666667

1.2083333333

1.283

HT

Sheet1

HT

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.5131.3041.2661.3761.3171.5391.3770.6571.5130.1131.6321.4130.1321.3411.1981.331

1.4881.4191.261.1191.3091.5461.6820.6491.4880.1271.8141.0560.1251.2771.1371.193

1.1811.4571.4791.2691.3781.4281.5870.4781.1870.1261.7591.3980.1211.0871.0871.229

1.3061.7391.2581.1241.491.4271.5930.3911.4920.1221.6091.530.1381.2321.1691.309

1.3261.581.2861.7851.7351.4251.5180.4271.5530.1131.8421.4090.1260.9941.321.265

1.4021.4241.4091.2581.271.3891.6040.6791.6650.1121.6441.5870.1241.2491.3391.371

1.36933333331.48716666671.32633333331.32183333331.41651.4591.56016666670.54683333331.4830.11883333331.71666666671.39883333330.12766666671.19666666671.20833333331.283

0.12421540430.15174638930.09431578160.24682092020.1739226840.06634756970.10381794960.1291703010.15915150020.00696897890.10101023050.1846352260.00621825270.12990252760.10100231020.0664289094

MCF

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CK(1)F2RfPPT

1.8391.561.2131.1951.3391.7961.5240.5321.6360.1271.8731.5320.1381.6051.2581.695

1.5382.2662.0611.451.6841.9451.6060.6291.8180.1231.7571.6260.131.7461.481.538

1.9041.7741.731.3641.3421.7041.8280.3641.3840.1691.5981.6280.1081.9541.581.404

1.6251.991.7511.4021.582.2541.9990.4271.3650.1381.4671.5290.1341.4641.834

1.8041.9881.5181.0581.291.70142.1020.5411.5820.131.4451.9880.1361.5081.935

1.3391.9341.6110.8791.7751.921.7590.4051.7830.1261.7061.5890.1291.2811.738

1.67483333331.91866666671.64733333331.22466666671.50166666671.88673333331.8030.4831.59466666670.13551.6411.64866666670.12916666671.76833333331.42851.6906666667

0.21454269190.23678738710.28120929340.22350809080.20535302940.20753743440.22227370510.10046093770.19201215240.01719011340.16855028920.17180182380.01092550530.17556860010.12962368610.1939006619

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

0.2110.2730.2880.2650.2370.2550.2670.2370.250.1140.3950.2780.1050.1610.2070.265

0.2670.2990.2810.2580.2840.2760.2450.280.0980.3970.2990.1020.1720.1910.27

0.2570.2780.2840.260.2820.2510.2380.2470.0880.3470.2880.1040.1620.233

0.2570.2760.2890.2650.2750.2460.2440.2770.0760.3370.2490.114

0.2390.2610.2570.2260.2780.2320.2230.2540.1020.3590.2870.131

0.2650.3060.2940.2640.2860.240.2260.2480.0720.3890.2730.115

0.2110.25966666670.28466666670.27833333330.25166666670.27666666670.2520.23550.25933333330.09166666670.37066666670.2790.11183333330.1650.1990.256

00.01184342290.01643978910.01436198690.01620699440.0113431330.0166252820.00913783340.01506873140.01607067720.02627292650.01721627140.01083358970.00608276250.01131370850.0200748599

NotreatMeOH0.5%Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.7851.9820.370.3480.7850.4811.581.2670.1370.7531.0530.150.9941.9432.026

1.0121.5750.2790.2870.3160.4781.7710.8360.1550.8750.5110.1381.1341.9152.073

1.4091.9350.2760.2550.2290.3991.2060.90.1351.5290.7270.1411.0922.0392.054

1.7961.9420.2050.2720.3460.5441.1240.8730.1210.7710.9130.1591.1831.9242.073

1.751.8610.2420.3990.4810.5761.0520.8330.120.6521.1330.140.8431.8362.011

1.8782.2130.8720.7891.2311.1431.5081.7350.1431.5561.1090.1681.1611.9422.272

1.6051.9180.3740.39166666670.56466666670.60351.37351.0740.13516666671.02266666670.90766666670.14933333331.06783333331.93316666672.0848333333

0.33301651610.20634146460.25005359430.20188478560.37996561250.2712686860.28719592620.36320022030.01333291670.40891889990.24594525140.01206095630.12876865560.06516261710.0950524417

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HT

Sheet2

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MCF

Sheet3

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FHC

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Chart6

1.6748333333

1.9186666667

1.6473333333

1.2246666667

1.5016666667

1.8867333333

1.803

0.483

1.5946666667

0.1355

1.641

1.6486666667

0.1291666667

1.7683333333

1.4285

1.6906666667

MCF

Sheet1

HT

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.5131.3041.2661.3761.3171.5391.3770.6571.5130.1131.6321.4130.1321.3411.1981.331

1.4881.4191.261.1191.3091.5461.6820.6491.4880.1271.8141.0560.1251.2771.1371.193

1.1811.4571.4791.2691.3781.4281.5870.4781.1870.1261.7591.3980.1211.0871.0871.229

1.3061.7391.2581.1241.491.4271.5930.3911.4920.1221.6091.530.1381.2321.1691.309

1.3261.581.2861.7851.7351.4251.5180.4271.5530.1131.8421.4090.1260.9941.321.265

1.4021.4241.4091.2581.271.3891.6040.6791.6650.1121.6441.5870.1241.2491.3391.371

1.36933333331.48716666671.32633333331.32183333331.41651.4591.56016666670.54683333331.4830.11883333331.71666666671.39883333330.12766666671.19666666671.20833333331.283

0.12421540430.15174638930.09431578160.24682092020.1739226840.06634756970.10381794960.1291703010.15915150020.00696897890.10101023050.1846352260.00621825270.12990252760.10100231020.0664289094

MCF

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CK(1)F2RfPPT

1.8391.561.2131.1951.3391.7961.5240.5321.6360.1271.8731.5320.1381.6051.2581.695

1.5382.2662.0611.451.6841.9451.6060.6291.8180.1231.7571.6260.131.7461.481.538

1.9041.7741.731.3641.3421.7041.8280.3641.3840.1691.5981.6280.1081.9541.581.404

1.6251.991.7511.4021.582.2541.9990.4271.3650.1381.4671.5290.1341.4641.834

1.8041.9881.5181.0581.291.70142.1020.5411.5820.131.4451.9880.1361.5081.935

1.3391.9341.6110.8791.7751.921.7590.4051.7830.1261.7061.5890.1291.2811.738

1.67483333331.91866666671.64733333331.22466666671.50166666671.88673333331.8030.4831.59466666670.13551.6411.64866666670.12916666671.76833333331.42851.6906666667

0.21454269190.23678738710.28120929340.22350809080.20535302940.20753743440.22227370510.10046093770.19201215240.01719011340.16855028920.17180182380.01092550530.17556860010.12962368610.1939006619

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

0.2110.2730.2880.2650.2370.2550.2670.2370.250.1140.3950.2780.1050.1610.2070.265

0.2670.2990.2810.2580.2840.2760.2450.280.0980.3970.2990.1020.1720.1910.27

0.2570.2780.2840.260.2820.2510.2380.2470.0880.3470.2880.1040.1620.233

0.2570.2760.2890.2650.2750.2460.2440.2770.0760.3370.2490.114

0.2390.2610.2570.2260.2780.2320.2230.2540.1020.3590.2870.131

0.2650.3060.2940.2640.2860.240.2260.2480.0720.3890.2730.115

0.2110.25966666670.28466666670.27833333330.25166666670.27666666670.2520.23550.25933333330.09166666670.37066666670.2790.11183333330.1650.1990.256

00.01184342290.01643978910.01436198690.01620699440.0113431330.0166252820.00913783340.01506873140.01607067720.02627292650.01721627140.01083358970.00608276250.01131370850.0200748599

NotreatMeOH0.5%Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.7851.9820.370.3480.7850.4811.581.2670.1370.7531.0530.150.9941.9432.026

1.0121.5750.2790.2870.3160.4781.7710.8360.1550.8750.5110.1381.1341.9152.073

1.4091.9350.2760.2550.2290.3991.2060.90.1351.5290.7270.1411.0922.0392.054

1.7961.9420.2050.2720.3460.5441.1240.8730.1210.7710.9130.1591.1831.9242.073

1.751.8610.2420.3990.4810.5761.0520.8330.120.6521.1330.140.8431.8362.011

1.8782.2130.8720.7891.2311.1431.5081.7350.1431.5561.1090.1681.1611.9422.272

1.6051.9180.3740.39166666670.56466666670.60351.37351.0740.13516666671.02266666670.90766666670.14933333331.06783333331.93316666672.0848333333

0.33301651610.20634146460.25005359430.20188478560.37996561250.2712686860.28719592620.36320022030.01333291670.40891889990.24594525140.01206095630.12876865560.06516261710.0950524417

Sheet1

HT

Sheet2

MCF

Sheet3

FHC

Chart6

1.6748333333

1.9186666667

1.6473333333

1.2246666667

1.5016666667

1.8867333333

1.803

0.483

1.5946666667

0.1355

1.641

1.6486666667

0.1291666667

1.7683333333

1.4285

1.6906666667

MCF

Sheet1

HT

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.5131.3041.2661.3761.3171.5391.3770.6571.5130.1131.6321.4130.1321.3411.1981.331

1.4881.4191.261.1191.3091.5461.6820.6491.4880.1271.8141.0560.1251.2771.1371.193

1.1811.4571.4791.2691.3781.4281.5870.4781.1870.1261.7591.3980.1211.0871.0871.229

1.3061.7391.2581.1241.491.4271.5930.3911.4920.1221.6091.530.1381.2321.1691.309

1.3261.581.2861.7851.7351.4251.5180.4271.5530.1131.8421.4090.1260.9941.321.265

1.4021.4241.4091.2581.271.3891.6040.6791.6650.1121.6441.5870.1241.2491.3391.371

1.36933333331.48716666671.32633333331.32183333331.41651.4591.56016666670.54683333331.4830.11883333331.71666666671.39883333330.12766666671.19666666671.20833333331.283

0.12421540430.15174638930.09431578160.24682092020.1739226840.06634756970.10381794960.1291703010.15915150020.00696897890.10101023050.1846352260.00621825270.12990252760.10100231020.0664289094

MCF

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CK(1)F2RfPPT

1.8391.561.2131.1951.3391.7961.5240.5321.6360.1271.8731.5320.1381.6051.2581.695

1.5382.2662.0611.451.6841.9451.6060.6291.8180.1231.7571.6260.131.7461.481.538

1.9041.7741.731.3641.3421.7041.8280.3641.3840.1691.5981.6280.1081.9541.581.404

1.6251.991.7511.4021.582.2541.9990.4271.3650.1381.4671.5290.1341.4641.834

1.8041.9881.5181.0581.291.70142.1020.5411.5820.131.4451.9880.1361.5081.935

1.3391.9341.6110.8791.7751.921.7590.4051.7830.1261.7061.5890.1291.2811.738

1.67483333331.91866666671.64733333331.22466666671.50166666671.88673333331.8030.4831.59466666670.13551.6411.64866666670.12916666671.76833333331.42851.6906666667

0.21454269190.23678738710.28120929340.22350809080.20535302940.20753743440.22227370510.10046093770.19201215240.01719011340.16855028920.17180182380.01092550530.17556860010.12962368610.1939006619

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

0.2110.2730.2880.2650.2370.2550.2670.2370.250.1140.3950.2780.1050.1610.2070.265

0.2670.2990.2810.2580.2840.2760.2450.280.0980.3970.2990.1020.1720.1910.27

0.2570.2780.2840.260.2820.2510.2380.2470.0880.3470.2880.1040.1620.233

0.2570.2760.2890.2650.2750.2460.2440.2770.0760.3370.2490.114

0.2390.2610.2570.2260.2780.2320.2230.2540.1020.3590.2870.131

0.2650.3060.2940.2640.2860.240.2260.2480.0720.3890.2730.115

0.2110.25966666670.28466666670.27833333330.25166666670.27666666670.2520.23550.25933333330.09166666670.37066666670.2790.11183333330.1650.1990.256

00.01184342290.01643978910.01436198690.01620699440.0113431330.0166252820.00913783340.01506873140.01607067720.02627292650.01721627140.01083358970.00608276250.01131370850.0200748599

NotreatMeOH0.5%Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.7851.9820.370.3480.7850.4811.581.2670.1370.7531.0530.150.9941.9432.026

1.0121.5750.2790.2870.3160.4781.7710.8360.1550.8750.5110.1381.1341.9152.073

1.4091.9350.2760.2550.2290.3991.2060.90.1351.5290.7270.1411.0922.0392.054

1.7961.9420.2050.2720.3460.5441.1240.8730.1210.7710.9130.1591.1831.9242.073

1.751.8610.2420.3990.4810.5761.0520.8330.120.6521.1330.140.8431.8362.011

1.8782.2130.8720.7891.2311.1431.5081.7350.1431.5561.1090.1681.1611.9422.272

1.6051.9180.3740.39166666670.56466666670.60351.37351.0740.13516666671.02266666670.90766666670.14933333331.06783333331.93316666672.0848333333

0.33301651610.20634146460.25005359430.20188478560.37996561250.2712686860.28719592620.36320022030.01333291670.40891889990.24594525140.01206095630.12876865560.06516261710.0950524417

Sheet1

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Sheet2

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Sheet3

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Chart7

0.211

0.2596666667

0.2846666667

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0.2516666667

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0.252

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0.2593333333

0.0916666667

0.3706666667

0.279

0.1118333333

0.165

0.199

0.256

FHC

Sheet1

HT

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.5131.3041.2661.3761.3171.5391.3770.6571.5130.1131.6321.4130.1321.3411.1981.331

1.4881.4191.261.1191.3091.5461.6820.6491.4880.1271.8141.0560.1251.2771.1371.193

1.1811.4571.4791.2691.3781.4281.5870.4781.1870.1261.7591.3980.1211.0871.0871.229

1.3061.7391.2581.1241.491.4271.5930.3911.4920.1221.6091.530.1381.2321.1691.309

1.3261.581.2861.7851.7351.4251.5180.4271.5530.1131.8421.4090.1260.9941.321.265

1.4021.4241.4091.2581.271.3891.6040.6791.6650.1121.6441.5870.1241.2491.3391.371

1.36933333331.48716666671.32633333331.32183333331.41651.4591.56016666670.54683333331.4830.11883333331.71666666671.39883333330.12766666671.19666666671.20833333331.283

0.12421540430.15174638930.09431578160.24682092020.1739226840.06634756970.10381794960.1291703010.15915150020.00696897890.10101023050.1846352260.00621825270.12990252760.10100231020.0664289094

MCF

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CK(1)F2RfPPT

1.8391.561.2131.1951.3391.7961.5240.5321.6360.1271.8731.5320.1381.6051.2581.695

1.5382.2662.0611.451.6841.9451.6060.6291.8180.1231.7571.6260.131.7461.481.538

1.9041.7741.731.3641.3421.7041.8280.3641.3840.1691.5981.6280.1081.9541.581.404

1.6251.991.7511.4021.582.2541.9990.4271.3650.1381.4671.5290.1341.4641.834

1.8041.9881.5181.0581.291.70142.1020.5411.5820.131.4451.9880.1361.5081.935

1.3391.9341.6110.8791.7751.921.7590.4051.7830.1261.7061.5890.1291.2811.738

1.67483333331.91866666671.64733333331.22466666671.50166666671.88673333331.8030.4831.59466666670.13551.6411.64866666670.12916666671.76833333331.42851.6906666667

0.21454269190.23678738710.28120929340.22350809080.20535302940.20753743440.22227370510.10046093770.19201215240.01719011340.16855028920.17180182380.01092550530.17556860010.12962368610.1939006619

Notreat0.5%MeOHRb1Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

0.2110.2730.2880.2650.2370.2550.2670.2370.250.1140.3950.2780.1050.1610.2070.265

0.2670.2990.2810.2580.2840.2760.2450.280.0980.3970.2990.1020.1720.1910.27

0.2570.2780.2840.260.2820.2510.2380.2470.0880.3470.2880.1040.1620.233

0.2570.2760.2890.2650.2750.2460.2440.2770.0760.3370.2490.114

0.2390.2610.2570.2260.2780.2320.2230.2540.1020.3590.2870.131

0.2650.3060.2940.2640.2860.240.2260.2480.0720.3890.2730.115

0.2110.25966666670.28466666670.27833333330.25166666670.27666666670.2520.23550.25933333330.09166666670.37066666670.2790.11183333330.1650.1990.256

00.01184342290.01643978910.01436198690.01620699440.0113431330.0166252820.00913783340.01506873140.01607067720.02627292650.01721627140.01083358970.00608276250.01131370850.0200748599

NotreatMeOH0.5%Rb2RcRdRg2Rh2RePPDRg3Rh1CKF2RfPPT

1.7851.9820.370.3480.7850.4811.581.2670.1370.7531.0530.150.9941.9432.026

1.0121.5750.2790.2870.3160.4781.7710.8360.1550.8750.5110.1381.1341.9152.073

1.4091.9350.2760.2550.2290.3991.2060.90.1351.5290.7270.1411.0922.0392.054

1.7961.9420.2050.2720.3460.5441.1240.8730.1210.7710.9130.1591.1831.9242.073

1.751.8610.2420.3990.4810.5761.0520.8330.120.6521.1330.140.8431.8362.011

1.8782.2130.8720.7891.2311.1431.5081.7350.1431.5561.1090.1681.1611.9422.272

1.6051.9180.3740.39166666670.56466666670.60351.37351.0740.13516666671.02266666670.90766666670.14933333331.06783333331.93316666672.0848333333

0.33301651610.20634146460.25005359430.20188478560.37996561250.2712686860.28719592620.36320022030.01333291670.40891889990.24594525140.01206095630.12876865560.06516261710.0950524417

Sheet1

HT

Sheet2

MCF

Sheet3

FHC

CompounK

Real time based cell analysis on HT-29 tumor cell death by compound K

ControlPredinisolone(3mg/Kg)Rh1(10)Rh1(20)Skin symptoms (after 4weeks) Effect of ginsenoside Rh1 on skin symptoms in atopic dermatitis model

SerumDraining lymph nodeSkin tissueIgE IL-6 IFN Foxp3Clinical Symptoms score Histological Inflammation Ear tissueEar swelling Effects of ginsenoside Rh1 (20mg/Kg) on atopic dermatitis-like skin lesions induce by hapten in hairless mice

Effect of ginseng and fermented ginseng on suppression of allergy in ovalbumin allergy model (in collaboration with Prof. Sung MK, Sookmyung Womens University)

Lowered markers by red ginseng and fermented red ginsengColon tissue mRNA

0.1% RG 0.3% RG 0.1% FRG 0.3% FRG IgEIgG1IgG2aOVA-IgEOVA-IgG1OVA-IgG2aIL-5TNF-IL-4IFN-IL-2BLGIL-4MMCP1TNF-iNOSCOX-2

Effect of fermented ginseng on skin symptoms in atopic dermatitis model

Lowered markers by red ginseng and fermented red ginseng

0.1% RG 0.3% RG 0.1% FRG 0.3% FRG 0.1% PF FRG0.3% PF FRGScratching behaviorMDCEOXTARCIgEIgG1IgG2aMast cellIFN-TNF-

Effect of fermented ginseng on serum glucose in db/db mice

OGTTHbA1c (%)

Histology of pancreas A, B ( ) , C , A . -

Water Intake

Serum fasting glucose , AUC (area under curve) glucose HbA1c , insulin , adiponectin

Liver PPAR-a mRNA , glucose-6-phosphatase mRNA , Glut-2 mRNA

Adipose tissue PPAR-g2 mRNA , TNF-a mRNA , IL-6 mRNA

Muscle Glut-4 mRNA , PPAR-a mRNA

Histology Inflammation

Effects of fermented ginseng in DB/DB mice model

(9-10 )

, ,

, , : IL-2, IFN-g, IL-4, IL-5

NK

, , : CD3, CD4, CD8, B220, CD11b, panNK

Body weight (g)Each value represents the mean S.E.M of 10 animals. The significance of differences between mean values was assessed by 1-way ANOVA coupled with Duncans multiple-range test at the 5% level of significance.**

Each value represents the mean S.E.M of 10 animals. The significance of differences between mean values was assessed by 1-way ANOVA coupled with Duncans multiple-range test at the 5% level of significance.*****Serum total cholesterol (mg/dl)

Each value represents the mean S.E.M of 10 animals. The significance of differences between mean values was assessed by 1-way ANOVA coupled with Duncans multiple-range test at the 5% level of significance.***Serum triglyceride (mg/dl)

, , , , inflammation

T

B

1) 18 55 2) 2 (Perennial)3) Skin Prick Test for Perennial allergen4) Nasal Symptom(, , , ) : 2 2

: , 1 2, 1 3, , 4 , , , Placebo

TNSS TNSS ********************************

TNSS ******************************** Effect of fermented ginseng on the rhinitis symptoms in double blind placebo controlled clinical experiment (in collaboration with Prof. Kang HR, Seoul Medical School)

TNSS TNSS ********************************

Skin Prick TestAtopic Index : .Atopic Score

Atopic Score( )(Placebo)p-value* N=29N=28Mean S.DMean S.D (8) (1)2.711.172.311.210.199 (3)2.320.882.251.080.779Difference0.391.000.060.790.168p-value**0.0450.705 (1)6.932.796.533.240.610 (3)6.292.567.435.640.314Difference0.642.36-0.903.790.065p-value**0.1470.213 (6) (1)0.621.080.831.670.555 (3)0.731.140.891.700.571Difference-0.110.64-0.050.680.743p-value**0.3630.683 (1)1.833.012.745.220.416 (3)1.912.862.795.400.435Difference-0.081.98-0.052.000.960p-value**0.8280.888

( )

RQoL(Rhinitis Quality of Life)**************

RQoL(Rhinitis Quality of Life)************

( ) : Cr, bil, ALT

0 4 T-bil(mg/ml)GPT(IU/L)T-bil(mg/ml)GPT(IU/L)T-bil(mg/ml)GPT(IU/L)A1.2131.3141.213B0.6270.6420.525C0.7360.843D1.0131.5111.115

1. .

2. . (atopic score)

3. .

4. .

Placebo (n=45)2wksBlood sampling 2Blood sampling1Random Double blindFermented ginseng (n=50)Analysis (SPSS; ANCOVA)* Endocrine analysisginseng20: placebo20 Match (age, hts,wt, BMI)

Effect of fermented ginseng on the elderly women (over 55 years old) in double blind placebo controlled experiment

1ACTH22.7+11.8(49)20.4+10.1(44)0.34pg/mL2HGH1.8+1.4(20)0.9+1.2(20)0.057ng/mL3TSH2.3+1.2(20)2.8+1.8(20)0.796IU/mL4free T41.04+0.11(20)1.05+0.13(20)0.985ng/dL5LH30.3+9.0(20)38.8+14.8(20)0.303mIU/mL6FSH68.0+18.5(20)74.0+24.3(20)0.679mIU/mL7E218.4+7.1(20)14.5+7.5(19)0.08pg/mL8Insulin6.2+2.9(48)6.4+3.6(43)0.039U/mL9Glucose87.4+7.1(49)89.6+6.8(44)0.31mg/dL10Creatinin0.918+0.086(49)0.900+0.089(44)0.71mg/dL11Apolipoprotein B89.6+16.4(49)86.7+17.0(44)0.321mg/dL12hs CRP0.57+1.01(49)0.73+1.62(44)0.545mg/L13Uric acid5.0+0.96(49)4.7+0.89(44)0.122mg/dL14total Chol217.7+30.9(49)216.2+31.6(44)0.137mg/dL15HDL53.6+11.2(49)54.6+10.6(44)0.128mg/dL16LDL141.3+30.1(49)139.1+31.6(44)0.078mg/dL17TG127.0+68.2(49)141.8+74.8(44)0.461mg/dL18FFA660.3+217.0(49)640.0+223.7(44)0.612Eq/L19BUN15.3+3.9(49)14.6+3.1(44)0.467mg/dL20WBC5.17+1.14(50)5.23+1.26(44)0.80910/21Neutrophil2580+874(50)2747+816(45)0.3/22Lymphocyte2102+493(50)2028+504(45)0.562/23Monocyte349+98(50)344+80(45)0.6/24Eosionphil116+83(50)119+92(45)0.775/25Basophil24+13(50)28+18(45)0.664/26HbA1c125.3+9.5(50)128.9+10.0(45)0.04mg/dL27pH(RU)5.9+0.8(49)5.7+0.8(43)0.022-28Hb13.0+0.76(50)13.2+0.86(44)0.334g/dL29DHEAS66.2+29.1(49)65.2+31.4(44)0.019g/dL30Cortisol10.8+3.8(49)11.2+3.5(44)0.516g/dL31Cortisol free19.8+14.0(47)22.5+15.8(41)0.96g/day32Aldosterone8.4+3.6(20)6.15+3.5(20)0.12ng/dL33ACE41.9+15.4(20)43.7+12.8(20)0.831U/L34Ccr60.2+25.1(47)64.8+20.0(41)0.823mL/min35PAI-126.2+20.7(40)20.4+10.2(35)0.171ng/mL36ADH0.84+0.19(20)0.81+0.17(20)0.928pg/mL37CBG6293+8469(20)6178+8294(20)0.948ng/mL38CRF0.45+1.0(20)0.18+0.1(20)0.166ng/mL39creatinemg/dL

HPG axisLuteinizing Hormone (LH)Follicle Stimulating Hormone (FSH)Estrdiol (E2)P=0.303P=0.679P=0.08

Chart6

30.39

38.814.8

mIU/mL

(n)(n)p

1ACTH22.7+11.8(49)20.4+10.1(44)0.34pg/mL

2HGH1.8+1.4(20)0.9+1.2(20)0.057ng/mL

3TSH2.3+1.2(20)2.8+1.8(20)0.796IU/mL

4free T41.04+0.11(20)1.05+0.13(20)0.985ng/dL

5LH30.3+9.0(20)38.8+14.8(20)0.303mIU/mL

6FSH68.0+18.5(20)74.0+24.3(20)0.679mIU/mL

7E218.4+7.1(20)14.5+7.5(19)0.08pg/mL

8Insulin6.2+2.9(48)6.4+3.6(43)0.039U/mL

9Glucose87.4+7.1(49)89.6+6.8(44)0.31mg/dL

10Creatinin0.918+0.086(49)0.900+0.089(44)0.71mg/dL

11Apolipoprotein B89.6+16.4(49)86.7+17.0(44)0.321mg/dL

12hs CRP0.57+1.01(49)0.73+1.62(44)0.545mg/L

13Uric acid5.0+0.96(49)4.7+0.89(44)0.122mg/dL

14total Chol217.7+30.9(49)216.2+31.6(44)0.137mg/dL

15HDL53.6+11.2(49)54.6+10.6(44)0.128mg/dL

16LDL141.3+30.1(49)139.1+31.6(44)0.078mg/dL

17TG127.0+68.2(49)141.8+74.8(44)0.461mg/dL

18FFA660.3+217.0(49)640.0+223.7(44)0.612Eq/L

19BUN15.3+3.9(49)14.6+3.1(44)0.467mg/dL

20WBC5.17+1.14(50)5.23+1.26(44)0.80910/

21Neutrophil2580+874(50)2747+816(45)0.3/

22Lymphocyte2102+493(50)2028+504(45)0.562/

23Monocyte349+98(50)344+80(45)0.6/

24Eosionphil116+83(50)119+92(45)0.775/

25Basophil24+13(50)28+18(45)0.664/

26HbA1c125.3+9.5(50)128.9+10.0(45)0.4mg/dL

27pH(RU)5.9+0.8(49)5.7+0.8(43)0.022-

28Hb13.0+0.76(50)13.2+0.86(44)0.334g/dL

29DHEAS66.2+29.1(49)65.2+31.4(44)0.019g/dL

30Cortisol10.8+3.8(49)11.2+3.5(44)0.516g/dL

31Cortisol free19.8+14.0(47)22.5+15.8(41)0.96g/day

32Aldosterone8.4+3.6(20)6.15+3.5(20)0.12ng/dL

33ACE41.9+15.4(20)43.7+12.8(20)0.831U/L

34Ccr60.2+25.1(47)64.8+20.0(41)0.823mL/min

35PAI-126.2+20.7(40)20.4+10.2(35)0.171ng/mL

36ADH0.84+0.19(20)0.81+0.17(20)0.928pg/mL

37CBG6293+8469(20)6178+8294(20)0.948ng/mL

38CRF0.45+1.0(20)0.18+0.1(20)0.166ng/mL

39creatinemg/dL

Axis(n)(n)p

HPA axisCRF0.45+1.0(20)0.18+0.1(20)0.166ng/mL

ACTH22.7+11.8(49)20.4+10.1(44)0.34pg/mL

Cortisol10.8+3.8(49)11.2+3.5(44)0.516g/dL

Cortisol free19.8+14.0(47)22.5+15.8(41)0.96g/day

CBG6293+8469(20)6178+8294(20)0.948ng/mL

HPG axisLH30.3+9.0(20)38.8+14.8(20)0.303mIU/mL

FSH68.0+18.5(20)74.0+24.3(20)0.679mIU/mL

E218.4+7.1(20)14.5+7.5(19)0.08pg/mL

HPT axisTSH2.3+1.2(20)2.8+1.8(20)0.796IU/mL

free T41.04+0.11(20)1.05+0.13(20)0.985ng/dL

growth hormoneHGH1.8+1.4(20)0.9+1.2(20)0.057ng/mL

DHEAS66.2+29.1(49)65.2+31.4(44)0.019g/dL

(n)(n)p

ACTH22.7+11.8(49)20.4+10.1(44)0.34pg/mL

Ginseng(20)Placebo(20)p

HPG axisLH30.338.8914.80.303mIU/mL2020

FSH687418.524.30.679mIU/mL2020

E218.414.57.17.50.08pg/mL2020

HPT axisTSH2.32.81.21.80.796IU/mL2020

free T41.041.050.110.130.985ng/dL2020

growth hormoneHGH1.80.91.41.20.057ng/mL2020

14414467490.8722020

DHEAS66.265.229.131.40.019g/dL4944

diabetesInsulin6.26.42.93.60.039U/mL4843

Glucose87.489.67.16.80.31mg/dL4944

HbA1c125.3128.99.5100.4mg/dL5045

HOMA

Lipidtotal Chol217.7216.230.931.60.137mg/dL4944

HDL53.654.611.210.60.128mg/dL4944

LDL141.3139.130.131.60.078mg/dL4944

TG127141.868.274.80.461mg/dL4944

FFA660.3640217223.70.612Eq/L4944

Apolipoprotein B89.686.716.4170.321mg/dL4944

Creatinin0.9180.90.0860.0890.71mg/dL4944

hs CRP0.570.731.011.620.545mg/L4944

Uric acid54.70.960.890.122mg/dL4944

total Chol217.7216.230.931.60.137mg/dL4944

BUN15.314.63.93.10.467mg/dL4944

ACE41.943.715.412.80.831U/L2020

Ccr60.264.825.1200.823mL/min4741

PAI-126.220.420.710.20.171ng/mL4035

creatinemg/dL

Aldosterone8.46.153.63.50.12ng/dL2020

WBC5.175.231.141.260.80910/5044

Neutrophil258027478748160.3/5045

Lymphocyte210220284935040.562/5045

Monocyte34934498800.6/5045

Eosionphil11611983920.775/5045

Basophil242813180.664/5045

urine Ca

serum Ca

9

14.8

mIU/mL

Chart7

6818.5

7424.3

mIU/mL

(n)(n)p

1ACTH22.7+11.8(49)20.4+10.1(44)0.34pg/mL

2HGH1.8+1.4(20)0.9+1.2(20)0.057ng/mL

3TSH2.3+1.2(20)2.8+1.8(20)0.796IU/mL

4free T41.04+0.11(20)1.05+0.13(20)0.985ng/dL

5LH30.3+9.0(20)38.8+14.8(20)0.303mIU/mL

6FSH68.0+18.5(20)74.0+24.3(20)0.679mIU/mL

7E218.4+7.1(20)14.5+7.5(19)0.08pg/mL

8Insulin6.2+2.9(48)6.4+3.6(43)0.039U/mL

9Glucose87.4+7.1(49)89.6+6.8(44)0.31mg/dL

10Creatinin0.918+0.086(49)0.900+0.089(44)0.71mg/dL

11Apolipoprotein B89.6+16.4(49)86.7+17.0(44)0.321mg/dL

12hs CRP0.57+1.01(49)0.73+1.62(44)0.545mg/L

13Uric acid5.0+0.96(49)4.7+0.89(44)0.122mg/dL

14total Chol217.7+30.9(49)216.2+31.6(44)0.137mg/dL

15HDL53.6+11.2(49)54.6+10.6(44)0.128mg/dL

16LDL141.3+30.1(49)139.1+31.6(44)0.078mg/dL

17TG127.0+68.2(49)141.8+74.8(44)0.461mg/dL

18FFA660.3+217.0(49)640.0+223.7(44)0.612Eq/L

19BUN15.3+3.9(49)14