15% 15% [Current Reproductive Endocrinology 27(3): (2000)] SBS % 15% [ ] n

Transcription

1 코드번호 D-03 (cgmp) (WHO ICH ) compound K 200 [ ] (infertility)/(subfertility) 1 1) 2) 3) , , % 5.8% % 2010 (73%) (66.7%) 30 (1 180 ) (1 50) , , % 11.3% 2-1 -

2 15% 15% [Current Reproductive Endocrinology 27(3): (2000)] SBS % 15% [ ] ng/ml 2 (radish leaves) [US patent ] retinoid agonist ( tarzarotene)[us patient ] retinoid agonist Vit. C, zinc, selenium, Vit. E, Vit. B12, L-carnithine, b-carotene [ ] 2~ % 1 FTA % 1990 IMF 1 [KIM, SLK et al. Panax ginseng protects the testis against 2,3,7,8-tetrachlorodibenzo-р-dioxin induced testicular damage in guinea pigs, BJU International, 83: (1999).] 1) 2) - 2 -

3 3) launching 1) 2) 3) 4) 5) 6) WHO tonic 6 ipet ipet 2011 Experimental Gerontology GINST Fig. 1 GINST Fig

4 GINST Fig. 3 MDA GINST Fig. 4 Figure 4. Effects of GINST on testis parameters related to spermatogenesis in aged rats. (A) Sperm number per tubule, (B) Sertoli cells per tubule, (C) Germ cells per tubule, and (D) Sertoli cell index. Statistical comparisons: YCR vs V-AR, * p< 0.05, ** p< 0.01; V-AR vs. GINST-AR, # p< 0.05, ## p< Values are mean ± SD (n=6)

5 Figure 5. Effects of GINST on testicular antioxidant enzymes in aged rats. (A) GST activity (μmol/min/mg of protein). (B) GPx activity (μmol/min/mg of protein). (C) GSH level, expressed as μg/mg protein. (D) MDA level, expressed as nmol/min/mg protein. Statistical comparisons: YCR vs. V-AR, ** p< 0.01; V-AR vs. GINST-AR, ## p< n=6. Figure 6. Histological analysis of seminiferous tubules in the rat testis. Representative images of tubular cross-sections of testis from young control rats (YCR, A), vehicle-treated aged rats (V-AR, B), and GINST-treated aged rats (GINST-AR, C). Sections were stained with H&E. The images are typical of those obtained in five independent experiments. Scale bar = 45 μm. LC, Leydig cell; PS, primary spermatocyte; SG, spermatogonium; SP, spermatozoa; SR, Sertoli cell; ST, spermatid. proteome - 5 -

6 Figure 7. Protein expression and confirmation of changes in expression in AR and GINST-AR. (A) Arrows indicate proteins that were differentially expressed in V-AR relative to GINST-AR. Protein levels were quantified from four independent experiments ( * p<0.05). The numbers indicated in the gels correspond to the numbers in Table 1. (B) Protein expression of GSTmu5 and PH-GPx in testis tissues. Tissue lysates from V-AR and GINST-AR testis were immunoblotted with anti-gstmu5 and -PH-GPx antibodies. The images are typical of those obtained in blots from three independent experiments. (C) GSTmu5 and PH-GPx mrna levels in testis from V-AR and GINST-AR. GAPDH served as a loading control. The images are representative of three independent experiments. 20 ppm (repeatable) (sensitive) GINST (oligospermia) (asthenospermia) GINST GINST (Prx, Gpx, SOD, CAT ) (inhibin-α, nectin-2) (FSHR, AR, LHR) mrna GINST (Prx, Gpx, SOD, CAT (inhibin-α, nectin-2) (FSHR, AR, LHR) mrna GINST (Prx, Gpx, SOD, CAT ) - 6 -

7 (inhibin-α, nectin-2) (FSHR, AR, LHR) mrna GINST GINST (Prx, Gpx, SOD, CAT ) (inhibin-α, nectin-2) (FSHR, AR, LHR) (testosterone, LH, FSH) Heat stress GINST (Prx, Gpx, SOD, CAT ) (inhibin-α, nectin-2) (FSHR, AR, LHR) (testosterone, LH, FSH) Immobilization stress GINST (Prx, Gpx, SOD, CAT ) (inhibin-α, nectin-2) (FSHR, AR, LHR) (testosterone, LH, FSH) validated ICH guideline Compound K validated (specification) 3 batch QC data( ) real time & accelerated stability CASA(computer aided sperm analyzer) microscope sperm morphology MDA analysis Halo-sperm study serum OT/PT (AST/ALT), serum FSH, LH, Testosterone, SHBG (brief sexual function inventory) (fatigue severity scale) (SF-36 health survey score) - 7 -

8 코드번호 D-04 10,306 2, (radish leaves) [US patent ] retinoid agonist ( tarzarotene)[us patient ] retinoid agonist Vit. C, zinc, selenium, Vit. E, Vit. B12, L-carnithine, b-carotene [ ] compound K GINST ICH WHO guideline (pharmacodynamics) (clinical study) (GMP) 350(medisobizanews, ) 15% 70% 10% ( vol.57) Dailymedi ( CMH Health International (4,000)

9 6500 (4,700) (5,476) (5,829) (6,042) 5 50% 600 (10~15 2~3% DDT 1970 zero tolerance positive system 1 CO 2 cost-, labor-, efficiency-effective GINST protopanaxadiol compound K ( ) C20 ginsenoside Rd Rg3 artifact ginsenoside Rg3 compound K C3 C20 compound K compound K - 9 -

10 OH OH H OH O 6' 5' 4' OH 3' 2' 1' OH 6'' OH O 5'' 4'' OH 1'' OH 3'' 2'' OH H Fig. 8. Ginsenoside Rg3( ) compound K( ) 코드번호 D-05 In vitro (ATCC) No. CRL-2196 Spermatocyte GC-2spd, No. CRL-1714 Leydig TM3, No. CRL-1715 sertoli TM4 cell (ATCC, USA) 37ºC 5% CO 2 incubator GC-2spd 10% fetal bovine serum (Gibco) 100 units/ml penicillin (Gibco), 100 ng/ml streptomycin (Gibco) Dulbecco s Modified Eagle s medium (DMEM; Gibco, USA) TM3 TM4 DMEM Ham s F12 (1:1, v/v) 5% horse serum (Gibco), 2.5% fetal bovine serum, 100-units/mL penicillin, 100 ng/ml streptomycin RNA Total RNA Invitrogen TRIZOL (KRG-WE) 100 μg /ml (CS) 10 μg /ml (NS) 90 μg /ml 2 Hydrogen peroxide 600 μm PBS 2 TRIZOL 0.4 ml 100 μl chloroform 2~3 14,000 rpm 20 2-propanol 1:1 14,000 rpm 10 75% ethanol DEPC 20 μl RNA (PCR) TRIZOL RNA M-MLV reverse transcriptase protocol cdna total RNA (1 g) 0.1 g oligo-(dt) 10 mm dntps 65 C 10 single strand 1X single strand

11 buffer, 0.5 mm DTT, 200 Unit M-MLV reverse transcriptase 25 C C C 15 PCR tube 250 ng cdna, 1xPCR buffer, 1 mm MgCl2, 200 mm dntps, 0.2 mm의 primer PCR 92 C C C cycling PCR 1.5 % agarose gel 6 μl mrna S (Glutathion S-transferase, GST) GSTm5 (Glutathion peroxidase, GPX) GPX4 PRX (Peroxiredoxin) PRX3 mrna primer GSTm5: TCA AGC TAG ATC TGG ACT TTC CTA A (Forward) GSTm5: ATC ATA GGT GAG AAA ATC CAC AAA G (Reverse) GPX4: AGG CAA AAC TGA CGT AAA CTA CAC T (Forward) GPX4: CGT TCT TAT CAA TGA GAA ACT TGG T (Reverse) PRX3: ATG AGT TTC ATG ACG TAA AC (Forward) PRX3: AAA GTA CTC TTT TGA AGC TG (Reverse) pathway mrna nectin-2 inhibin-α mrna mrna mrna Nectin-2: AGT GAC CTG GCT CAG AGT CA (Forward) Nectin-2: TAG GTA CCA GTT GTC ATC AT (Reverse) Inhibin-α: TTG ACT CTA CAG GAT GTG GA (Forward) Inhibin-α: ACA TAA GTG AAG AGA CCT TC (Reverse) receptor mrna receptor Androgen receptor (AR) Follicle-stimulating hormone receptor (FSHR) Luteinizing hormone receptor (LHR) mrna mrna mrna AR : CTG GAC TAC CTG GAT CTC TAC CTT A (Forward) AR : CCT GGG CTG TAG TTT TAT TGT ACT T (Reverse) FSHR : ATG CTG CTG GCT TTT TCA CT (Forward) FSHR : TCT TGG TGT CGC TTG ATG AG (Reverse) LHR : GCA TTC AAT GGG ACG ACT CT (Forward) LHR : CCT CAA AGA TGG CGG AAT AA (Reverse) In vivo

12 23 ± 2 C 55 ± 5% 12 SPF (Specific Pathogen Free) 12 (male, Sprague Dawley) 4 AIN-76A (YC, Young control, 7) (OC, Old control) GINST 200mg/kg b.w. (GINST-AR, 200 mg/kg b.w.) 9 4 (male, Sprague Dawley) 6 AIN-76A (NC, Normal control) (HC, Heating control) GINST 100mg/kg b.w. (HG100, 100mg/kg b.w.) GINST 200mg/kg b.w. (HG200, 200mg/kg b.w.) 10 4 (male, Sprague Dawley) 6 AIN-76A (NC, Normal control) (IC, Immobilization control) GINST 100mg/kg b.w. (IG100, 100mg/kg b.w.) GINST 200mg/kg b.w. (IG200, 200mg/kg b.w.) 10 GINST GINST rat 1 200mg/kg b.w , 200mg/kg b.w , 200mg.kg b.w (Blood Chemistry Panel) (Glu: glucose, T-cho: total cholesterol, LDL: LDL-cholesterol, HDL: HDL-cholesterol, TG: triacylgrycerol) (testosterone, LH, FSH) (Hitachi Medical Co., Ltd., Model 747, Tokyo, Japan) 10% buffered formalin 48 automatic tissue processor(citable 2000) Hematoxylin & Eosin(H&E) Johnsen's score seminiferous tubule 10 (Table 1)[4]

13 Table 1. Parameter for testicular biopsy score counting system by Johnson's method Score 10 9 Description of scoring system Complete spermatogenesis with many spermatozoa (determined by head form) Germinal epithelium organized in regular thickness leaving an open lumen Many spermatozoa present but germinal epithelium disorganized with marked sloughing or obliteration of lumen 8 Only a few spermatozoa present (<5 to 10) 7 No spermatozoa but many spermatids present 6 No spermatozoa and only a few spermatids present (<5 to 10) 5 No spermatozoa and no spermatids but several or many spermatocytes present Only a few spermatocytes (<5) but no spermatids or spermatozoa 4 present 3 Spermatogonia are the only germ cells present 2 No germ cells, but Sertoli cells are present 1 No cells in tubular section Data from Johnsen's SC: Hormones 1:2, RT-PCR RNA Total RNA Invitrogen TRIZOL 50 μ g TRIZOL 0.5 ml 150 μl chloroform 2~3 14,000 rpm 20 TRIZOL 0.5 ml chloroform 150 μl 14,000 rpm 20 2-propanol 1:1 14,000 rpm 10 75% ethanol DEPC 60 μl RNA (PCR) TRIZOL RNA M-MLV reverse transcriptase protocol cdna total RNA (1 g) 0.1 g oligo-(dt), 10 mm dntps 65 C 10 single strand 1X single strand buffer, 0.5 mm DTT, 200 Unit M-MLV reverse transcriptase 25 C C C 15 PCR tube 250 ng cdna, 1xPCR buffer, 1 mm MgCl2, 200 mm dntps, 0.2 mm의 primer PCR 92 C C C cycling primer PCR 2% agarose gel 5 μl loading

14 mrna S (Glutathion S-transferase, GST) GSTm5 (Glutathion peroxidase, GPX) GPX4 PRX (Peroxiredoxin) PRX3 mrna GSTm5: TCA AGC TAG ATC TGG ACT TTC CTA A (Forward) GSTm5: ATC ATA GGT GAG AAA ATC CAC AAA G (Reverse) GPX4: AGG CAA AAC TGA CGT AAA CTA CAC T (Forward) GPX4: CGT TCT TAT CAA TGA GAA ACT TGG T (Reverse) PRX3: ATG AGT TTC ATG ACG TAA AC (Forward) PRX3: AAA GTA CTC TTT TGA AGC TG (Reverse) pathway mrna nectin-2 inhibin-α mrna mrna mrna Nectin-2: AGT GAC CTG GCT CAG AGT CA (Forward) Nectin-2: TAG GTA CCA GTT GTC ATC AT (Reverse) Inhibin-α: TTG ACT CTA CAG GAT GTG GA (Forward) Inhibin-α: ACA TAA GTG AAG AGA CCT TC (Reverse) receptor mrna receptor androgen receptor (AR) follicle-stimulating hormone receptor (FSHR) luteinizing hormone receptor (LHR) mrna mrna mrna AR : CTG GAC TAC CTG GAT CTC TAC CTT A (Forward) AR : CCT GGG CTG TAG TTT TAT TGT ACT T (Reverse) FSHR : ATG CTG CTG GCT TTT TCA CT (Forward) FSHR : TCT TGG TGT CGC TTG ATG AG (Reverse) LHR : GCA TTC AAT GGG ACG ACT CT (Forward) LHR : CCT CAA AGA TGG CGG AAT AA (Reverse) Western blot standard buffer (RIPA buffer 980 ml + protease inhibitor 10 ml + phosphatase inhibitor 10 ml) lysis (14,000 rpm, 20 min, 4 C)

15 loading buffer (5X) 10% SDS-PAGE PVDF membrane transfer 5% skim milk 1 blocking GSTm5, PRX2, nectin-2, inhibin-α, actin 1 overnight ECL detection (Thermo) SPSS package (standard deviation, SD) one way analysis of variance (ANOVA) p<0.05 Duncan s multiple range test 1 ICH WHO IH901 RM (100% method HPLC peak purity 94%) HPLC (ODS, 20 x 250 mm YMC-Pack, 203 nm, 55% CH 3 CN) IH901 peak IH901 RM 1 H NMR spectroscopy, 13 C NMR spectroscopy, 1 H- 1 H COSY spectroscopy, HMQC spectroscopy, HMBC spectroscopy, NOESY spectroscopy, FT-IR spectroscopy, Mass spectrometry, UV Spectrophotometry Melting point, Elemental analysis 3 HPLC Method, Water content, Residual solvent content, Optical rotation, Thin-layer chromatographic purity test 3 HPLC Method Validation ICH guideline (LOD & LOQ)

16 2 Gas chromatography, HPLC 2 total motile sperm count 0.5x mmHg 90mmHg 1 BMI > 30 BMI < 18 CASA (computer aided sperm analyzer ) Ejaculated volume, sperm count, total motile sperm count, motility, morphology, total leukocyte count open label mg/ 1.92g/ ) 500mg/( 0.96g/ ) 3 block randomization 3 CASA (computer aided sperm analyzer )& microscopic examination Ejaculated volume, sperm count, total motile sperm count, motility, morphology, total leukocyte count

17 Sperm chromatin assessment (Halo sperm assay) (Seminal fluid, oxidative stress Malondialdehyde(MDA) analysis Total testesterone & free testosterone index, FSH, LH, Inhibin B, OT (ALT), PT(AST) (SF-36) (Brief sexual function inventory) (fatigue severity scale) (IRB No ) < 32% 140mmHg 90mmHg 1 BMI > 30 BMI < 18 CASA (computer aided sperm analyzer Ejaculated volume, sperm count, total motile sperm count, motility, morphology, total leukocyte count 500mg/ mg/ 0.96g/ 3 3 CASA (computer aided sperm

18 analyzer & microscopic examination Ejaculated volume, sperm count, total motile sperm count, motility, morphology, total leukocyte count Total testesterone & free testosterone index, FSH, LH, Inhibin B, OT (ALT), PT(AST) (SF-36) (Brief sexual function inventory) (fatigue severity scale) 12 (IRB No ) < 32% 140mmHg 90mmHg 1 BMI > 30 BMI < 18 CASA (computer aided sperm analyzer Ejaculated volume, sperm count, total motile sperm count, motility, morphology, total leukocyte count double blinded mg/ 0.96g/ placebo

19 CASA (computer aided sperm analyzer & microscopic examination Ejaculated volume, sperm count, total motile sperm count, motility, morphology, total leukocyte count Sperm chromatin assessment (Halo sperm assay) (Seminal fluid oxidative stress Malondialdehyde (MDA) analysis Total testesterone & free testosterone index, FSH, LH, Inhibin B, OT (ALT), PT(AST) (SF-36) (Brief sexual function inventory) (fatigue severity scale) 1 GINST GINST Compound K in vitro doxorubicin 16 in vivo CRO 1 validation cgmp real time Batch analysis data WHO 2 (serum AST/ALT, serum FSH, LH, testosterone, SHBG, Inhibin β) CASA(computer aided sperm analyzer) oxidative stress

20 1 in vitro GINST mrna GINST compound K (CK) mrna Mouse GC-2spd 600 μm PRx3, PRx4, GPx4 GSTm5 mrna (p<0.01~0.05) mrna GINST CK GINST 50 mg/ml 100 mg/ml CK 10 μm, 20 μm 2 PRx3, PRx4 GPx4 GINST mrna (p<0.01~0.05) GSTm5 GINST CK (p<0.05). Fig 9. GC-2spd cells mrna *, GINST compound K ** Significantly different from control at p<0.05 and p<0.01, respectively., Significantly different from H 2 O 2 at p<0.05 and p<0.01, respectively. Leydig cell TM3 300 μm

21 mrna (p<0.01~0.05) GINST (p<0.01~0.05) CK Fig 10. TM3 cells mrna *, GINST compound K ** Significantly different from control at p<0.05 and p<0.01, respectively. Significantly different from H 2 O 2 at p<0.01, respectively. mrna GC-2spd pathway mrna fig μm inhibin-α mrna (p<0.01) GINST CK (p<0.01) fig. 12 TM3 inhibin-α mrna

22 Fig. 11. GC-2spd cells receptor mrna GINST compound K * Significantly different from control at p<0.05, respectively., Significantly different from H 2 O 2 at p<0.05 and p<0.01, respectively. TM3 300 μm receptor (p<0.01~0.05) GINST (p<0.01~0.05) CK FSHR LHR (p<0.01~0.05) Fig. 12. TM3 cells receptor mrna *, GINST compound K ** Significantly different from control at p<0.05 and p<0.01, respectively., Significantly different from H 2 O 2 at p<0.05 and p<0.01, respectively

23 (doxorubicin) GINST mrna Sertoli, Leydig) GINST compound K (CK) mrna GC-2spd cell line doxorubicin 500 μm GC-2spd cell line mrna doxorubicin 500 μm DMSO doxorubicin DMEM doxorubicin 500 μm doxorubicin Fig. 13. Effect of GINST on the mrna expression level of antioxidant enzymes in doxorubicin-exposed GC-2spd cells. GC-2spd (sperm) cells were treated with GINST (100 mg/ml) for 3 hr before exposing the cells to doxorubicin (500 μm, in DMSO). The band intensities of PRx3, GPx4 and GSTm5 were analyzed by using an NIH ImageJ package and normalized to GAPDH value. mrna Doxorubicin (GC-2spd cells) pathway biomarker (inhibin-α, nectin-2) mrna doxorubicin

24 Fig. 14. Effect of GINST on the mrna expression level of spermatogenesis related molecules in doxorubicin-exposed GC-2spd cells. GC-2spd (sperm) cells were treated with GINST (100 mg/ml) for 3 hr before the exposure to doxorubicin 500 μm. The band intensities of inhibin-α and nectin-2 were analyzed by using an NIH ImageJ package and normalized to GAPDH value. TM3, TM4 cells TM3, TM4 cells doxorubicin MTT assay Fig μg/ml bio-markers doxorubicin Fig. 15. doxorubicin MTT assay GINST GC-2spd, TM3, TM4 cells MTT assay Fig. 16 cell viability

25 Fig. 16. Dose-dependent effect of GINST on viability of GC-2spd, TM3 and TM4 cells. mrna GINST mrna GC-2spd, TM3, TM4 cell line GSTm5, GPx4 cell line TM4의 cell line (p<0.05) PRx4 cell line Fig. 17. Dose-dependent effect of GINST on mrna expression levels for antioxidant enzymes in GC-2spd, TM3 and TM4 cells. mrna GC-2spd, TM3, TM4 pathway mrna mrna

26 Fig. 18. Dose-dependent effect of GINST on mrna expression levels for spermatogenesis related molecules in GC-2spd, TM3 and TM4 cells. mrna receptor AR mrna GC-2spd cell GINST LHR FSHR mrna Fig. 19. Dose-dependent effect of GINST on mrna expression levels for sex hormone receptors in GC-2spd, TM3 and TM4 cells. in vivo

27 GINST GINST Fig. 20 (aged rat, V-AR) testosterone YCR GINST testosterone AR (p<0.05) FSH LH (YCR) AR (p<0.05) GINST (GINST-AR) AR FSH LH (p<0.05~0.01) Fig. 20. GINST * Significantly different #, ## from YCR at p<0.01, respectively. Significantly different from V-AR at p<0.05 and p<0.01, respectively. Fig. 21 YCR V-AR (Sertoli cell) GINST-AR V-AR V-AR

28 A. YCR B. V-AR C. GINST-AR Fig. 21. GINST LC, Leydig cell; PS, primary spermatocyte; SG, spermatogonium; SP, spermatozoa; SR, Sertoli cell; ST, spermatid. Fig. 22-A YCR V-AR (p<0.05) GINST (p<0.01) Fig. 22-B, C D Sertoli cell germ cell SCI V-AR (p<0.01) GINST-AR V-AR (p<0.01) Fig. 22. GINST *, ** Significantly different from YCR at p<0.05 and p<0.01, respectively. ## Significantly different from V-AR at p<0.01, respectively. Fig GINST MDA GSH level SOD, GPx, GR GST MDA YCR V-AR MDA (p<0.01) GINST-AR MDA (p<0.01)

29 V-AR GSH (p<0.01) GINST-AR (p<0.01) Fig. 23. MDA GSH level GINST Significantly different from YCR at p<0.01. Significantly different from V-AR at p<0.01. ** YCR V-AR (p<0.01) GINST-AR (p<0.01) SOD YCR GINST-AR Fig. 24. GINST Significantly different from YCR at p<0.01., Significantly different from V-AR at p<0.05 and p<0.01, respectively. ** Fig. 25 GINST V-AR MDA level V-AR YCR GSH

30 (p<0.01) GINST (p<0.01) Fig. 25. MDA GSH level GINST different from YCR at p<0.01. Significantly different from V-AR at p<0.01. ** Significantly Fig. 26 GR, GPx, SOD, GST GINST (p<0.01) Fig. 26. GINST *, ** Significantly different from YCR at p<0.05 and p<0.01, respectively., Significantly different from V-AR at p<0.01, respectively. GINST Table 2 spleen heart heat stress GINST (p<0.01) GINST NC (p<0.01) heat stress GINST NC HC

31 (p<0.05) GINST Table 2. Effect of GINST in rat organ weight of heat-induced stress. Organ Group NC HC HG100 HG200 Liver Spleen Kidney Testis Heart Epidydimis ± ± ± ± ± ± ± ± ± ± ** ± ± ± ± ± ± ± ± ± ± ± ± * ± ± Adrenal gland ± ± ± ± Data are expressed as the mean ± S.E.M. (n=10). Statistical analysis was performed by ANOVA followed by Duncan's t-test using the SAS ver. 9.1 (SAS Inc., Cary, NC, USA). NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. GOT heat stress (p<0.05) HG200 (p<0.05) γ-gpt heat stress GINST ALB GINST (Table 3) Table 3. Effect of GINST on liver-related blood chemistry panels. Group GOT γ-gpt ALB NC 77.3 ± ± ± 0.1 HC ± 23.5** 22.5 ± ± 0.2 HG ± ± ± 0.1 HG ± ± ± 0.2 Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group

32 GINST NC HC GINST (p<0.05) Table 4. Effect of GINST on glucose and lipid metabolism-related blood chemistry panels. Group T-CHO TG LDL-C GLU NC 89.3 ± ± ± ± 10.5 HC 66.3 ± ± ± ± 15.5** HG ± ± ± ± 16.1 HG ± ± ± ± 27.4 Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. T-Cho: total cholesterol, LDL-C: low density lipoprotein-cholesterol, HDL: high density lipoprotein-cholesterol TG: triacylglycerol. NC HC (motility) (progressive) (p<0.01) HG200 (p<0.05) average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straitneness (STR), linearity (LIN) elongation GINST Table 5. Effect of GINST on sperm kinematic values of heat-induced stress. Average Group NC HC Motile Progressive VAP VSL VCL STR LIN Elongation ± ± ± ± ± ± ± ± 20.68* 8.27* ± ± ± ± ± ± ± ±

33 HG ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± HG Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. VAP; average path velocity, VSL; straight line velocity, VCL; curvilinear velocity, LIN; linearity = VSL/VCL 100, STR; straightness = VSL/VAP 100. mrna S (glutathion S-transferase, GST) GSTm5 PRx (Peroxiredoxin) PRx4 RT-PCR GPx4, GSTm5 PRx4 heat stress (p<0.01) GINST (p<0.05) Fig. 27. Effect of GINST on mrna expression of antioxidant enzymes in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group

34 p<0.01 compared with HC group. p<0.05 compared with HC group. mrna pathway CREB-1 Nectin-2 mrna heat stress (p<0.01) GINST heat stress CREB-1 Nectin-2 mrna (p<0.05) Fig. 28. Effect of GINST on mrna expression levels of spermatogenesis-related molecules in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. mrna Fig. 29 heat stress (luteonizing hormone receptor, LHR) (follicle stimulating hormone receptor, FSHR) (androgen receptor, AR) (p<0.01) GINST heat stress

35 Fig. 29. Effect of GINST on mrna expression of sex hormone receptors in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. Western blot Fig. 30 mrna GPx4, GSTm5 PRx4 heat stress (p<0.01) GINST (p<0.05) GINST heat stress

36 Fig. 30. Effect of GINST on protein expression of antioxidant enzymes in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. biomarker pathway CREB-1 Nectin-2 protein mrna heat stress (p<0.05~0.01) GINST heat stress Nectin-2 CREB Fig. 31. Effect of GINST on protein expression level of spermatogenesis-related biomarker molecules in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group

37 Fig. 32 LHR, FSHR, AR heat stress (p<0.05~0.01) GINST heat stress (p<0.05~0.01) androgen receptor 1/8 GINST 80% sensitivity Fig. 32. Effect of GINST on protein expression of sex hormone receptors in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. GINST heat stress pathway biomarker molecules GINST GINST Table 6 immobilization stress

38 GINST GINST IC GINST Organ Group NC IC Table 6. Effect of GINST in rat organ weight of immobilization-induced stress. IG100 Liver Spleen Kidney Testis Heart Epidydimis ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Adrenal gland ± ± ± IG ± ± ± ± ± ± ± Data are expressed as the mean ± S.E.M. (n=10). Statistical analysis was performed by ANOVA followed by Duncan's t-test using the SAS ver. 9.1 (SAS Inc., Cary, NC, USA). NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. (blood chemistry panel) ALT immobilization stress (p<0.05) IG ALP immobilization stress IG100 ALB GINST (Table 7) Table 7. Effect of GINST on liver-related blood chemistry panels. Group ALT ALP ALB NC 31.0 ± ± ± 0.08 IC 24.6 ± 4.2* ± ± 0.12 IG ± ± ± 0.11 IG ± ± ± 0.12 Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<

39 compared with HC group. p<0.05 compared with HC group. GINST NC HC GINST (p<0.05) Table 8. Effect of GINST on glucose and lipid metabolism-related blood chemistry panels. Group T-CHO TG LDL-C GLU NC ± ± ± ± 27.2 IC ± ± ± ± 16.9 IG ± ± ± ± 13.3 IG ± ± ± ± 9.9 Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; HC: heat-stressed control; HG100: heat-stressed and received GINST 100 mg/kg b.w.; HG200: heat-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. T-Cho: total cholesterol, LDL-C: low density lipoprotein-cholesterol, HDL: high density lipoprotein-cholesterol TG: triacylglycerol. NC IC (motility) (progressive) (p<0.05) IG (p<0.01~0.05) average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straitneness (STR), linearity (LIN) elongation GINST Table 9. Effect of GINST on sperm kinematic values of immobilization-induced stress. Average Motile Progressive VAP VSL VCL STR LIN Elongation Group ± 6.00 ± ± ± ± ± ± ± NC IC ± 3.63 ± ± ± ± ± ± ±

40 11.17* 1.36* IG ± 5.88 ± ± ± ± ± ± ± IG ± 7.13 ± ± ± ± ± ± ± Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. *p<0.01 compared with NC group. **p<0.05 compared with NC group. p<0.01 compared with HC group. p<0.05 compared with HC group. VAP; average path velocity, VSL; straight line velocity, VCL; curvilinear velocity, LIN; linearity = VSL/VCL 100, STR; straightness = VSL/VAP 100. mrna S (glutathion S-transferase, GST) GSTm5 PRx (Peroxiredoxin) PRx4 RT-PCR GSTm5 immobilization stress (p<0.05) GINST (p<0.05) Fig. 33. Effect of GINST on mrna expression of antioxidant enzymes in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC

41 group. p<0.01 compared with IC group. p<0.05 compared with IC group. mrna CREB-1 Inhibin-α immobilization stress (p<0.05) GINST (p<0.01~0.05) Fig. 34. Effect of GINST on mrna expression levels of spermatogenesis-related molecules in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with IC group. p<0.05 compared with IC group. mrna Immobilization stress (luteonizing hormone receptor, LHR) (follicle stimulating hormone receptor, FSHR) (androgen receptor, AR) (p<0.01~0.05) GINST

42 Fig. 35. Effect of GINST on mrna expression of sex hormone receptors in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with IC group. p<0.05 compared with IC group. Western blot GPx4, GSTm5 PRx4 immobilization stress GINST Fig. 36. Effect of GINST on protein expression of antioxidant enzymes in rat testis

43 Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with IC group. p<0.05 compared with IC group. biomarker Inhibin-α Immobilization stress mrna mrna GINST Fig. 37. Effect of GINST on protein expression level of spermatogenesis-related biomarker molecules in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with IC group. p<0.05 compared with IC group. AR, LHR, FSHR mrna immobilization stress (p<0.05~0.01) GINST heat stress (p<0.05~0.01)

44 Fig. 38. Effect of GINST on protein expression of sex hormone receptors in rat testis. Data are expressed as mean ± S.E.M. (n=10). Statistical analysis was carried out by Student's t-test and one way ANOVA using GraphPad Prism version 4.0. NC: normal control; IC: immobilization-stressed control; IG100: immobilization-stressed and received GINST 100 mg/kg b.w.; IG200: immobilization-stressed and received GINST 200 mg/kg b.w. * p<0.01 compared with NC group. ** p<0.05 compared with NC group. p<0.01 compared with IC group. p<0.05 compared with IC group

45 Fig. 39. Fig. 40. Fig. 41. GINST Table 10. Panax ginseng C.A Meyer PanaxschinsengNees Ginseng Ginseng Radix Alba rea.go.kr/ KISS, DBpia or or and or in vitro in vivo ginsenoside Total Total

46 Pubmed (ginseng or red ginseng or ginsenoside) and (sperm motility or sexual function) in vitro in vivo Total ginsenoside Total Table g/ 1.5 ~ 3.0 g/ ( ) Table 12. Rg1 Rg1 Rb1 3~ Rb1 80 mg 0.8~34 mg/g Ginsenoside Rg1 (" mg/ 11) ( g/ 6) 1 Ligustilide 2 Eleutheroside E g/day 3 Ginsenoside Rg1 Rb1 (" ) 4 Baicalin

47 2 Rg1, Rb1 Rg3 3~80 mg Rg1, Rb1 Rg3 2.4~80 mg Rg1, Rb1 Rg3 25 ~ 80mg/ 3 g/ Rg1, Rb1 Rg ( ) ( ) (CRO; Contact Research Organization) (Source Data Verification: SDV) template follow-up monitoring visit check Source Data Verification (SDV)

48 routine monitoring source data source document Fig. 42. issues, checklist results data monitoring SDV Table /03/ /03/ /03/ /06/ /11/ /01/ /11/ /05/ /08/ /05/ /08/ /02/ /04/ /07/ /11/ /08/ /11/ /04/ /07/ /07/ /11/ /04/ /07/ /09/ /11/ /06/ /11/ /01/ /01/ /07/ /03/ /05/ /08/ /11/ /04/

49 Fig. 43. Table

50 3 LOT 3 GLP Table 15. Panax ginseng C.A.Meyer Ginseng, Korean ginseng, Compound K Compound K 5 mg/g mg/kg mg/kg

51 mg/kg 0.5 mg/kg cfu/ml Compound K HPLC, 203 nm 58 DB DB 180 mmhg 960 mg/ Rg1 Rb1 3~80 mg 1.5~10 g 30 g 1 9~11 3~4g 6~8g 5~6 4g 20~24g Rg1 Rb1 3~80 mg Rg1 Rb1 0.8~34 mg 960 mg/day 2 6 g/day g/day

52 3 100mg/kg receptor 960~970mg/d 0.96~4g/d mg/ 2000 mg/kg 250 mg/kg 500 mg/kg 1000 mg/kg Chinese Hamster Lung(CHL/IU) mg/kg (MTD) 500 mg/kg (NOAEL) 500 mg/kg 180 mmhg 960 mg/ Mouse GC-2spd mrna PRx3, PRx4, GPx4, GSTm5 mrna mrna

53 inhibin-α mrna receptor mrna AR, FSHR, LHR mrna (GINST) [J Ginseng Res Apr;40(2): ] (aged rat, V-AR, 12-mo-old) 200mg/kg 4 protein PRx3, GPx4, GSTm5 protein mrna PRx3, PRx4, GPx4, GSTm5 mrna protein Nectin-2, CREB-1 protein mrna Nectin-2 mrna receptor protein, mrna AR, LHR, FSHR protein mrna (GINST) [J Ginseng Res Apr;40(2): ] (aged rat, V-AR, 12-mo-old), 200mg/kg, 4 V-AR V-AR VAP, VCL, LIN testosterone LH, FSH Sertoli cell, germ cell, SCI GST, GPx, GSH MDA (GINST) [Exp Gerontol May;53: ] (heat stress in rat), 100, 200mg/kg, 9 (GSTm5, GPx4) (CREB-1, inhibin-α) receptor(ar, LHR, FSHR) protein, mrna

54 (GINST) (heat stress in rat), 100, 200mg/kg, 6 receptor protein, mrna (GINST) (n= 60), 960 mg/ 12 oxidative stress MDA Total testosterone, Sex Hormone binding globulin, Free androgen index (FAI), free testosterone, FSH, LH, Inhibin B, OT (ALT), PT(AST) (GINST) 960mg/g ICH WHO GINST EU Eurofins Dr. Specht Laboratorien EU Table 16. Dr. Specht Laboratorien

55 Pb, Cd EU As, Hg AflatoxinB1 593 EU Table 17. 구분 Parameter Measurement 중금속 곰팡이 독소 잔류 농약 미생물 Dimensio n Result RL* MRL* * Lead(Pb)## ICP-MS mg/kg Cadmium(Cd)## ICP-MS mg/kg Arsenic(As)## ICP-MS mg/kg n.n Mercury(Hg)## ICP-MS mg/kg n.n AflatoxinB1## IAC,HPLC-FLD µg/kg n.n. 1 - AflatoxinB2## IAC,HPLC-FLD µg/kg n.n. 1 - AflatoxinG1## IAC,HPLC-FLD µg/kg n.n. 1 - AflatoxinG2## IAC,HPLC-FLD µg/kg n.n. 1 - Quintozene(sum) GC-ECD mg/kg Pentachloroaniline GC-ECD mg/kg Tebuconazole LC-MS/MS mg/kg Trifloxystrobin LC-MS/MS mg/kg Propamocarb LC-MS/MS mg/kg Pyraclostrobin LC-MS/MS mg/kg Pyrimethanil LC-MS/MS mg/kg Metalaxyl LC-MS/MS mg/kg Azoxystrobin LC-MS/MS mg/kg Fenhexamid LC-MS/MS mg/kg Fludioxonil LC-MS/MS mg/kg Cyprodinil LC-MS/MS mg/kg Difenoconazole LC-MS/MS mg/kg Dimethomorph LC-MS/MS mg/kg AerobicPlateCount30 C Microbiological cfu/g <10 - Escherichiacoli## Microbiological cfu/g < No further pesticides/parameters of above mentioned scope of analysis are detectable. *RL=Reporting Limit () *MRL=Maximum Residue Level according to Regulation (EC) NO396/2005 (EU ) 비고

56 Fig 44. Eurofins / Dr.SpechtLaboratorien clinical formulae Placebo formulae GINST 960 mg mg GINST data 960 mg 1920 mg Tablet GINST 500 mg Tablet 32% 160 mg soft capsule Table (960 mg/day % mg GINST Table (1920 mg/day % mg GINST

57 1000 Table 20. Placebo % mg Placebo RP corp. batch analysis data (3 batches) 3 batch batch 80 kg 3 batch Fig batch data

58 Fig 46. Batch GENERAL CHARACTERISTICS IH901 (ginsenoside K) 20-O-β-D-Glucopyranoside, (3β, 12β)-3,12-dihydroxydammar-24-en-20yl (9CI) IH CAS Compound K C 36 H 62 O g/molar ([M+Na] ) C C 69.42%, H 10.03%, O 20.55% (Found C 69.48%, H 10.04%, O 20.48%) (Panax ginseng C.A. Meyer, Appendix 1) Main metabolite of ginsenoside in human and rat Produced from saponins of Gynostemma pentaphyllum by endogenous glycosidases C C) [α]d (c, 1.0 in MeOH) [α]d (c, 0.5 in pyridine) Compound K (IH901) RM IH901 RM (100% method HPLC peak purity 94%) HPLC (ODS, 20 x 250 mm YMC-Pack, 203 nm, 55% CH 3 CN) IH901 peak (ultra pure distilled water) IH901RM

59 IH901 RM F. Gaedeke et. al. (Appendix 2) (Appendix 3) UV, FT-IR, HR-FAB/MS NMR IH901 RM IH901 RM HPLC TLC fingerprint 3 HPLC 100% (mass balance) HPLC IH901 1 H NMR spectroscopy 1 H NMR spectroscopy (20 mg/ml pyridine-d 5, 500 MHz, TMS as the external reference) IH901 RM 1 H(Appendix4), 13 C(Appendix5), 1 H- 1 HCOSY(Appendix6), HMQC(Appendix7), HMBC(Appendix8) NOESY (Appendix 9) spectra 1 H signals assign (Table 15) proton signal chemical shift (ppm), multiplicity coupling constant NMR spectra 1 HNMR data IH901 RM ginsenoside K Table 21, 22 proton chemical shifts assign NMR IH901 RM chemical shift IH901 RM proton chemical shifts IH901 RM 1 H NMR spectrum ginsenoside K IH901 RM [*L. N. Atopkina et. al., Chemistry of Natural Compounds, Vol. 42 (4), (2006), Appendix 10] Triterpen aglycon glucose moiety resonances 1 H NMR spectrum 1 H 13 C NMR spectroscopy 13 C NMR spectrum deuterated pyridine-d MHz NMR spectrometer (JEOL, Japan) Calibration TMS signal chemical shift 0.00 ppm 13 CNMR spectrum signal Table 23, 24 IH901 RM carbon resonances data (Appendix 10) IH901 RM ginsenoside K ginsenoside analogs 1 H 13 C NMR spectra NMR spectra

60 Fig. 47 IH901 RM Fig. 47. Chemical structure of IH901 Table H NMR data for the IH901 IH901 Ref. literature* No. Chemical shift (ppm) Chemical shift (ppm) Chemical shift (ppm) m ; 1H 0.92 m ; 1H m ; 1H 1.84 m ; 1H m ; 1H OH 5.76 d (J=4.5 Hz) ; 1H d (J=11.0 Hz) ; 1H m ; 1H 1.46 m ; 1H m ; 1H 1.26 m ; 1H m ; 1H m ; 1H 1.54 m ; 1H m ; 1H 12-OH 5.61 s ; 1H t (J=11.0 Hz) ; 1H m ; 1H 1.01 m ; 1H m ; 1H 1.37 m ; 1H m ; 1H s ; 3H s ; 3H s ; 3H m ; 1H 1.84 m ; 1H m ; 1H

61 2.26 m ; 1H t (J=6.5 Hz) ; 1H s ; 3H s ; 3H s ; 3H s ; 3H s ; 3H Table H NMR data for the IH901 Glucose 1 at C d (J=7.5 Hz) ; 1H t (J=8.0 Hz) ; 1H t (J=8.5 Hz) ; 1H t (J=8.5 Hz) ; 1H m ; 1H br d (J=11.5 Hz) ; 1H br d (J=11.5 Hz) ; 1H OH 6.14 s ; 1H Abbreviation: s singlet, d doublet, t triplet, dd doublet of doublets, m multiplet, brs broad singlet. Chemical shifts are expressed in ppm and referenced to the tetramethylsilane (TMS) signal at 0.00 ppm. *L. N. Atopkina and V. A. Denisenko : Synthesis of 20S-protopanaxadiol 20-O-β-D-glucopyranoside, a metabolite of Panax ginseng glycosides, and compounds related to it. Chemistry of Natural Compounds, Vol. 42 (4), (2006), (Appendix 10). Table C NMR data for the IH901 RM C atom Chemical shift (ppm) IH901 Ref. literature

62 Table C NMR data for the IH901 RM Glucose 1 at C Chemical shifts are expressed in ppm and referenced to TMS signal at 0.00 ppm. *L. N. Atopkina and V. A. Denisenko : Synthesis of 20S-protopanaxadiol 20-O-β -D-glucopyranoside, a metabolite of Panax ginseng glycosides, and compounds related to it. Chemistry of Natural Compounds, Vol. 42 (4), (2006), (Appendix 10). IUPAC nomenclature system numbering NMR signals 1 H- 1 H COSYspectroscopy Proton-proton connectivity ( 3 J HH ) 1 H- 1 H COSY spectrum (Appendix6) 1 H- 1 H COSY spectrum cross-peaks IH901 (ginsenoside K) HMQC spectroscopy Direct 1 H- 13 C connectivity( 1 J CH ) HMQC (heteronuclear multiple-quantum coherence) spectrum (Appendix 7) Table 23, 24 HMQC spectrum assign IH901 RM chemical shifts [*L. N. Atopkina et. al., Chemistry of Natural Compounds, Vol. 42 (4), (2006), Appendix 10] ginsenoside K carbon chemical shifts HMBC spectroscopy Two or three-bonded 1 H- 13 Cconnectivity( 2,3 J CH ) HMBC spectrum (Appendix 8)

63 HMBC spectrum IH901 RM [*L. N. Atopkina et. al., Chemistry of Natural Compounds, Vol. 42 (4), (2006), ; Appendix 10] ginsenoside K proton carbon resonances assignment NOESY spectroscopy NOE data IH901 RM NOE data (Appendix 10) NOE data ginsenoside K FT-IR spectroscopy IH901 RM Ge-coated KBr pellet (3.0 mg/g) IR spectroscopy (Mattson IR spectroscopy, model Galaxy 7020A) IR spectrum specific band Table 25 band IR spectrum IH901 RM FT-IR spectrum Appendix 11 IR spectrum IH901 RM (L. N. Atopkina and V.A. Denisenko, 2006) IR spectrum (Appendix 12) Table 25. FT-IR spectral data for IH901 RM Chemical moiety Sample (Wavenumber: -1 ) Ref* ν (O-H): O-H stretching vibration ν (C-H): C-H stretching vibration ν (C=C) : C=C stretching vibration δ (C-H): C-H bending vibration ν (O-H): O-H stretching vibration ν: stretching vibration, δ: bending vibration. FT-IR spectrometer: Mattson Instruments, Inc., Galaxy 7020A, Spectral range: 4000 to 400cm -1, Beam splitter: Ge coated on KBr, detector: DTGS, resolution: 0.25cm -1 (step selectable); *L. N. Atopkina and V. A. Denisenko : Synthesis of 20S-protopanaxadiol 20-O-β-D-glucopyranoside, a metabolite of Panax ginseng glycosides, and compounds related to it. Chemistry of Natural Compounds, Vol. 42 (4), (2006), (Appendix 10). Mass spectrometry IH901 RM positive negative mode HPLC/ESI (LC/MS; LCQ DECA XP, Thermo Finnigan) 10 mg 10 ml MeOH 5 ml LC/MS positive negative mode FAB/MS [JMS-700 Mstation FAB/MS spectrometer, JEOL Ltd.] HR-FAB/MS

64 Conditions for HPLC/MS analysis: Mass spectrometer Equipment : Ionization source : Polarity : Analyzing type : Mass range : Scan mode : HPLC: Pump: Injector: Column oven: Column: Detector: Integrator: Mobile phase: Flow rate: Injection volume: Detection: Temperature: Pump program: LCQ DECA XP, Thermo Finnigan Electrospray ionization (ESI) positive & negative ion trap analyzer 100~2000 m/z mass Waters Alliance 2690 (Waters) Alliance 2690 auto sampler (Waters) Alliance 2690 (Waters) Steel column, 4.9 x 250 mm, C18, 5 μm (Supelco) W 996 photodiode array (Waters) Quattro Ultima tandem mass spectrometer (Micromass) Workstation with MassLynx softrware (Micromass) A; acetonitrile B; water 1.0 ml/min 5 μl UV 203 nm 40 C Flow rate [ml/min] Time [min] Eluent A [%] Eluent B [%] Electrospray ionization mass spectrometry LC/MS IH901 RM (Appendix 13) IH901 RM (Appendix 3) Positive mode m/z peak [M+Na] + (Appendix 13) IH901 RM sugars moiety cleavage Positive negative modes fragment ions Table 26 Negative mode m/z peak [M-H] - m/z 621.6, , [M-H] -,[M-H-C 6 H 10 O 5 ] - [C 6 H 10 O 5 -H] - fragment ions (Appendix 14)

65 Table 26. Possible correlation of the main product ions of IH901 determined by ESI-MS. Correlation IH901 Ref. Fragment m/z Positive mode [M+Na] Negative mode [M-H] [621.6-C 6 H 10 O 5 ] [C 6 H 10 O 5 -H] Meng C., Fengrui S., Zhiqiang L. and Shuyimg L. Metal ion adducts in the structural analysis of ginsenosides by electrospray ionization with multi-stage mass spectrometry. Rapid Commun. Mass Spectrumm. 2001; 15: (Appendix 15) Fast atomic bombardment mass spectrometry (FAB/MS) FAB/MS IH901 RM main product ions Table 27 spectra product ion spectra ginsenoside K IH901 RM ginsenoside K (Appendix 16, 17) HR-FAB-MS C 36 H 62 O 8 ginsenoside K (Appendix 18) Conditions for FAB/MS analysis: Equipment : HR-FAB, JMS-700 Mstation, JEOL Ltd Analyzing type : ion trap analyzer Mass range : 100~1500 m/z FAB mode : mass Table 27. Possible correlation of the main product ions of IH901 RM in FAB/MS Correlation IH901 Ref. Fragment m/z Positive mode [M+Na] Negative mode [M-H] [M-H-C 6 H 10 O 5 ] UV Spectrophotometry IH901 RM UV spectrum UV/VIS spectrophotometer (JASCO, V-530) 0.5 mg/ml (MeOH) 200 ~ 500 nm (Appendix 19) IH901 RM UV 203 nm IH901 RM UV spectrum direct maximum 230 nm UV spectrum IH901 RM HPLC (Hewlett Packard, photodiode array detector, 203 nm) IH901 RM UV (Appendix 20)

66 IH901 RM Melting point IH901 RM 39 1 Manstead/Electrothermal melting point analyzer, IA9100 IH901 RM C (Appendix 20) (Appendix 12) C IH901 RM Elemental analysis 2 mg IH901 RM (Flash EA 1112 series/ce Instrument) 1,100 C 3 (Table 28) Table 28. Elemental composition of IH901 RM reference material. Element Measured value (%) 1 차 2 차 3 차 mean Theoretical value (%) * Carbon Hydrogen Oxygen IH-901 (ginsenoside K): C 36 H 62 O 8 ; (69.42%) (10.03%) (20.55%) 100% Method 1 (HPLC) 100% HPLC Method 1 HPLC fingerprint IH901 RM (Appendix 21) Appendix 30 HPLC method 1 IH901 RM HPLC (specificity) HPLC peak purity peak purity value (purity angle = ; 0.400) ; IH901 RM peak spectrally homogeneous (Appendix 22) 100% Method 2 (HPLC) 100% HPLC Method 2 HPLC fingerprint IH901 RM (Appendix 23) Appendix 23 HPLC method 2 IH901 RM HPLC HPLC peak purity peak purity value (purity angle = ; 0.400) ; 0.457)

67 IH901 RM peak spectrally homogenous (Appendix 24) 100% Method 3 (HPLC) 100% HPLC Method 3 HPLC fingerprint IH901 RM (Appendix 25) evaporative light scattering detector (ELSD, Sedex model 55, SEDERE, Alfortville, France) ginsenosides UV Kwon (Appendix 26) ginsenoside ELSD HPLC HPLC ginsenosides carbohydrate column IH901 RM Rt 7.01 IH901 RM HPLC 3 Water content IH901 RM 26 micro-scale IH901 RM 0.583% (Appendix 28) Residual solvent content IH901 RM Optical rotation IH901 RM [a] D JASCO P-1020 polarimeter (Tokyo, Japan; filter 589 mm, cylindrical glass cell: 10 x 100 mm) IH901 RM MeOH (Table 29) ginsenoside K [a] D IH901 RM [a] D (c, 1.0 in MeOH) Table 29. Optical rotation of IH901 RM Sample No. IH901 RM Average IH901 (c, 1.0 in MeOH); Analytical Equipment: JASCO P-1020 Polarimeter (Tokyo, Japan); Filter589mm, Cylindrical glass cell: 10x100mm

68 Thin-layer chromatographic purity test (TLC IH901 RM TLC fingerprint (Appendix 28) IH901 RM 5 mg 10 ml methanol (0.5 mg/ml) TLC TLC fingerprint Stationary phase: HPTLC plate with silica gel 60 F 254 (20 20cm) Mobile phase: CHCl 3 -MeOH-D/W(9:3:1,v/v/v, lower phase) Applied volume: 10 μl, 20 μl Developing distance: 10 cm Impregation: spray with 20% sulfuric acid, heated at 115 C/15 min Detection: pinkish band appears at Rf value approximately 0.42 IH901 RM Rf value 0.42 TLC fingerprint ginsenosides IH901 RM 100% HPLC method 3 HPLC systems ICH Validation of analytical procedure: methodology (Appendix 29) (validation) HPLC 100% 1 1 IH901 RM 10 mg IH901 RM 10 ml (1,000 mg /ml) HPLC methanol mg/ml 5 HPLC 100% 1 Equipment : Hewlett Packard 1100 Column : C18, mm, 5 μm, Waters symmetry Detector : UV/VIS detector 203 nm Integrator : Chemstation for LC 3D Temperature : room temperature Injection volume : 20 μl Mobile phase A: acetonitrile B: water

69 Flow rate : Pump program : 1.6 ml/min gradient system Flow rate [ml/min] Time [min] Eluent A [%] Eluent B [%] HPLC 100% 1 IH901 RM 100% HPLC method 100% HPLC IH901 RM [Appendix 30] 6 IH901 RM 100% 6 (Table 30) HPLC 100% method 1 IH901 RM HPLC 100% 1 blank HPLC fingerprint (Appendix 31) IH901 RM IH901 RM Peak purity spectrally homogenous (Appendix 22) 0.5 mg/ml 6 (precision; repeatability, Table 30) ± % Table 30. Precision of HPLC 100% method 1 for IH901 RM reference material Measurement Peak areas Content (%) Peak area mean ± Mean value: 100% Standard deviation:

70 Variation coefficient: --- Confidence interval of the mean value (n=6, P=95%): --- Found IH-901 (ginsenoside K)content: 100% HPLC IH901 RM 1 mg/ml ~ mg/ml (two-fold dilution) 5 (Appendix 32) (Table 31) Table 31. Linearity of gradient method 1 for IH901 reference material Concentration IH901 RM [mg/ml] Peak areas mg/ml ~ mg/ml Y = A R 2 = Y A IH901 RM [ppm] Validation of analytical procedures: methodology (Appendix 29) (ICH) (Section c) HPLC method 1 IH901 RM HPLC 100% 2 2 IH901 RM 10 mg IH901 RM 10 ml (1,000 mg /ml) HPLC methanol mg/ml 5 HPLC 100% 2 Equipment : Hewlett Packard 1100 Column : C18, mm, 5 μm, Waters symmetry Detector : UV/VIS detector 203 nm Integrator : Chemstation for LC 3D Temperature : room temperature Injection volume : 20 μl Mobile phase A: acetonitrile containing 0.1% HCOOH

71 Flow rate : Pump program : B: water containing 0.1% HCOOH 1.6 ml/min gradient system Flow rate [ml/min] Time [min] Eluent A [%] Eluent B [%] HPLC 100% 2 IH901 RM 100% HPLC method 100% HPLC IH901 RM [Appendix 33] 6 IH901 RM 100% 6 Table 32 HPLC 100% method 2 IH901 RM HPLC 100% 2 blank HPLC fingerprint (Appendix 33) IH901 RM IH901 RM Peak purity spectrally homogeneous (Appendix 24) 0.5 mg/ml 6 (precision; repeatability) ± % (Table 32) Table 32. Precision of HPLC 100% method 2 for IH901 reference material Measurement Peak areas Content (%) Peak area mean ± Mean value: 100% Standard deviation:

72 Variation coefficient: --- Confidence interval of the mean value (n=6, P=95%): --- Found IH-901 (ginsenoside K)content: 100% HPLC IH901 RM 1 mg/ml ~ mg/ml (two-fold dilution) 5 (Appendix 35) (Table 33) Table 33. Linearity of gradient method 2 for IH901 reference material Concentration IH901 RM [mg/ml] Peak areas mg/ml ~ mg/ml Y = A R 2 = Y A IH901 RM [ppm] Validation of analytical procedures: methodology (Appendix 29) (ICH) (Section c) HPLC method 2 IH901 RM HPLC 100% method 3 3 IH901 RM 10 mg IH901 RM 10 ml (1,000 mg/ml) HPLC methanol mg/ml 5 HPLC 100% 3 Equipment: Waters Alltech Model ELSD 2000 Integrater: Breeze Column: Alltech Prevail Carbohydrate ES column (p/n 35101) Mobile phase A: acetonitrile/h 2 O/isopropyl alcohol=80/5/15 B: acetonitrile/h 2 O/isopropyl alcohol=80/15/15 Flow rate: 1.0 ml/min Temperature: room temperature

73 Injection volume: 10 μl Detector: Alltech ELSD 2000 (gain 8) Gas flow rate: Drift Tube Temp: Pump program: 2.0 SLPM 90 C gradient system Flow rate [ml/min] Time [min] Eluent A [%] Eluent B [%] HPLC 100% 3 IH901 RM HPLC 100% method 3 100% HPLC IH901 RM 7.03 [Appendix 36] 6 IH901 RM 100% IH901 RM 6 (Table 34) HPLC 100% 3 blank HPLC fingerprint (Appendix 36, 37) IH901 RM IH901 RM (Appendix 36) 0.5 mg/ml 6 (precision; repeatability) ± % (Table 34) Table 34. Precision of 100% method 3 of IH901 reference material Measurement Peak areas Content (%) Peak area mean ± Mean value: 100% Standard deviation: --- Variation coefficient:

74 Confidence interval of the mean value (n=6, P=95%): --- Found IH-901 (ginsenoside K)content: 100% HPLC IH901 RM 1 mg/ml ~ mg/ml (two-fold dilution) 5 (Appendix 38) (Table 35) Table 35. Linearity of gradient method 3 for IH901 reference material Concentration IH901 RM [mg/ml] Peak areas mg/ml ~ mg/ml Y = 6E + 06A R 2 = ELSD 2 2 sensitivity ELSD IH901 RM carbohydrate column IH Validation of analytical procedures: methodology (Appendix 32) (ICH) (Section c) HPLC method 3 IH901 RM IH901 RM 3 1 HPLC 100% % HPLC 1 2 IH901 RM IH901 RM HPLC 100% IH901 RM (mass balance) HPLC 100% 0.583% %

75 [100.00% - (%) (%)] x HPLC (%) / 100 IH901 (mass balance) % Appendix 1~38 ( ) 1. Picture of Panax ginseng C.A. Meyer 2. Literature cited for the documentation of reference material 3. Physico-chemical information for IH-901 obtained from D/B 4. 1 H NMR spectrum of IH-901 reference material C NMR spectrum of IH-901 reference material 6. 1 H- 1 H COSY NMR spectrum of IH-901 reference material 7. 1 HMQC NMR spectrum of IH-901 reference material 8. HMBC spectrum of IH-901 reference material 9. NOESY spectrum of IH-901 reference material 10. Reference literature for NMR assignment of IH FT-IR spectrum for IH-901 reference material 12. Reference literature for IR spectrum of IH-901RM 13. HPLC/MS spectrum for IH-901RM (positive mode) 14. HPLC/MS spectrum for IH-901RM (negative mode) 15. Reference literature for LC/Mass spectrum of IH-901RM 16. HR-FAB/MS spectrum for IH-901RM (positive mode) 17. HR-FAB/MS spectrum for IH-901RM (negative mode) 18. Elemental composition determined by HR-FAB/MS 19. UV spectrum for IH-901RM 20. UV spectrum of the peak corresponding to IH-901RM in HPLC photodiode array system 21. HPLC fingerprint 1 for IH-901 reference material 22. Peak purity analysis for IH-901 reference material in HPLC system HPLC fingerprint 2 for IH-901 reference material 24. Peak purity analysis for IH-901 reference material in HPLC system HPLC fingerprint 3 for IH-901 reference material 26. Reference literature for the LC separation of less polar ginsenosides 27. Reference literature for optical rotation of IH-901RM 28. TLC fingerprint for IH-901RM 29. ICH guideline for validation of analytical procedure: methodology 30. HPLC fingerprint 1 for IH-901RM 31. HPLC fingerprint of test material solvent for IH-901RM 32. Standard curve for IH-901RM in HPLC method

76 33. Chromatogram of HPLC 100% method 2 for IH-901RM 34. HPLC fingerprint 2 of test material solvent for IH-901RM 35. Standard curve for IH-901RM in HPLC 100% method Chromatogram of HPLC 100% method 3 for IH-901RM 37. HPLC fingerprint 3 of test material solvent for IH-901RM 38. Standard curve for IH-901RM in HPLC 100% method 3 Validation GINST 1.00 g 10 ml 3 8 ml 3 partition chromatography pooling HPLC 0.45 μm millipore filter HPLC/DAD HPLC column C18 (Supelco 4.6 mm 250 mm, 5 μm) water (A) acetonitrile (B) 1.6 ml/min 20 μl (gradient) HPLC/DAD Table 36 Table 36. HPLC gradient condition Column Mobile Phase Gradient condition Flow rate column temperature Injection volume Detector Supelco C18 (4.6 mm I.D. 250 mm, 5 mm), 또는동등한칼람 A: water B: acetonitrile time (min) Mobile phase (%) A B ml/min room temperature 20 ml 203 nm (DAD 190~ 400 nm) (LOD & LOQ) Compound K Compound K LOD (Limit of detection) mg/ml, LOQ (Limit of Quantification) mg/ml

77 C18 compound K R 2.0 Rt DAD 203 nm Fig. 49. HPLC chromatogram of compound K. (Linearity) Linearity CK 2.0~0.125 mg/ml 2-fold dilution (standard curve) (Fig. 50) R Fig. 50. Linearity of compound. (repeatability) (intermediate precision) (repeatability) 100% 3 (0.5 mg/ml, 1 mg/ml, 2 mg/ml) 3 RSD 1.40, 0.66, 0.31%

78 Table 37. Repeatability data of compound K Treatment 0.5 mg/ml 1.0 mg/ml 2.0 mg/ml Mean (peak area) S.D RSD (%) (intermediate precision) (intermediate precision) 100% HPLC peak area (1.0 mg/ml) 4 C 0, 24, 48 3 %RSD 0.66, 0.28, 2.37% Table 38. Intermediate precision data of compound K Treatment 0 Hour 24 Hour 48 Hour Mean Inter-day SD Inter-day RSD(%) (Accuracy) Spiking/recovery compound K compound K 0.5, 1.0, 2.0 mg/g 95.59, , % %RSD 1.36, 1.10, 0.79 % Table 39. Accuracy data of compound K Treatment Recovery (%) 0.5 mg/g 1.0 mg/g 2.0 mg/g Mean S.D RSD (%)

79 6 Si-Kwan Kim et al., Two-phase partition chromatography using soybean oil eliminates pesticide residues in aqueous ginseng extract, J. Chromatography A. 1042: (2004) 6 32 GC HPLC Rt 4 10% (10 ml) 2.0 ppm spiking 6 7 ml vortexing, spinning GC (ECD/NPD) HPLC ± % ± % ± % ± % ± % ± % (Table 40) Table 40. Recovery of pesticide residues in the oil phase by 6 different vegetable oils. Recovery Retention Pesticides Group Time Soybean Canola Corn Grape Olive Sunflower Chlorothalonil Dieldrin Organophosphorus Tolclofos-methyl Average SD PCNB PCTA PCA DDE Endrin DDD Organochlorides DDT a-bhc b-bhc r-bhc d-bhc Aldrin Average

80 SD Diethofencarb Carbosulfan Carbamates Carbofuran Average SD Tefluthrin Cyfluthrin Pyrethroids Bifenthrin Average SD Hexaconazole Flusilazole Triazoles Fluquinconazole Average SD Cyprodinil Pyrimidines Pyrimethanil Average SD Kresoxim-methyl Strobilurins Pyraclostrobin Average SD Dimethomorph Morpholines Average SD Sethoxydim Cyclohexenoxims Average SD Cyazofamid Amides Methalaxyl Average SD Total average Total SD

81 Fig. 51. GC fingerprint of organochloride pesticides in canola oil phase (A) and its corresponding aqueous ginseng extract phase (B). Ginsenoside cost-, labor- time-effectiveness ginsenoside ginsenoside profile TLC HPLC ginsenosides ginsenosides [Rg1, Re, Rf, Rh1, Rg2(S), Rg2(R), Rb1, Rc, Rb2, Rd, Rg3(S), Rg3(R), Rh2(S), Rh2(R)] 97.78, , , , , 90.59, 98.30, 90.31, 93.96, 93.23, 94.09, , 99.41, 95.80, mg/g 30% Fig. 52. Ginsenosides TLC profiles in aqueous ginseng extract layer before and after canola oil treatment. FG: fresh ginseng before treatment, TG: fresh ginseng after canola oil treatment