Anti-inflammatory and immunomodulatory effects of ginsenosides from Korea red ginseng April 26 th, 2018 Presenter : Le Ba Vinh, Ph.D student Superviser : Prof. Young Ho Kim College of Pharmacy Natural Products Chemistry Laboratory

Contents 3. Evaluation of biological activities 2. Extraction and isolation 1. Introduction 4. Conclusions 2. Structure elucidation Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY

I. Introduction and proposal for study Plant name : Panax ginseng Mayer. Family : Araliaceae Part used : Leaves, flower, and root. Chemical components: Triterpenoid saponin, fatty acid… Distribution : Common in Korea, China, and Vietnam. Is the most traditionally used as an important herbal medicine and treatment so many diseases. Natural Products Chemistry Laboratory Plant information

Others products from Korean red ginseng Honey ginseng Beverage Candy Herbs tea Korean red ginseng Extract For baby

Previous studies on Korean red ginseng Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY I. INTRODUCTION

Purposes of study Isolation chemical compounds from Korean red ginseng. Identification chemical structures of isolated compounds. Evaluation of isolated compounds on anti-inflammation activity. Evaluation of isolated compounds on immunomodulatory activity. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY I. INTRODUCTION

Plant material Collection Identification Store Korean red ginseng was provided by Korea red ginseng Cooperation Daejeon, Korea 2016 Taxonomically identified by Prof. Young Ho Kim. A voucher specimen, CNU 16006, was deposited at the Herbarium of College of Pharmacy, Chungnam National University, Daejeon, Korea Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY II. EXTRACTION AND ISOLATION

Scheme 1. Extraction and isolation of compounds 1–11 from Korean red ginseng. A: Acetone, D: Dichloromethane, M: Methanol, W: Distilled water, CC: Column chromatography. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY II. EXTRACTION AND ISOLATION

Figure 1. Structures of ginsenosides isolated from Korean red ginseng. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY III. STRUCTURE ELUCIDATION

1D-NMR ( 1 H, 13 C), 2D-NMR (HMBC, HSQC, COSY, ROESY ) [  ] D, IR, HR-ESI MS, chemical reaction Data base: DNP, SciFinder,… III. STRUCTURE ELUCIDATION

Figure 1. IR spectrum of compound 1 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION The IR spectrum of 1 showed strong absorptions of hydroxyl and carbonyl groups, and double bond.

Figure 2. HR-ESI-MS spectrum of compound 1 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION The molecular formula of 1 was identified to be C 36 H 60 O 9 from the sodium adduct molecular ion peak at m/z [M+Na] + according to the HR-ESI-MS spectra.

Figure 3. 1 H-NMR spectrum of compound 1 in CD 3 OD (600 MHz) Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION 8 singlet methyls (CH 3 ) δ 0.91, 3H, s 0.92, 3H, s 1.19, 3H, s 1.42, 3H, s 1.67, 3H, s 1.72, 3H, s 1.80, 3H, s Anomeric, δ 5.05 d, J=7.5

Figure C-NMR spectrum of compound 1 in CD 3 OD (150 MHz) Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION The 13C NMR spectrum of 1 showed the signals of 36 carbon; Dammarane-type triterpene aglycone

Figure 5. HMQC spectrum of compound 1 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION

Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Figure 6. HMBC spectrum of compound 1

Figure 7. COSY spectrum of compound 1 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION

Figure 8. NOESY spectrum of compound 1 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 1 III. STRUCTURE ELUCIDATION

Detailed structure elucidation of new compound (1)  Sugar determination Hydrolysis by HCl 1N, 80 o C, 3h Separation with EtOAc Trimethylsilyimidazole L-cysteine methyl ester Extract with n-Hexane The retention time (t R min) was confirm D-glucose. (Standard D-glucose derivative t R min, L-glucose derivative t R min). t R = t Peak - t Hexane Trimethylsilylimidazole derivatives Aqueous residue Comp. 1, 2 (2 mg) Reaction residue GC analysis Column: SPB-1 (0.25 mm x 30 m) Detector: FID Column temperature: 210 o C Injector temperature: 270 o C Detector temperature: 300 o C Carrier gas: He (2 mL/min) GC conditions

Figure 9. HR-ESI-MS spectrum of compound 2 The molecular formula of 2 was deduced to be C 16 H 12 O 10 from a quasi-molecular ion peak at m/z [M-H] - according to the HR-ESI-MS spectra. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION

Figure H-NMR spectrum of compound 2 in C 5 H 5 N-d 5 (600 MHz) Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION

Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION Figure C-NMR spectrum of compound 2 in C 5 H 5 N-d 5 (150 MHz)

Figure 12. HMQC spectrum of compound 2 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION

Figure 13. HMBC spectrum of compound 2 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION

Figure 13. COSY spectrum of compound 2 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION

Figure 14. NOESY spectrum of compound 2 Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION

Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Structure elucidation of new compound 2 III. STRUCTURE ELUCIDATION

Anti-inflammation Our assay Inflammation  Cancer  Cardiovascular diseases  Neurological disorders  Diabetic complications  Metabolic disorder complications  Bone, Muscular and Skeletal diseases  Inflammatory bowel disease IL-6 TNF-α COX-2, and iNOS Anti-inflammation activity Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY IV. EVALUATION OF BIOLOGICAL ACTIVITIES

Effects on the LPS-stimulated production of IL-6 and TNF-α in RAW cells cultured at 10 5 cells/mL in 48-well plates h The cells were treated with various concentrations of compounds (0.4, 2.0 and 10.0 μM) in the presence of 1μg/ml LPS for 18 h. RAW cells were obtained from Korean Cell Line Bank Maintained in RPMI 1640 supplemented 10% fetal bovine serum (FBS) 100 U/mL penicillin at 37°C in humidified incubator containing 5% CO 2 Cell culture and stimulation MTT was added to the cell culture medium for 2 hr The supernatant was removed and the formazan crystals were dissolved in DMSO Absorbance was measured at 450 nm. Cell viability Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Anti-inflammation activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

 The plated were washed and HRP substrate solution to each wells.  The plated were washed and TMB solution added to the each wells.  A stop solution was then added to the plates  The optical density of 450 nm was read. RAW264.7 cells seeded in 6-well plates Griess reagent 1% sulfanilamide 5% phosphoric acid 0.1% naphthylethylenediamine-HCl The absorbance was measured at 520 nm using a microplate reader. Incubated at room temperature for 20 min Western blot analysis A capture antibody solution 0.05% Tween-20 in PBS 100 uL of the culture supernatants blocked for 1h at room temperature washed three times added incubated at room temperature washed and detection antibody solution was incubated at room temperature for 1h Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Anti-inflammation activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

Figure 14. The anti-inflammatory effects of compounds 1–11 on the IL-6. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Anti-inflammation activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

Figure 15. Effect of compounds 1 on regulating of inflammatory mediators in LPS-induced RAW cells. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Anti-inflammation activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

Figure 16. Effects of compounds 1 on the expression of COX- 2 and iNOS in LPS-induced RAW cells. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Anti-inflammation activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

Figure 16. Effects of compounds on IL- 2 production in PMA/Io-induced EL-4T cells. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Immunology activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

Figure 16.. Effects of compounds 5, 7, and 9 on NF-AT and NF-κB promoter activities in PMA/Io- activated EL-4T cells. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Immunology activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

Figure 16. Effects of compounds 5, 7, and 9 on viability of EL-4 T cells. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY Immunology activity IV. EVALUATION OF BIOLOGICAL ACTIVITIES

 From the roots of Korean red ginseng, 11 compounds were isolated. Structure of isolated compounds were elucidated using 1D-, 2D-NMR, HR ESI MS spectroscopic methods, and GC analysis. Among them, compound 1 and 2 were new compounds.  The effects of compounds 1 ‒ 11 on LPS-induced production of the inflammatory mediators IL-6 and TNF-α by RAW cells. Compound 1 significantly decreased TNF-α release from activated RAW264.7 cells in a dose dependent manner and suggest that compound 1 decreased the production of proinflammatory cytokines, IL-6 and TNF-α from macrophage cells.  Moreover, compound 1 significantly inhibited COX-2 and iNOS gene expression in LPS-activated. The inhibitory effect of compound 1 is more significantly than that of Celecoxib (positive control of COX-2). Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY V. CONCLUSION

 This is the first study of the chemical constituents from Korean red ginseng that have activates T cell function via the regulation of NF-AT mediated IL-2 production  Given the results for compound 5,7 and 9, these compounds are candidates for further investigation to elucidate their molecular mechanisms of action on specific immunomodulatory target. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY V. CONCLUSION

 Korea Ginseng Research Institute, Korea Ginseng Corporation, Daejeon.  Professor Young Ran Kim, College of Pharmacy and Research Institute of Drug Development, Chonnam National University.  Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology. Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY V. Acknowledgements

Natural Products Chemistry Laboratory 충남대학교 CHUNGNAM NATIONAL UNIVERSITY V. CONCLUSION Under review

Natural Products Chemistry Laboratory CHUNGNAM NATIONAL UNIVERSITY