Immunostimulating anticaner polysaccharides from natural products 2006 12

면역활성과 항암활성을 갖는 천연물유래 다당체 Immuno-stimulating & anti-caner polysaccharides from natural products 2006. 12 신 광 순 경기대학교 식품생물공학과 www. kyonggi. ac. kr

v Introduction Several Biologically Active Materials in Plants Low M. W materials Alkaloids Flavonoids Terpenoids High M. W. materials Proteins Polysaccharides Tannins b-Glucan Cellulose Hemicellulose Pectic materials Arabinoxylan, Xyloglucan etc. Dept. of Food Sci. & Biotech.

v Plant cell wall polysaccharides Dept. of Food Sci. & Biotech.

v A proposed structure of pectins and pectic polysaccharides Pectin Poly-Gal. A RG-II Poly-Gal. A Homo-polygalacturonan region (Poly-Gal. A) - -D-Gal. Ap-(1 4)- -D-Gal. Apn=to 70 COOR R=H, CH 3, Na+ and/or Ca++ Rhamnogalacturonan I region (RG-I) - -D-Rhap -(1 4)- -D-Gal. Ap-(1 2)- -L-Rhap- (1 4)- -D-Gal. Apn Rhamnogalacturonan II region (RG- II) R 1 - -D-Gal. Ap-(1 4)- -D-Gal. Ap n=8 to 15 R 2 R 1=arabinan, galactan, arabino-3, 6 -galactan and/or other oligosaccharides R 2=KDO containing disaccharide, DHA containing disaccharide, Ace. A containing oligosaccharide and uronic acid rich oligosaccharide ` Dept. of Food Sci. & Biotech.

v Pectin components A. Homogalacturonan B. Rhamnogalacturonan I (RG-I) C. Rhamnogalacturonan II (RG-II) Dept. of Food Sci. & Biotech.

Pharmacological activity of pectins isolated from plants Immunostimulating activity - Complement system activating activity - Mitogenic activity - Auto-immmue disease curing activity (Immune complex clearance activity) - Macrophage phagocytosis potentiating activity Anti-ulcer activity Anti-metastasis activity Hypoglycemic activity Cholesterol decreasing effect Other applications - Vaccine for typhoid fever - Drug delivery Dept. of Food Sci. & Biotech.

v Anti-metastasis activity B 16 -BL 6 Cancer-metastasis occcurring mechanism and the assay for anti-metastasis activity Prostate cancer cell Target organ 14 days Galectin : galactose Galactose containing binding protein oligosaccharide Remove lungs and fix in Bouin’s soln Dept. of Food Sci. & Biotech.

v Anti-metastasis activity of modified citrus pectin (RG-I) Citrus pectin Acid treatment MCP Prostate adenocacinoma MAT-Ly. Lu B 16 -F 1 Target organ Epithelial cell Inhibition of cell-cell interaction Dept. of Food Sci. & Biotech.

v Anti-metastasis activity Anti-metastatic activity of MB-40 prepared by Shin’s group Control 4 mg/h 20 mg/h 100 mg/h 500 mg/h 2 mg/h Dept. of Food Sci. & Biotech.

v Anti-metastasis activity Effect of MB and MB-40 on the inhibition of tumor metastasis produced by colon 26 -M 3. 1 cells S T S +14 -2 0 +1 Kill & colony count Dept. of Food Sci. & Biotech.

v Anti-metastasis activity Effect of MB-40 on the prophylactic and therapeutic inhibition of metastasis produced by colon 26 -M 3. 1 cells Anti-AGM 1 S A T S S +14 -3 -2 -1 0 +1 +4 Kill & colony count Dept. of Food Sci. & Biotech.

MB-40에 의한 면역세포의 signaling pathway Dept. of Food Sci. & Biotech.

v Complement activation The pathways of complement activation Membrane attack complex (MAC, x 100, 000) Dept. of Food Sci. & Biotech.

v Complement activation Complement system activating pectins The roots of Panax ginseng (인삼) The roots of Bupleurum falcatum (시호) The roots of Glycyrrhiza uralensis (감초) The roots of Angelica acutiloba (당귀) Pectins AR-2 IIa AR-2 IIb AR-2 IIc AR-2 IId Pectic arabinogalactan PG-1 : RG-I PG-2 : RG-II PG-3 : Oligo-Gal. A AGIIa AGIIb-1 Dept. of Food Sci. & Biotech.

v Complement activation Anti-complementary activity of enzymic digestion products from the pectins of Angelica acutiloba Product Concentration (mg/ml) 1000 500 Product 100 Concentration (mg/ml) 1000 Inhibition of TCH 50 (%) AR-2 IIa Original PG-1 PG-2 PG-3 a. Not 100 Inhibition of TCH 50 (%) AR-2 IIc 59. 5 82. 5 38. 0 19. 2 30. 8 74. 5 23. 8 0. 6 25. 0 2. 0 AR-2 IIb Original PG-1 PG-2 PG-3 500 Original PG-1 PG-2 PG-3 54. 3 84. 5 28. 5 1. 8 29. 2 82. 8 3. 0 41. 8 84. 5 48. 8 33. 0 n. d. 85. 0 81. 8 10. 0 n. d. 52. 5 40. 0 AR-2 IId 62. 6 82. 2 22. 0 4. 8 31. 8 74. 5 2. 0 1. 5 2. 0 25. 5 Original PG-1 PG-2 PG-3 n. d. determined. PG-1, ramified region; PG-3, oligogalacturonides Dept. of Food Sci. & Biotech.

v Complement activation potency of the ramified region and its neutral oligosaccharides from the pectins of Angelica acutiloba Dept. of Food Sci. & Biotech.

v Complement activation Complement system activation mechanism of pectins from Angelica acutiloba Poly-Gal. A - Activity : ++++ - Essential for activity expression - Complement activation through both pathways Poly-Gal. A Arabinogalactan Galactan Arabinan Modulation of activity RG-I Modulation of activity - Activity : + or - Modulation of activation through the classical pathway - Activity : ++ - Complement activation through the only alternative pathway Dept. of Food Sci. & Biotech.

v Immune complex clearance activity Formation of immune complexes in human body and the related diseases (auto-immune disease) Circulating in blood stream Ag Ab Ag. Ab Immune complex Deposition in tissue : Receptor : Physical binding : Electrical binding : Absorption in collagen Disease Reticuloendotherial system Liver : Kupffer cell Lung : Alveolar Cell Kidney : Mesangial Cell Brain : Microgrial cell Spleen : Macrophage Blood : Monocyte/Neutrop. Macrophage Lymph node : Macrophage (resident or circulating) Inflammation Hypersensitivity III Autoimmune disease* Remove SLE Rheumatoides arthritis Dept. of Food Sci. & Biotech.

v Immune complex clearance activity Determination of Fc. R mediated immune complex binding to macrophage Dept. of Food Sci. & Biotech.

v Immune complex clearance activity The roots of Panax ginseng C. A. Meyer : One of the most famous medicinal plants in oriental counties : In 1958, The FDA gave Panax ginseng GRAS status : A main active component - Ginseng saponin (ginsenoside) : Biological activity - Immuno-stimulating activity - Anti-tumor activity - Hypoglycemic activity - Anti-cholesterolemic activity, etc. Roots : widely used clinically in oriental countries, but very expensive (4 - 6 years for harvest) Leaves : can be harvested every year, but Korean cultivator have usually discarded them ** If the polysaccharide from the leaves have a new biological activity or similar effects as the roots have, the leaves will be valuable Dept. of Food Sci. & Biotech.

v Immune complex clearance activity Purification scheme of immune complex clearance enhancing polysaccharide from the hot water extracts of ginseng leaves Water-soluble crude polysaccharide fraction from ginseng leaves (GL-2) Cetavlon Precipitate (GL-3) Supernatant Addeed 1% boric acid Adjusted to p. H 8. 8 Precipitate (GL-4) Supernatant (GL-5) DEAE- Sepharose CL-6 b (Cl- form) Unabsorbed fraction (GL-4 I) 0. 1 M (GL-4 IIa) 0. 2 M (GL-4 IIb 1) Absorbed fraction Stepwise elution with Na. Cl 0. 3 M (GL-4 IIc) 0. 4 M 0. 5 - 2. 0 M (GL-4 IId) (GL-4 IIe - h) (GL-4 IIb 2) QAE- Sepharose, stepwise elution with Na. Cl Bio-gel P-30 Main fraction (GL-4 IIb 2') Dept. of Food Sci. & Biotech.

v Immune complex clearance activity Effect of GS-4 IIb 2 and periodate oxidation product on immune complex clearance activity GS-3 c-II Periodate oxidized GS-3 c-II Dept. of Food Sci. & Biotech.

v Immune complex clearance activity Physico-chemical properties of GL 4 IIb 2 Dept. of Food Sci. & Biotech.

v Immune complex clearance activity A proposed structure of RG-II portion in GL-4 IIb 2 Uro. A-rich octasaccharide chain Ace. A-containing nonasaccharide chain Dept. of Food Sci. & Biotech.

v Immune complex clearance activity Structural differences between GL-4 IIb 2' and RGII GL-4 IIb 2’ Preparation dp Pharmacological activity Obtained without treatment of endo-PGase RG-II Released from primary cell walls upon digestion with endo-PGase 60 30 Relatively strong immune complex clearance activity Not reported Structural feature [ Rha-(1 5)-KDO : Ara-(1 5)-DHA ] 5: 1 Linkage difference [ Ace. A containing oligosaccharide ] R 1 = Rha R 2 = Ara 1: 1 R 1 = OH R 2 = OH R 1 1 3 R 2 -(1 2)-Rha-(1 2)-Ara-(1 4)-Gal-(1 2)-Ace. A-(1 3)-Rha-(1 3’)-Api 2 1 2 -O-Me-Fuc Dept. of Food Sci. & Biotech.

v Immune complex clearance activity Comparison of TBA-reaction positive material content in polysaccharide fraction from roots and leaves of Panax ginseng P. ginseng roots (GR) P. ginseng leaves (GL) Crude polysaccharide fraction (GR-2, GL-2) cetavion fractionation strong acidic fr. (GL-3) weak acidic fr. (GL-4) neutral fr. (GL-5) Dept. of Food Sci. & Biotech.

v Cytokine production Effect of RG-IIs from the leaves of Panax ginseng on IL-6 production of macrophage Cytokine production of macrophages Signal pattern recognition receptors; Toll like Receptors Cytokines (IL-6, IL-12) Macrophages were treated with 10 mg/ml of sample Dept. of Food Sci. & Biotech.

Dept. of Food Sci. & Biotech.

v Cytokine production Effect of monomerization and artificial re-dimerization of GL-RIII on IL-6 production enhancing activity. Dept. of Food Sci. & Biotech.

v Conclusion Rha-KDO Ace. A oligosaccharie Rha-KDO Activation of Marcrophage function Rha-KDO IL-6 M Fc. R Ara-DHA Uro. A octasaccharide Ace. A oligosaccharie Uro. A octasaccharide Ara-DHA Ace. A oligosaccharie p. H<2. 0 Uro. A octasaccharide Ara-DHA O O Rha-KDO O B O Core Dept. of Food Sci. & Biotech.

v Other immunostimulating activity Echinacea purpurea : Echinacea-polysaccharide II (MW 75, 000, RG-I) Cytotoxicity against tumor cell (TNF-a, IL-6, interferon-b 2, oxygen radicals) Glycyrrhiza uralensis : Mitogenic activity (RG-I) Panax notoginseng Angelica acutiloba Glycyrrhiza uralensis Reticloendothelial system activation : arabino-3, 6 -galactan Dept. of Food Sci. & Biotech.

v NK-cell activation Natural killer (NK) and LAK cells and their activation NK-Cell u known as large granular lymphocytes (LGL) u kill infected and malignant cells : NK cell interacting with a tumor cell not expressing MHC-I molecules u activated by IL 2 and IFN to become LAK cells Dept. of Food Sci. & Biotech.

v NK-cell activation Cytotoxicity assay by chromium release Target cell Effector cell 51 Cr Plate out Spin and wash Co-culture 4 -16 hr Measure 51 Cr in supernatant Dept. of Food Sci. & Biotech.

v NK-cell activation Anti-tumor activity of rhamnogalacturonan I from Viscum album (겨우살이 ) : RG-I : Not activated the killing potential of NK-cell : ** Synergistic interaction between NK-cell and tumor cell Target antigen Tumor Cell NK-cell **Bridge Effect Rha residue Gal residue RG-I Dept. of Food Sci. & Biotech.

v NK-cell activation Effect of AAA-1 on the enhancement of NK-cell activity Dept. of Food Sci. & Biotech.

v Anti-ulcer activity Bupleurum falcatum : Buleuran 2 IIb & Buleuran 2 IIc (MW 63, 000, pectin, Poly-Gal. A + RG-II) : Oral, subcutaneous, and intraperitoneal ad. (a systemic action in the stomach) Reduction of gastric lesion : Et. OH induced, indometacin/HCl induced, pyrolus ligated, Ac. OH induced and water immersion stress ulcer : anti-secretory activity on acid and pepsin protective coating radical scarvenging effect Dept. of Food Sci. & Biotech.

v Anti-ulcer activity Effects of bupleural 2 IIb and 2 IIc on HCl/ethanol induced gastric lesions in mice Dept. of Food Sci. & Biotech.

v Polysaccharide absorption mechanism How can the polysaccharide be absorbed to the body after oral administration ? Dept. of Food Sci. & Biotech.

v Polysaccharide absorption mechanism Distribution of orally administrated bupleuran 2 IIc in mouse liver. (after 1 week) 39 Dept. of Food Sci. & Biotech.

v Intestinal immune system modulating activity An ileal Peyer's patch in the gut associated lymphoid tissues (GALT) Peyer’s Patch * Specialized M-cell * The lymphocytes from one region of the gut circulate via the mesenteric lymph nodes. Dept. of Food Sci. & Biotech.

v Intestinal immune system modulating activity Assay of intestinal immune system modulating activity by measurement of bone marrow cell proliferation PP cell + samples Culture 5 days Cervical C 3 H/He dislocation mice Obtain PP Supernatant PP cells in RPMI 1640 6 (2 x 10 cells/m. L) Culture 6 days BM cells C 3 H/He mice BM cells in RPMI 1640 TM Alamar Blue (2. 5 x 105 cells/m. L) Measurement of bone marrow cells proliferation by fluorospectrometer Dept. of Food Sci. & Biotech.

v Intestinal immune system modulating activity Polysaccharides with intestinal immune system modulating activity : Atractylodes lancea DC (창출) arabino-3, 6 -galactan rhamnogalacturonan II : Pinus densiflora (솔잎) methanol extracts : Citrus unshiu (귤피) pectic polysaccharide flavone glycoside : Bifidobacterium sp. cell wall fraction Dept. of Food Sci. & Biotech.

v Utilization as a vaccine Cross reactivity of Vi antiserum against Vi and OAc. Pec Vi antiserum COOH O OAc O O O NAc O OAc n n Capsular Polysaccharide (Vi) of Salmonella typhi O-acetylated pectin (OAc. Pec) Vaccine : OAc. Pec-tetanus toxoid conjugate 43 Dept. of Food Sci. & Biotech.

v Drug carrier Utilization for hepatic drug carrier Groman, E. V. et al : Arabinogalactan from Larix accidentalis : Intravenous injection liver half life [ 3 H]-arabinogalactan (53. 5%) (3. 42 days) : Not toxic (mouse, 5000 mg/kg) (rat, 500 mg/kg/day, 90 days) Asialoglycoprotein receptor Arabinogalactan Hepatocytes 44 Dept. of Food Sci. & Biotech.

v Conclusion Biological activities of structural unit on pectin Ramified region (RG-I) Vaccine for typhoid fever Drug delivery Anti-metastasis activity Anti-ulcer activity (leaves of Panax ginseng) (roots of Bupleurum falcarum) Immunostimulating activity - Complement system activation - Auto-immmue diease curing activity (leaves of Panax ginseng) (roots of Bupleurum falcarum) - Macrophage phagocytosis (activation of RES) - IL-6 production - Intestinal immune modulating activity Anti-tumor activity RG-II Galacturonan region + + + + Dept. of Food Sci. & Biotech.