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Biologically Active Phenolic Constituents from Korean Medicinal Plants

Biologically Active Phenolic Constituents from Korean Medicinal Plants. Jinwoong Kim College of Pharmacy Seoul National University. Table of Contents. 1. Introduction. 2. Hepatoprotective Activity against CCl 4 - or H 2 O 2 - induced Hepatotoxicity.

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Biologically Active Phenolic Constituents from Korean Medicinal Plants

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  1. Biologically Active Phenolic Constituents from KoreanMedicinal Plants Jinwoong Kim College of Pharmacy Seoul National University

  2. Table of Contents 1. Introduction 2. Hepatoprotective Activity against CCl4- or H2O2- induced Hepatotoxicity 3. Neuroprotective Activity against Glutamate-induced Neurotoxicity 4. Anti HIV-1 Reverse Transcriptase and Protease Activities 5. PLC1 Inhibitory Activity 6. Conclusion

  3. Introduction

  4. Introduction General Properties of Plant Phenolics • Derived from the intermediates of the shikimic acid biosynthetic pathway. • Have an aromatic ring that contains various attached substituent groups, such as hydroxy, carboxy and methoxy, and often other non-aromatic ring structures. • Take parts in plants as phytoalexins, antiherbivorics, antioxidants, UV protectants, and pigments. • Possess a number of biological and enzyme inhibitory activities - antibacterial, antifungal, immunosuppressive, anti-inflammatory and anti-tumor activities - 5-lipoxygenase, cAMP-phosphodiesterase, aldose reductase and protein kinase C inhibitory activities

  5. Introduction Responsible for the biosynthesis of a variety of plant phenolic compounds O H O M e O O H H O O OH O O O M e O O G l c O e O M O O H O Coumarins Phenylpropanoids Flavonoids Benzoic acid O O HO HO O O OCH3 O HO OH O O HO OH M e O O M e O O M e Lignans Tannins Stilbenoids

  6. Hepatoprotective Activity against CCl4-or H2O2- induced Hepatotoxicity Scrophularia buergeriana (玄蔘) Cynanchum wilfordii (白首烏)

  7. Assay method for Hepatoprotective activity Primary cultured rat hepatocytes Add compounds for test Intoxicated with CCl4 or H2O2 Measurement of GPT activity

  8. Scrophularia buergeriana • Parts used : Roots • Traditional uses : Fever, swelling, constipation, neuritis, and laryngitis • Description : A perennial herb with a leafy, erect, qurdangular, and smooth stem • Chemical components : Iridoid glycoside, terpenoid, phenylpropanoid, flavonoid Scrophularia buergeriana Miq. ( Scrophulariaceae )

  9. Compound R1 R2 R3 R4 13 OH H H OCH3 14 OH H OCH3 H 15 OH OH H H 16 OCH3 H H OCH3 17 OH H H OH 18 H H H H Scrophularia buergeriana Structures of compounds Isolated from S. buergeriana Phenylpropanoids Compound R1 R2 R3 R4 R5 O R 1 1 O-4--L-rha H H OCH3 H 2 OH H H H H 3 OH H H OCH3 H 4 OH H OCH3 H H 5 OH OH H H H R 6 OCH3 H H OCH3 H 2 7 OH H H OH H 8 H H H H H 9 OH H OH OH H R R 3 5 10 OH H OCH3 OH H R 11 OH H OH OCH3 H 4 12 OH H OCH3 OH OCH3 Acetophenones O R 1 R R 2 4 R 3

  10. Scrophularia buergeriana Effects of compounds1-18 on the release of GPT from primary cultures of CCl4-injured rat hepatocytes Compound Relative protection (%) 1 mM 5 mM Control 100.0  4.9 100.0  4.8 0.0  10.4 0.0  10.4 CCl4-treated 72.3  16.1*** 69.7  4.0 *** 4-O-E-p-methoxycinnamoyl-a-L-rhamnopyranoside ester (1) Cinnamic acid (2) 39.3  10.3 20.2  4.8 p-methoxycinnamic acid (3) 57.0  7.4 *** 69.0  4.8 *** m-methoxycinnamic acid (4) 48.8  13.2 * 54.6 15.0 * o-methoxycinnamic acid (5) 0.0  14.4 22.9  7.2 p-methoxycinnamoyl methyl ester (6) 220.  7.0 34.6  7.6 p-coumaric acid (7) 12.1  3.9 38.4  7.6 Cinnamaldehyde (8) 8.2  9.8 5.0  8.5 Caffeic acid (9) 20.6  4.1 50.7  6.0 * Ferulic acid (10) 15.4  11.2 27.0  9.7 Isoferulic acid (11) 48.9  3.2 * 64.1  7.2 *** 3,5-dimethoxy-4-hydroxy-cinnamic acid (12) 22.7  9.0 19.7  8.2 4-hydroxyacetophenone (13) 7.5  9.7 0.9  6.0 3,5-dimethoxyacetophenone (14) 2.3  4.1 0.0  8.4 3,5-dimethoxy-4-hydroxyacetophenone (15) 35.5  10.2 28.0  7.5 Acetovanillone (16) 8.5  4.3 19.7  7.8 -9.6  1.6 Syringic acid (17) 11.8  2.7 Syringaldehyde (18) -6.2  7.4 -0.5  4.2 Silybin 19.6  4.5 21.2  5.0

  11. Scrophularia buergeriana • 4-O-E-p-Methoxycinnamoyl--L-rhamnopyranoside ester (1) and p-methoxycinnamic acid(3) significantly blocked the release of GPT at 1 M and 5 M. • Cinnamaldehyde (8) and acetophenones (13-18) showed very little activity  ,-unsaturated carboxy moiety is important to exhibit hepatoprotective activity. • Compounds with aromatic p-methoxy group linked to ,-unsaturated carboxy moiety showed stronger activity than either unsubstituted or p-hydroxy substituted compounds (2,7,9)

  12. Scrophularia buergeriana Effects of compounds 1, 3, and 11 on hepatic GSH level in primary cultures of CCl4-injured rat hepatocytes 1 3 11 O O O H H H O O H O H R h a H H H O O C H O C H O C H 3 3 3 H H H GSSG/ Total GSH Concentration (mM) Total GSH (nmol/mg protein) Reduced GSH (nmol/mg protein) Compound Control 59.74  2.13 41.39  1.92 0.307 CCl4-treated 9.44  1.02 4.08  0.08 0.621 Compound 1 5 14.36  1.34 ** 9.53  1.82 * 0.337 Compound 3 5 13.32  0.68 ** 10.45  0.83 ** 0.235 Compound 11 5 14.70  2.21 * 9.42  0.12 *** 0.359 Silybin 50 26.51  4.23 ** 21.65  1.46 *** 0.301

  13. Scrophularia buergeriana • 4-O-E-p-Methoxycinnamoyl--L-rhamnopyranoside ester (1),p-methoxycinnamic acid(3), and isoferulic acid (11) significantly preserved the level of total glutathione (GSH) and prevented the decrease in GSH level by CCl4 at a concentration of 5 M. • Glutathione (GSH) participates in the reductive processes that are essential for the protection of cells through quenching the reactive intermediates and the radicals generated during oxidative toxicity caused by CCl4.

  14. Scrophularia buergeriana Effects of compounds 1, 3, and 11 on hepatic enzymes related to the GSH redox pathway in primary cultures of CCl4-injured rat hepatocytes 1 3 11 O O O H H H O O H O H R h a H H H O O C H O C H O C H 3 3 3 H H H GST (nmol/mg protein) GPx (nmol/mg protein/min) GR (nmol/mg protein/min) Concentration (mM) Compound Control 1.416  0.018 91.74  11.70 0.755  0.135 CCl4-treated 0.812  0.032 48.73  3.37 1.110  0.036 Compound 1 5 0.855  0.035 62.60  7.02 * 1.286  0.036 *** Compound 3 5 0.831  0.049 68.83  1.24 *** 1.466  0.177 ** Compound 11 5 0.851  0.033 68.33  4.70 *** 1.416  0.034 *** 1.134  0.086 Silybin 50 0.891  0.024 *** 60.01  3.73 **

  15. Scrophularia buergeriana • 4-O-E-p-Methoxycinnamoyl--L-rhamnopyranoside ester (1),p-methoxycinnamic acid(3), and isoferulic acid (11) significantly preserved the activity of glutathione disulfide reductase (GR), but exerted little effect on glutathione peroxidase (GPx). • The hepatic glutathione–S-transferase (GST) activity was significantly preserved by the treatment with compounds 1,3, and 11. • GST is a soluble cytosolic enzyme responsible to the detoxification of xenobiotics.

  16. Scrophularia buergeriana • 4-O-E-p-Methoxycinnamoyl--L-rhamnopyranoside ester (1), p-methoxycinnamic acid(3) and isoferulic acid(11) showed substantial and almost equal hepatoprotective activity to silybin at a concentration 10 ~ 50 times lower. • The hepatoprotective activities of these three phenyl-propanoids are likely to be attributed to the maintenance of the GSH redox system in CCl4-injured rat hepatocytes. • The phenylpropanoids of S. buergeriana might be of significant therapeutic value in the prevention of hepatic diseases.

  17. Cynanchum wilfordii • Parts used : Roots • Traditional uses : Tonic, scrofula, and cold knee • Description : Glabrous, scandent, perennial herb, 1-3 m tall. Leaves opposite, deltoid. • Chemical components : Sarcostine, deacylcynanchogennin- deacylmetaplexigenin-cymarosides, wilforine, cynamoyl-tigloylsarcostine Cynanchum wilfordii (Max.) Hemsl. ( Asclepiadaceae )

  18. Cynanchum wilfordii Effect of cynandione A on the activities of glutamic pyruvic transaminase and sorbitol dehydrogenase released from primary cultures of CCl4-injured rat hepatocytes O H O C H 3 O H O C H 3 O H H O Glutamic pyruvic transaminase activity ( int. units L-1) Sorbitol dehydrogenase value ( units mL-1) Group Control 29.5  2.5 2.1  0.1 CCl4-treated 82.4  3.4 48.1  0.5 CCl4 + cynandione A 1 uM 79.6  3.7 44.9  2.8 CCl4 + cynandione A 5 uM 75.2  5.4 41.2  2.4 CCl4 + cynandione A 10 uM 66.3  4.2 * 34.3  1.9 * CCl4 + cynandione A 50 uM 59.2  4.0 ** 27.5  2.1 ** CCl4 + cynandione A 100 uM 59.9  5.8 ** 28.3  3.8 ** CCl4 + sylibin 100 uM 51.8  2.8 *** 21.8  1.3 ***

  19. Cynanchum wilfordii • Cynandione A significantly decreased the levels of glutamic pyruvic tranaminase (GPT) and sorbitol dehydrogenase released to the culture medium, and the effect was most potent at a concentration of 50 M.

  20. O H O C H 3 O H O C H 3 O H H O Cynanchum wilfordii Effect of cynandione A on glutathione levels in primary cultures of CCl4-injured rat hepatocytes Total glutathione ( nmol (mg protein)-1 ) Reduced glutathione ( nmol (mg protein)-1 ) Glutathione disulphide / total glutathione Group 0.27  0.01 19.3  1.4 14.0  0.8 Control 0.48  0.02 4.7  0.1 2.5  0.1 CCl4-treated 0.28  0.05 ** 10.6  2.7 * 7.7  0.2 CCl4 + cynandione A

  21. Cynanchum wilfordii • Cynandione A(50 M) significantly preserved levels of total glutathione and prevented the decrease in glutathione levels caused by CCl4.

  22. O H O C H 3 O H O C H 3 O H H O Cynanchum wilfordii Effect of cynandione A on hepatic antioxidant enzymes in primary cultures of CCl4-injured rat hepatocytes Glutathione disulphide reductase (mol NADPH oxidised (min-1 (mg protein)-1 ) Catalase (mol H2O2 consumed (min-1 (mg protein)-1 ) Superoxide dismutase (units mL-1) Malondialdehyde (nmol(mg protein)-1 ) Group 2.40  0.3 52.7  4.2 682.7  21.9 27.4  1.8 Control 3.41  0.2 22.2  1.7 312.7  9.2 15.3  1.3 CCl4-treated 2.93  0.3 * 31.4  5.7 563.2  37.9 * 21.3  1.5 ** CCl4 + cynandione A

  23. Cynanchum wilfordii • Cynandione A significantly preserved the activities of the antioxidative enzymes such as superoxide dismutase (SOD) and catalase. • However, it showed only a slight effect on glutathione disulphide reductase (GR) activity. • Cynandione A lowered the level of malondialdehyde significantly.

  24. Cynanchum wilfordii • Cynandione A(50 M) significantly reduced (approximately 50%) the release into the culture medium of glutamic pyruvic transaminase and sorbitol dehydrogenase from the primary cultures of rat hepatocytes exposed to CCl4 . • Cynandione A appeared to protect primary cultured rat hepatocytes exposed to CCl4 from significant drops in the levels of specific markers, such as GSH, SOD, and catalase. Cynandione A protected the hepatocytes from CCl4-injury by maintaining the level of glutathione and by inhibiting the production of malonaldehyde, due to its radical scavenging properties.

  25. Neuroprotective Activity against Glutamate-induced Neurotoxicity Torreya nucifera (榧子) Hedyotis diffusa (白花蛇舌草) Inula britannica (金佛草)

  26. Assay method for Neuroprotective activity Primary cultured rat cortical cells Add compounds for test (pre-treatment if necessary) Intoxicated with Glutamate Add compounds for test (post-treatment) Neurotoxicity measured by LDH Assay

  27. Torreya nucifera • Parts used : Fruits • Traditional uses : Treatment of tapeworm infection • Description : A pyramidal silhouette and long, graceful branches clothed with glossy, dark green leaves • Chemical components : Palmitic acid, stearic acid, oleic acid, linoleic acid Torreya nuciferaSieb. & Zucc. ( Cephalotaxaceae )

  28. Torreya nucifera Neuroprotective activity of arctigenin on glutamate-induced neruotoxicity in primary cultures of rat cortical cells O M e O O H O H O M e O M e Protection (%) Concentration 0.01 mM 0.10 mM 1.00 mM 10.0 mM Control 100.0  1.9c 0.0  0.6 Glutamate-injuredd Pre-treatmenta paradigm 1 hr before 20.1  2.1 42.5  2.3 ** 57.5  1.2 *** 40.0  1.9 * Post-treatmentb paradigm Immediately after 13.2  1.4 38.1  1.2 ** 43.8  1.2 ** 43.1  1.4 ** 30 min after 13.0  2.2 28.3  1.7 * 35.7  0.7 ** 5.3  1.6 1 hr after 8.1  1.8 19.3  1.3 * 18.1  1.9 * -3.7  1.2 3 hr after 5.5  0.7 17.5  1.0 * -1.3  0.3 -18.3  0.8

  29. Torreya nucifera • Arctigenin significantly attenuated neurotoxicity induced by glutamate in pre-treatment paradigm at concentrations ranging from 100 nM to 1 M. • Arctigenin also showed significant neuroprotective activity when administered immediately or 30 min after glutamate insult at concentrations ranging from 100 nM to 1 M.

  30. Torreya nucifera The neuroprotective effect of arctigenin in glutamate-intoxicated primary cultures of rat hippocampal neurons. A B Neurons exposed to 10 uM glutamate Control C D Neurons exposed to 10 uM glutamate & 10 uM arctigenin Neurons exposed to 10 uM glutamate & 1 uM arctigenin

  31. Torreya nucifera • Arctigenin decreased glutamate-induced morphological signs of necrosis including swelling of cell bodies and neurite fragmentation at a concentration of 1 M.

  32. Protection (%) NMDA-injured (a) KA-injured (a) Concentration (mM) Control 100.0  4.2 0.0  1.3 Excitatory-injured 10.3  3.9 Arctigenin 0.01 6.6  2.7 0.10 21.2  1.3 * 54.6  4.3 *** † † 1.00 41.0  1.8 ** 73.8  2.5 *** † † 57.0  3.1 ** † Torreya nucifera Neuroprotective activity on arctigenin on N-methyl-D-aspartate (NMDA) or Kainate (KA)-induced rat cortical cells

  33. Torreya nucifera • Arctigenin protected neurons more selectively against KA-induced than NMDA-induced neurotoxicity in primary cultured cortical cells. • Arctigenin significantly inhibited the binding of [3H]-kainate to its receptor.

  34. Torreya nucifera • Arctigenin significantly attenuated glutamate-induced neurotoxicity when added prior to or after an excitotoxic glutamate challenge. • Arctigenin protected cultured neuronal cells more selectively from neurotoxicity induced by KA than NMDA. • Arctigenin directly scavenged free radicals generated by excess glutamate and reduced the level of cellular peroxide in cultured neurons. The neuroprotective activities of arctigenin are likely to be attributed to the directly binding of KA receptors and partly scavenging of free radicals.

  35. Hedyotis diffusa • Parts used : Rhizome • Traditional uses : Pneumonia in children, appendicitis, pelvis and some tumors in Korea and China • Description : An annual herb has leaves that look like the snake's tongue and white flowery patterns • Chemical components : Acylated iridoid glycosides anthraqunone derivitives Hedyotis diffusa Willd. ( Rubiaceae )

  36. R R1 R2 gal  glc  feruroyl gal  glc  feruroyl gal  glc  feruroyl gal  glc gal  glc H OH OH H OH 28 29 30 31 32 24 25 26 27 6-O-Z-p-methoxycinnamoyl 6-O-E-p-methoxycinnamoyl 6-O-Z-p-coumaroyl 6-O-E-p-coumaroyl Hedyotis diffusa Structures of compounds Isolated from H. diffusa R 1 O H C O O C H 3 R H O O O R 2 H O H C O g l c 2 O H O

  37. Hedyotis diffusa Neuroprotective activities of compound 24-32 on primary cultures of rat cortical cells injured by glutamate Cell viability (%) Compound 0.1 mM 1 mM 10 mM 24 28.8  4.6 * 54.6  2.9 ** 23.7  4.4 * 25 65.4  4.1 *** 71.8  2.8 *** 52.8  3.9 ** 26 6.9  0.9 25.7  2.0 * 6.3  3.5 27 14.5  1.0 62.2  4.0 *** 26.8  3.5 * 28 25.2  3.2 * 66.9  5.8 *** 25.2  3.6 * 29 62.7  1.1 *** 72.9  3.3 *** 75.0  0.2 *** 30 62.9  2.9 *** 71.5  1.6 *** 73.7  2.7 *** 31 4.6  3.6 27.0  4.9 * 25.2  3.6 * 32 48.2  0.7 ** 66.0  1.6 *** 54.0  2.9 ***

  38. R 1 O H C O O C H 3 R H O O O R 2 H O H C O g l c 2 O H O Hedyotis diffusa SAR of Flavonoid and Iridoid Glycosides O di-OH group H C O 3 acyl moiety (feruloyl) H O O trans-configuration p-methoxy group in aromatic ring O C H 3

  39. Hedyotis diffusa • Among the flavonoid glycosides, compounds 29,30 showed the strongest neuroprotective activity against glutamate- induced neurotoxicity. This results indicate that the presence of di-OH in the B ring and an acyl moiety might be crucial for the biological response. • Among the iridoid glycosides, compound 25 showed the strongest neuroprotective activity against glutamate-induced neurotoxicity. This results indicate that the p-methoxy group in the aromatic ring and a trans double bond in the acyl moiety play an essential role against glutamate-induced damage.

  40. Inula britannica • Parts used : Aerial parts • Traditional uses : Digestive disorders, bronchitis, and inflammation • Description : Biennial or perennial herb with an erect and appresed hairs or almost hairless stem • Chemical components : Sesquiterpene lactone (britanin, inulicin) Inula britannica L. ( Compositae )

  41. Inula britannica Structures of flavonoids Isolated from I. britannica O H B R O 4 R 2 A C R R 3 1 O H O

  42. Inula britannica Effects of flavonoids isolated from n-BuOH fraction of I. britannica on cell viability of glutamate-injured cortical cell cultures.

  43. Inula britannica • Amongthe 12 flavonoids tested, only patuletin (1), nepetin (6), and axillarin (10) showed significant neuroprotective activities against glutamate-induced toxicity at a concentrations ranging from 1 to 50M in a dose-dependent manner. • Patuletin (1) maintained the cellular viability of 70.6% at a concentration of 50M.

  44. Inula britannica Effects of patuletin, nepetin and axillarin on intracellular [Ca2+] and cellular peroxide in glutamate-injured rat cortical cells. O H O H O H H O O H O O H O O O H O H O H H C O O H H C O H C O O C H 3 3 3 3 O H O O H O O H O Patuletin (1) Axillarin (10) Nepetin (6) Cellular peroxide (arbitrary unit) Compound Concentration (mM) Intracellular [ Ca 2+ ]i (nM) 75.0  15.0 95.5  13.0 Control 423.0  20.0 403.3  10.4 Glutamate-treated 1.0 372.9  12.3 304.8  4.3 * Patuletin 10.0 329.1  10.2 * 249.8  16.5 ** 50.0 225.9  28.7 ** 172.4  3.1 *** 1.0 386.2  6.1 * 389.2  3.7 Nepetin 10.0 379.0  14.7 352.7  4.2 50.0 345.0  5.9 * 332.6  11.3 * 1.0 337.2  9.1 341.4  3.7 Axillarin 10.0 277.4  18.2 ** 292.2  7.6 ** 50.0 199.8  17.8 *** 180.3  1.8 ***

  45. Inula britannica • By pretreatment with patuletin (1) and axillarin (10) at a concentrations of 50 M, the content of intracellular [Ca2+] was maintained at 44.4% and 35.6%, respectively. • Furthermore, patuletin (1) and axillarin (10) effectively reduced cellular peroxides up to 24 h after glutamate challenge by pretreatment.

  46. Inula britannica Effects of patuletin, nepetin and axillarin on the activities of catalase, GSH-px, and GSSG-R in glutamate-injured cortical cultures. O H O H O H H O O H O O H O O O H O H O H H C O O H H C O H C O O C H 3 3 3 3 O H O O H O O H O Patuletin (1) Axillarin (10) Nepetin (6) Catalase ( umol H2O2 consumed / min/ mg protein GSH-px ( umol NADPH consumed /min /mg protein) GSSH-R ( umol NADPH consumed/ min/ mg /protein) Compound Control 41.5 ± 3.6 18.1 ± 2.4 13.2 ± 0.8 Glu-insulted 18.1 ± 4.2 7.7 ± 1.1 5.2 ± 1.0 Glu. + Patuletin 36.8 ± 4.8 ** 14.6 ± 0.9 ** 11.9 ± 0.9 *** Glu. + Nepetin 32.2 ± 5.7 * 12.9 ± 1.2 ** 10.3 ± 1.3 *** Glu. + Axillarin 33.3 ± 4.2 * 13.3 ± 1.1 ** 10.8 ± 0.9 ***

  47. Inula britannica • Amongthe 12 flavonoids tested, only patuletin (1), nepetin (6), and axillarin (10) protected primary cultured of rat cortical cells from oxidative stress induced by glutamate. - the 6-methoxy group in the A ring and the 3’,4’-hydroxy groupsin the B ring are crucil for the protection. • Patuletin (1), nepetin (6), and axillarin (10) exerted significant neuroprotective activity when they were administered either before or after the glutamate insult. These flavonoids maintained the activities of antioxidant enzymes such as catalase, GPx, GR • These flavonoids showed significant neuroprotective activity at least in part by an antioxidant effect.

  48. Anti HIV-1 Reverse Transcriptase and Protease activities Ecklonia cava Terminalia chebula Euphorbia pekinensis (大戟)

  49. Ecklonia cava • Description : A perennial marine brown algae that grows around 2~10m coastline in the gradual deepening zone. • Traditional uses : Antitumor, anticoagulant and antithrombin • Chemical components : Phlorotannin, fucodian, alginates, laminarans, organic minerals Ecklonia cava Kjellman. ( Phaeophyceae )

  50. H O O H H O O H O H O O H O H O H O O O O H H O O O O H O O H H O O H O O O H O H H O O H H O O H H O O H O H O O H O O O H O O H O O H O O H O O H O O H O H O H O H O O O O H O H H O O O H O H Ecklonia cava Structures of Compounds Isolated from Ecklonia cava 1 Eckol 2 8,8’-bieckol 3 8,4’’’-dieckol 4 phlorofucofuroeckol

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