Biofunctional Molecules from Molecules from Biofunctional Several - PDF document

Workshop Japan-Egypt “Pharmacognosy and Traditional Medicine” (July 20~23, 2010) Biofunctional Molecules from Molecules from Biofunctional Several Egyptian Herbal Medicines Several Egyptian Herbal Medicines – Black Cumin, Colocynth, Black Pepper Black Cumin, Colocynth, Black Pepper – – – Hisashi Matsuda and Masayuki Yoshikawa Department of Pharmacognosy, Kyoto Pharmaceutical University Search for Biofunctional Molecules from Other Egyptian Herbal Medicines - 1 1. St. Mary’s Flower （ the whole plant of Anastatica hierochuntica ） OH Hepatoprotective activity … Flavonolignans HO O O Antiinflammatory activity (NO production-inhibitory activity) … Neolignans HO Anti-melanogenesis activity … Flavonoids, Neolignans OH O HOH 2 C CH 2 OCH 3 2. Crinum yemense ( C. album ), bulbs O OH HO Antiinflammatory activity … Amaryllidaceae alkaloids OCH 3 (NO production-inhibitory activity) OAc OH O 3. Dichrocephala integrifolia , aerial parts N O HO Antiinflammatory activity … Sesquiterpenes (NO production-inhibitory activity) H OH HO O O O OH O H O O 4. Boswellia carterri , resin O O OH O Differentiation-inducing activity … Triterpenes OH

Search for Biofunctional Molecules from Other Egyptian Herbal Medicines - 2 5. Cyperus longus , whole plants OH OH Antiinflammatory activity … Stilbene dimers HO (NO production-inhibitory activity) O Hepatoprotective activity … Sesquiterpenes HO OH 6. Guggul-gum ( Balsamodendron mukul ), regin CH 2 OH Antiinflammatory activity … triterpenenes OH 7. Fenugreek ( Trigonella foenum-graecum ), OH CH 3 H seeds O O HO O Immunological adjuvant activity HO OH HO O H … triterpenene glycosides HO O OH OH HO H OH 8. Moroheiya ( Corchorus olitorius ), O HO O CH 3 CH 3 leaves, seeds O H HO O CH 3 HO OH O OH Anti-allergy, Antiinflammation (NO production-inhibitory HO activity) … fatty acids, ionone glucosides OH Black Cumin the seeds of Nigella sativa (Ranunculaceae) Origin Egypt, southwestern Asia, Mediterranean areas Distribution essential oils, fatty acids, saponins, alkaloids Constituents traditional Egyptian medicine for Application influenza, asthma, conjunctivitis, etc. Pharmacology antitumor, antiinflammation, antihypertension, hypoglycemia

Nigella sativa L. (Seeds) MeOH, ! a) SiO 2 column ( n -Hexane-AcOEt, CHCl 3 -MeOH-H 2 O) MeOH ext. (17.4%) b) ODS column (MeOH-H 2 O) c) HPLC (ODS, MeOH-H 2 O) EtOAc / H 2 O HP-20 (MeOH–H 2 O) EtOAc layer (10.1%) H 2 O eluate (2.6%) MeOH eluate (4.7%) a) b) c) a) b) c) 17 (1.15%) nigellamine A 1 (1, 0.0096%) 18 (0.15%) nigellamine A 2 (2, 0.0078%) nigellamine B 1 (3, 0.0012%) nigellamine B 2 (4, 0.0036%) nigellamine A 3 (5, 0.0005%) COO COO nigellamine A 4 (6, 0.0002%) O O nigellamine A 5 (7, 0.0002%) CH 2 OH CH 2 OH HO HO nigellamine B 3 (8, 0.0002%) O O OH OH HO O HO O nigellamine C (9, 0.0003%) H O O H O O nigellamine D (10, 0.0001%) OH OH OH OH O O HO HO carvacrol (11, 0.0170%) O O O H HO H O H HO H HO HO O O thymoquinol (12, 0.0256%) OH OH OH OH H H oleic acid (13, 0.20%) O OH HO OH H H linoleic acid (14, 0.46%) O HO OH HO OH OH linoleic acid methyl ester 17 18 HO H (15, 0.01%) OH 1 H-NMR (500MHz, CDCl 3 , # ) A white powder nigellamine A 1 (1) 27 -23.4° ( c =1.20, CHCl 3 ) [ ! ] D 13 C-NMR (125MHz, CDCl 3 , # c ) High resolution Pos. FAB-MS Calcd for C 40 H 44 NO 7 (M+H) + : 650.3118 3.08 Found : 650.3123 65.5 (brd, J = ca . 10) UV (MeOH, nm, log " ) : 264 (3.73), 226 (4.61) O IR (KBr, cm -1 ): 1717, 1647, 1636, 1592, 1541, 5.74 1509, 1277, 1069, 756, 712 N (dd, J =0.9, 10.4) 7 123.8 5.70 3 (brdd, J = ca . 6, 13) 75.4 5.45 O 10 (d, J =10.4) H-H COSY O 73.1 2 O HMBC 7' 7'' H 15 O O 12 18 0.1% NaOMe-MeOH r. t. O O 7''' 4.93 (d, J =11.0) methyl nicotinate (i) 5.29 (d, J =11.0) + methyl benzoate (ii) HO OH 67.2 H HO HPLC analysis 1a

Absolute Stereostructure of Nigellamine A 1 (1) O O O 4 N O 1) 0.1% NaOMe-MeOH, 0°C 3 O HO 2 2) HPLC separation O BzO HOH BzO HOH + O 1 O HO BzO Bz: benzoyl Nic: nicotinoyl 1b 1c 1 BzO 1 2(3) 4 H A white powder 24 +36.3° (c=0.4, CHCl 3 ) [ ! ] D High resolution Pos. FAB-MS Calcd for C 27 H 37 O 5 (M+H) + : 441.2641 Found : 441.2650 1a CD (MeOH, nm , "# "# ): 246 (–1.31) New Dolabellane-type Diterpenes Isolated from N. sativa O O O O N N N N O O O O O O O O O O O O H H H H O O O O O O O O N N N O O O O nigellamine A 1 (1) nigellamine A 3 (3) nigellamine A 2 (2) nigellamine A 4 (4) O O O O N N N N O O O O O O O O O O O O H H H H O O O O O O O O N N N OOH OOH OOH O O O O nigellamine B 3 (8) nigellamine B 2 (7) nigellamine B 1 (6) nigellamine A 5 (5) O O N N O O O O O O H O H O O N N CH 2 OH O nigellamine C (9) nigellamine D (10) Org. Lett ., 6 , 869–872 (2004); Chem. Pharm. Bull ., 52 , 494–497 (2004).

Promotion of Triglyceride (TG) Metabolism in Primary-Cultured Mouse Hepatocytes TG: % of control at 0.1 µ M Mouse hepatocytes + Test Sample N =4, p <0.01 in William's E medium with 10% FCS (8x10 4 cells/200 µ L/well) O O O O N N N O O O 2 O HO O HO O HO HO HOH O O O O O O HO N N O O O Incubation for 20 h 1 2 5 1a 64±4 ** 95±7 67±5 ** 102±4 Remove the medium and add water O O O N N N O O O O HO O HO O HO O O O O O O N N OOH OOH OH 18 O O O Sonication 6 7 7a 70±2 ** 79±2 ** 99±6 Centrifugation clofibrate Determination of TG CH 3 PPAR- ! agonist in the supernatant Cl O COOC 2 H 5 CH 3 64±5 ** Colocynth Normal Control Normal Control

Citrullus colocynthis (fruit, Egypt) MeOH, ! * SiO 2 column ODS column (MeOH-H 2 O) MeOH ext. (13.9%) HPLC (MeOH-H 2 O) EtOAc/H 2 0 n -BuOH H 2 O fraction (4.8%) n -BuOH fraction (2.8%) EtOAc fraction (6.3%) * * colocynthoside A (1, 0.0036%) cucurbitacin E colocynthoside B (2, 0.0095%) 2- O - " - D -glucopyranoside (4, 3.08%) (22,27)-hexanorcucurbitacin I 2- O - " - D -gulucopyranoside (3, 0.0020%) 3',4-Dihydroxy-3- cucurbitacin E 2- O - " - D -glucopyranoside (4, 0.0077%) methoxypropiophenone (0.0008%) cucurbitacin I 2- O - " - D -glucopyranoside (5, 0.150%) cucurbitacin J 2- O - " - D -glucopyranoside (6, 0.0015%) cucurbitacin K 2- O - " - D -glucopyranoside (7, 0.00073%) cucurbitacin L 2- O - " - D -glucopyranoside (8, 0.032%) isoorientin 3'-methyl ether (0.0032%) isovitexin (0.039%) isosaponarin (0.00051%) gastrodin (0.015%) benzyl " - D -glucopyranoside (0.00057%) 4-( " - D -glucopyranosyloxy)-benzaldehyde (0.00094%) 4-hydroxybenzyl- " - D -glucopyranoside (0.00094%) New and Known Constituents of C. colocynthis L. New Compounds O OH H HO O OH O O O CH 3 HO O OH H O HO O H O O OH H OH OH O O OH O O OH H OH CH 3 OH H colocynthoside A (1) colocynthoside B (2) OH OH O O HO HO O H H OAc OH H O OH O OH O OH H H H GlcO GlcO GlcO O O O (22-27)-hexanorcucurbitacin I cucurbitacin E cucurbitacin I 2- O - ! -D-glucopyranoside (3) 2- O - ! -D-glucopyranoside (4) 2- O - ! -D-glucopyranoside (5) O OH O OH O HO HO HO H OH H OH H OH O OH O OH O OH H H H GlcO GlcO GlcO O O O cucurbitacin J cucurbitacin K cucurbitacin L 2- O - ! -D-glucopyranoside (6) 2- O - ! -D-glucopyranoside (7) 2- O - ! -D-glucopyranoside (8) Glc = ! - D-glucopyranose

(O.D. at 620 nm) Leakage of dye ( N =5~10, * p <0.05, ** p <0.01, N : nomal, C : control) Chem. Pharm. Bull ., 55 , 428–434 (2004). Inhibitory Effects of Cucurbitacin E and Related Compounds on Proliferation in Human Leukemia U937 Cells (WST-1 Assay) O OH O O OH IC 50 at 72 h OH OH OH H OAc OAc H H O OH O OH O OH H H H Enhance HO HO HO O O O Reduce cucurbitacin J (10) dihydrocucurbitacin E (9) cucurbitacin E (4a) 2.4 µ M 0.016 µ M 3.3 µ M O O OH OH OAc OAc H H O OH O OH H H HO HO O O O OH cucurbitacin B (11) dihydrocucurbitacin B (12) H OAc O OH 0.0092 µ M 0.219 µ M H GlcO O O O OH H cucurbitacin E H OH O OH O OH 2- O - ! -D-glucopyranoside (4) H H HO ca . 60 µ M HO O O hexanorcucurbitacin D (14) cucurbitacin D (13) 2.4 µ M 0.33 µ M

Structure-Activity Relationships of Inhibitory Effects of Cucurbitacins on Proliferation in HT1080 and HL60 Cells IC 50 in HT1080 cell ( ! M) HT1080 : Human fibrosarcoma cell line IC 50 in HL60 cell ( ! M) HL60 : Human leukemic cell line (anaplastic) : enhance : reduce O O O HO OH OH OH OAc OAc H H H O OH O OH O OH H H H HO HO HO O O O 0.50 0.29 0.027 0.013 1.2 0.28 cucurbitacin D ( 13 ) cucurbitacin B ( 11 ) dihydrocucurbitacin B ( 12 ) O O O HO HO HO OH H OAc OAc H H O OH O OH O OH H H H HO HO HO 0.47 0.10 0.040 0.018 9.6 3.1 O O O cucurbitacin I ( 15 ) cucurbitacin E ( 4a ) dihydrocucurbitacin E ( 9 ) Chem. Pharm. Bull ., 58 , 747–751 (2010); Bioorg. Med. Chem . Lett ., 20 ,2994–2997 (2010). JAK/STAT3 Signaling Pathway and Effects of Cucurbitacins B (11) and E (4a) on Activation of STAT3 in U937 Cells Cytokines Cytokine receptor Tyk2 JAK2 Cucurbitacin B ( 11 ) Cucurbitacin E ( 4a ) Y705 P Control 10 100 10 100 ( µ M) STAT3 STAT3 STAT3 p-STAT3 P Y705 (Y705) STAT3 STAT3 p-ERK P Y705 mTOR MAPK JAK2 P Y705 P S727 STAT3 p-JAK2 STAT3 Transcription P Y705 DNA