Iridoid And Acyclic Monoterpene Glycosides, Kankanosides L, M, N, O, And P From Cistanche Tubulosa

Cistanche tubulosa (SCHRENK) R. WIGHT (Orobanchaceae) is a perennial parasitic plant growing on roots of Salvadora or Calotropis species, distributed in North Africa, Arabia, and Asian countries.1) Stems of this plant (Kanka-nikujuyou in Japanese) have traditionally been used for the treatment of impotence, sterility, lumbago, and body weakness as well as a promoting agent of blood circulation.1,2) During our studies on bioactive constituents from stems of C. tubulosa, 3-6) we previously reported twenty-four phenylethanoid aminoglycosides including kankanosides H1, H2, I, J1, J2, K1, and K2, and two acylated oligosugars from fresh stems of C. tubulosa. 5,6) Furthermore, principal phenylethanoid glycosides, echinacoside, acteoside, and isoacteoside, were found to inhibit increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (salt) levels in the mice's injured liver induced by D-galactosamine (D-GalN)/ lipopolysaccharide at doses of 25-100 mg/kg per os (p.o.). Structural requirements of the phenylethanoid glycosides for the hepatoprotective activity were also elucidated.5) In this continuing study on constituents in the fresh stems of C. tubulosa, we further isolated eleven iridoid glycosides including kankanosides L (1), M (2), and N (3), seven acyclic monoterpene glycosides including kankanosides O (4) and P (5), three phenylpropanoids, and four lignans. This paper deals with the isolation and structure elucidation of five new compounds (1-5).

NATURAL CISTANCHE TUBULOSA FOR RELIEVE SEXUAL DYSFUNCTION PHGS75% ECH 30% ACT 12%

Fresh stems of C. tubulosa (cultivated in Urumqi, Xinjiang Province, China) were extracted with methanol under reflux to yield a methanolic extract (8.36% from the fresh stems). From the methanolic extract, H2O- and MeOH-eluted fractions (5.63% and 2.73%, respectively) were obtained by Diaion HP-20 column chromatography (H2O→MeOH) as described previously.5) The MeOH-eluted fraction was subjected to SiO2 and ODS column chromatographies and finally HPLC to furnish kankanosides L (1, 0.0026%), M (2, 0.0001%), N (3, 0.0007%), O (4, 0.0020%), and P (5, 0.0002%), 6-deoxycatalpol3,7) (6, 0.197%), bartsioside3,7) (7, 0.0583%), gluroside3,7) (8, 0.0443%), kankanoiside A3) (9, 22.3 mg, 0.0010%), mussaenosidic acid3,7) (10, 0.0056%), 8- epiloganic acid3,8) (11, 0.0023%), 8-epideoxyloganic acid3,7) (12, 0.0004%), geniposidic acid3,7) (13, 0.0040%), kankanoside E3) (14, 0.0026%), (2E,6Z)-8-b-D-glucopyranosyloxy-2,6-dimethyl-2,6-octadienoic acid3,9) (15, 31.0 mg, 0.0014%), (2E,6E)-3,7-dimethyl-8-hydroxyoctadien-1-yl-O-b-D-glucopyranoside10) (16, 0.0082%), 8-hydroxygeraniol 8-O-bD-glucopyranoside11) (17, 0.0044%), betulalbuside A12) (18, 0.0004%), coniferin13) (19, 0.0002%), syringin13) (20, 0.0015%), sinapic aldehyde 4-O-b-D-glucopyranoside14) (21, 0.0001%), ( )-pinoresinol O-b-D-glucopyranoside4,8,15) (22, 0.0010%), eucommin A16) (23, 0.0002%), isoeucommin A17) (24, 0.0010%), and ( )-syringaresinol O-b-D-glucopyranoside4,8,18) (25, 0.0044%).

Structures of Kankanosides L (1), M (2), and N (3) 

Kankanoside L (1) was obtained as a white powder with negative optical rotation ([a]D 26 - 45.7 in MeOH). Its IR spectrum showed a strong absorption band at 3433 and 1080 cm- 1 suggestive of a glycoside moiety. The fast atom bombardment (FAB)-MS of 1 run in the positive- and negative-ion modes showed quasimolecular ion peaks at m/z 371 [M Na] and 347 [M- H]-, respectively, and the molecular formula was determined as C15H24O9 by high-resolution FABMS measurement. Acid hydrolysis of 1 with 1.0 M hydrochloric acid (HCl) liberated D-glucose, which was identified by HPLC analysis using an optical rotation detector.3-6) The 1 H- and 13C-NMR spectra of 1 (CD3OD, Tables 1, 2), which were assigned by various NMR experiments,19) showed signals assignable to four methylenes [d 1.43 (1H, br dd, J= ca. 5, 13 Hz, 4a-H), 1.73 (1H, br dd, J= ca. 12, 14 Hz, 6a-H), 1.85 (1H, m, 4b-H), 1.93 (1H, br dd, J= ca. 8, 14 Hz, 6b-H), 3.50 (1H, ddd, J= 2.4, 12.5, 13.0 Hz, 3a-H), 3.83 (1H, br dd, J=

ca. 5, 13 Hz, 3b-H), 3.78, 4.12 (1H each, both d, J= 13.1 Hz, 10-H2)], two methines [d 2.15 (1H, dd, J= 7.2, 8.9 Hz, 9-H) and 2.23 (1H, m, 5-H)], and an acetal group [d 4.81 (1H, d, J= 8.9 Hz, 1-H)] together with a b-glucopyranosyl moiety [d 4.70 (d, J= 7.9 Hz, 1 -H)]. As shown in Fig. 1, the 1 H–1 H correlation spectroscopy (1 H–1 H COSY) experiment on 1 indicated the presence of partial structures written in bold lines. In the heteronuclear multiple-bond correlation (HMBC) experiment on 1, long-range correlations were observed between the following protons and carbons (1-H and 3-C, 8-C; 3-H and 1-C; 7-H and 8-C, 10-C; 9-H and 8-C; 10-H2 and 7-C, 8-C; 1 -H and 1-C) as shown in Fig. 1. Next, the relative stereostructure of 1 was characterized by phasesensitive nuclear Overhauser enhancement spectroscopy (phase-sensitive NOESY) experiment, which showed NOE correlations between the following proton pairs (1-H and 3aH; 3a-H and 4a-H; 3b-H and 4b-H; 4b-H and 5-H, 9-H; 5- H and 6b-H, 9-H; 6a-H and 7-H; 7-H and 10-H2), as shown in Fig. 1. The 1 H- and 13C-NMR spectra of 1 were superimposable on those of principal iridoid constituent 6-deoxycatalpol (6), except for the signals due to the saturated d-lactol moiety. Finally, hydrogenation of 6 gave 1, so the stereostructure of kankanoside L was elucidated to be 3,4- dihydro-6-deoxycatalpol (1).

NATURAL CISTANCHE TUBULOSA FOR RELIEVE MAIL AROUSAL DISORDER PHGS75% ECH 30% ACT 12%

Kankanoside M (2) was obtained as a white powder with negative optical rotation ([a]D 26 - 18.7 in MeOH). The IR spectrum of 2 showed absorption bands at 3433, 1736, 1655, and 1076 cm- 1 ascribable to hydroxyl, d-lactone, olefin, and ether moieties. The positive-ion FAB-MS spectrum of 2 showed a quasimolecular ion peak at m/z 353 [M Na] , and the molecular formula was determined as C15H22O8 by high-resolution positive-ion FAB-MS measurement. Acid hydrolysis of 2 with 1.0 M HCl liberated D-glucose. The 1 H- and 13CNMR spectra of 2 (CD3OD, Tables 1, 2) showed signals assignable to four methylenes [d 1.66, 2.11 (1H each, both m, 4-H2), 2.15, 2.75 (1H each, both m, 6-H2), 4.29 (1H, ddd, J= 2.8, 8.4, 14.3 Hz, 3b-H), 4.32, 4.51 (1H each, both d, J= 13.1 Hz, 10-H2), 4.35 (1H, ddd, J= 3.1, 6.7, 14.3 Hz, 3a-H)], two methines [d 2.97 (1H, m, 5-H), 3.82 (1H, br s, 9-H)], an olefin [d 5.94 (1H, m, 7-H)], and a saturated lactone group (d C 174.9) together with a b-D-glucopyranosyl moiety [d 4.32 (1H, d, J= 7.9 Hz, 1 -H)]. As shown in Fig. 1, the 1 H–1 H COSY experiment on 2 indicated the presence of partial structures written in bold lines and, in the HMBC experiment, long-range correlations were observed between the following proton and carbon pairs (3-H and 1-C; 7-H and 9-C; 9-H and 1-C, 8-C; 10-H2 and 7-C, 8-C, 9-C; 1 -H and 10-C). The relative stereostructure of 2 was characterized by a phase-sensitive NOESY experiment, which showed NOE correlations between the following proton pairs (3a-H and 4a-H; 3b-H and 4b-H; 4b-H and 5-H; 5-H and 6b-H, 9-H) as shown in Fig. 1. Thus, the stereostructure of 2 was elucidated as shown.

Kankanoside N (3) was isolated as a white powder with negative optical rotation ([a]D 25 - 24.6 in MeOH). In the positive-ion FAB-MS of 3, a quasimolecular ion peak was observed at m/z 371 [M Na] . The molecular formula C16H28O8 was determined by high-resolution FAB-MS measurement. Acid hydrolysis of 3 with 1.0 M HCl liberated D-glucose. The 1 H- and 13C-NMR data (CD3OD, Tables 1, 2) showed signals assignable to a methyl [d 1.07 (3H, d, J= 7.2 Hz, 10-H3)], four methylenes {d 1.37, 1.87 (1H each, both m, 7-H2), 1.65, 1.81 (1H each, both m, 6-H2), [3.65 (1H, dd, J= 9.1, 9.8 Hz), 3.90 (1H, dd, J= 5.9, 9.8 Hz), 11-H2], and [3.70 (1H, dd, J= 3.3, 12.0 Hz), 3.88 (1H, m), 3-H2]}, four methines [d 1.69 (1H, m, 4-H), 1.75 (1H, m, 9-H), 2.06 (1H, m, 8-H), 2.16 (1H, m, 5-H)], and a hemiacetal group [d 4.67 (1H, d, J= 7.4 Hz, 1-H)] together with a b-D-glucopyranosyl moiety [d 4.26 (1H, d, J= 7.9 Hz, 1 -H)]. The iridoid structure of 3 was clarified by 1 H–1 H COSY and HMBC experiments and the relative stereostructure was characterized by a phase-sensitive NOESY experiment as shown in Fig. 1. Consequently, the sterostructure of 3 was elucidated as shown.

NATURAL CISTANCHE TUBULOSA FOR SOLVING SEXUAL PROBLEM PHGS75% ECH 30% ACT 12%

Structures of Kankanosides O (4) and P (5) 

Kankanosides O (4) and P (5), C16H26O8, were also obtained as white powders with negative optical rotations (4: [a]D 23 - 26.1; 5: [a]D 21 - 32.7 both in MeOH). The IR spectra of 4 and 5 showed absorption bands at 3433, 1696, 1647, and 1076 cm- 1 for 4, and 3434, 1701, 1647, and 1076 cm- 1 for 5, ascribable to glycosidic, carboxyl, and olefin functions. Their UV spectra showed a common absorption maximum at 217 nm indicating the presence of an a,b-unsaturated carboxylic acid moiety in both of them. Acid hydrolysis of 4 and 5 liberated D-glucose, whereas by the enzymatic hydrolysis with glucosidase, 4 and 5 gave (2E,6E)-8-hydroxy-2,6-dimethyl- 2,6-octadienoic acid20) (4a) and (2E,6E)-8-hydroxy-3,7- dimethyl-2,6-octadienoic acid21) (5a), respectively. 1 H- and 13C-NMR data of 4 (CD3OD, Tables 2, 3) showed signals assignable to two methyls [d 1.71 (3H, br s, 10-H3), 1.81 (3H, d, J= 1.0 Hz, 9-H3)], three methylenes {d 2.19 (2H, br t, J= ca. 7 Hz, 5-H2), 2.36 (2H, m, 4-H2), [4.24 (1H, dd, J= 7.6, 12.0 Hz), 4.33 (1H, dd, J= 6.2, 12.0 Hz), 8-H2]}, and two trisubstituted olefins [d 5.41 (1H, ddd, J= 1.2, 6.2, 7.6 Hz, 7- H), 6.75 (1H, tq, J= 7.2, 1.0 Hz, 3-H)] together with a b-Dglucopyranosyl part [d 4.34 (d, J= 7.8 Hz, 1 -H)]. By the comparison of carbon signals in the 13C-NMR spectrum of 4 

with those of 4a, a glycosylation shift was observed at the 8- position (d C 4: 65.5; 4a: 59.4). The position of the glucoside linkage was also confirmed by HMBC experiments as shown in Fig. 2. Consequently, the stereostructure of 4 was clarified to be (2E,6E)-8-b-D-glucopyranosyloxy-2,6-dimethyl-2,6-octadienoic acid. On the other hand, the 1 H- and 13C-NMR data of 5 (CD3OD, Tables 2, 3) indicated the presence of a (2E,6E)-8-hydroxy-3,7-dimethyl-2,6-octadienoic acid moiety [d 1.70 (3H, br s, 10-H3), 2.14 (3H, br s, 9-H3), 2.24 (2H, m, 4-H2), 2.27 (2H, m, 5-H2), 4.05, 4.20 (1H each, both br d, J= ca. 12 Hz, 8-H2), 5.47 (1H, tq, J= 7.1, 0.9 Hz, 6-H), 5.67 (1H, br s, 1-H)] together with a b-D-glucopyranosyl part [d 4.23 (d, J= 7.7 Hz, 1 -H)]. The connectivity of the b-D-glucopyranosyl moiety in 5 was elucidated based on HMBC experiments as shown in Fig. 2. Furthermore, a typical glycosylation shift was observed for the signals at 8-position (d C 5: 75.6; 5a: 68.8). based on the above-mentioned evidence, the stereostructure of 5 was determined to be (2E,6E)-8-b-D-glucopyranosyloxy-3,7-dimethyl-2,6-octadienoic acid.

Effects of the Constituents on Tumor Necrosis Factor-a (TNF-a)-induced Cytotoxicity in L929 Cells 

TNF-a is known to mediate a variety of organ injury through its induction of cellular apoptosis. In the case of the liver, the biological effects of TNF-a have been implicated in hepatic injury induced by hepatic toxins, ischemia/reperfusion, viral hepatitis, and alcohol.22-24) Therefore, TNF-a is considered to be an important target to discover anti-inflammatory and hepatoprotective agents. Based on the above-mentioned concept, we investigated protective constituents from naturally occurring products on TNF-a-induced cell death in L929 cells, a TNF-a-sensitive cell line.25) Previously, we have reported

that several constituents from Piper chaba, 26-29) Boesenbergia rotunda, 30,31) Punica granatum, 32) Helichrysum arenarium, 33-35) and Sapindus rarak, 36-38) were found to show inhibitory effects of TNF-a-induced cytotoxicity in L929 cells. Since the phenylethanoid constituents of C. tubulosa (e.g. echinacoside, acteoside, and isoacteoside, etc.) 5) also inhibited this cytotoxicity, we further examined iridoid, phenylpropanoid, and lignan constituents as shown in Table 4. As a result, kankanoside A (9, inhibition: 16.3± 2.0% at 100 mM), mussaenosidic acid (10, 44.7± 8.7%), 8-epigamic acid (11, 10.7± 0.4%), 8-hydroxy geraniol 8-O-b-D-glucopyranoside (17, 21.3± 2.4%), and ( )-pinoresinol O-b-D-glucopyranoside (22, 22.3± 1.6%), were found to show significant activity. Although their activities were weaker than those of echinacoside (IC50= 31.1 mM), acteoside (17.8 mM), and isoaceteoside (22.7 mM), the principle phenylethanoid constituents.5)

NATURAL CISTANCHE TUBULOSA FOR IMPROVING SEXUAL FUNCTION PHGS75% ECH 30% ACT 12%