Phenylethanoid Oligoglycosides And Acylated Oligosugars With Vasorelaxant Activity From Cistanche Tubulosa
Mar 15, 2022
for more information:ali.ma@wecistanche.com
Masayuki Yoshikawa et al
Abstract
The methanolic extract from the dried stems of Cistanche tubulosa (Schrenk) R. Wight was found to show an inhibitory effect on contractions induced by noradrenaline in isolated rat aortic strips. From the extract, new phenylethanoid aminoglycoside constituents, kankanosides F and G, and an acylated oligo sugar, kankanose, were isolated together with 14 known compounds. The structures of these new compounds were determined on the basis of their chemical and physicochemical evidence. In addition, principal constituents, kankanoside F, kankanose, echinacoside, acteoside, and cistanoside F, showed vasorelaxant activity, and several structural requirements for the activity were clarified. 2006 Elsevier Ltd. All rights reserved.
Click to Cistanche effect products
Introduction
The Orobanchaceae parasitic plant, Cistanche tubulosa(Schrenk) R. Wight, is widely distributed in North Africa, Arabia, and Asian countries, and the stems of Cistanche tubulosa, as well as C. salsa and C. deserticola, have been traditionally used as a promoting agent of blood circulation and treatment of impotence, sterility, lumbago, body weakness, and tonic.2,3 During the course of our characterization studies on bioactive constituents from Chinese natural medicines,1,4–14 we have reported the isolation and structure elucidation of five iridoids, kankanosides A–D and kankanol, and a monoterpene glycoside, kankanoside E, together with 16 known compounds from the stems of Cistanche tubulosa1 As a continuation of the study on this herbal medicine, the methanolic extract was found to show an inhibitory effect on contractions induced by noradrenaline in isolated rat thoracic aortic strips. From this methanolic extract, we additionally isolated phenylethanoid oligoglycosides, kankanosides F (1) and G (2), and an acylated oligo sugar, kankanose (3), and 14 known compounds, echinacoside (4), acteoside (5), isoacteoside (6), 20 -acetylacteoside (7), tubulosides A (8) and B (9), cistanoside F (10), salidroside (11), (2R,3R)-butane-2,3- diol 2-O-b-D-glucopyranoside, Meso-butane-2,3-diol 2-O-b-D-glucopyranoside, ethyl b-D-glucopyranoside, 3-methyl butanol b-D-glucopyranoside, (+)-pinoresinol 40 -O-b-D-glucopyranoside, and (+)-syringaresinol 40 -O-b-D-glucopyranoside. This paper deals with the structure elucidation of new compounds (1–3) as well as the vasorelaxant effects of the principal constituents (1, 3– 6,10, and 11) on contractions induced by noradrenaline in isolated rat aortic strips based on the traditional application for blood circulation (Fig. 1).
Results and discussion
2.1. Vasorelaxant effect of methanolic extract from the stems of Cistanche tubulosa on the contraction induced by noradrenaline in isolated rat thoracic aorta
As shown in Table 1, the methanolic extract from the stems of Cistanche tubulosa (purchased in Urumqi, Xinjiang Province, China) was found to show vasorelaxant activity on contractions induced by noradrenaline in isolated rat aortic strips, time- and concentration-dependently (30–300 lg/mL). The methanolic extract was further purified by normal- and reverse-phase column chromatography and finally HPLC to give 1 (0.016%), 2 (0.025%), 3 (0.020%), 4 (3.09%),3 5 (0.68%),3 6 (0.096%),3 7 (0.017%),3 8 (0.052%),3 9 (0.0036%),3 10 (0.025%),15 11 (0.044%),16 (2R,3R)-butane-2,3-diol 2-O-b-D-glucopyranoside (0.0087%),17 meso-butane-2,3-diol 2-O-b-D-glucopyranoside (0.0065%),17 ethyl b-D-glucopyranoside (0.055%),17 3-methylbutanol b-D-glucopyranoside (0.0030%),17 (+)-pinoresinol 40 -O-b-D-glucopyranoside (0.016%),18 and (+)-syringaresinol 40 -O-b-D-glucopyranoside (0.022%).15
2.2. Structures of kankanosides F (1) and G (2)
Kankanoside F (1) was obtained as a white powder with negative optical rotation (½a -28 D -44.8- in MeOH). The IR spectrum of 1 showed absorption bands at 1560, 1508, and 1458 cm-1 ascribable to an aromatic ring and strong absorption bands at 3432, 1075, and 1044 cm-1, suggestive of an aminoglycoside structure. In the positive- and negative-ion FAB-MS of 1, quasimolecular ion peaks were observed at m/z 647 (M+Na)+ and 623 (M-H), and high-resolution FAB-MS analysis revealed the molecular formula of 1 to be C26H40O17. The 1 H and 13C NMR (CD3OD, Table 2) spectra of 1, which were assigned by various NMR experiments,19 showed signals assignable to two methylenes [d 2.78 (2H, m, H2-7), 3.70, 4.00 (1H each, both m, H2-8)] and ortho- and meta-coupled ABC-type aromatic protons [d 6.56 (1H, dd, J = 1.9, 7.9 Hz, H-6), 6.67 (1H, d, J = 7.9 Hz, H-5), 6.69 (1H, d, J = 1.9 Hz, H-2)] together with three glucopyranose moieties [d 4.30 (1H, d, J = 8.0 Hz, H-10 ), 4.37 (1H, d, J = 8.0 Hz, H-1000), 5.16 (1H, br s, H-100)]. The aminoglycoside structure in 1 was characterized by heteronuclear multiple bond connectivity (HMBC) experiment, which showed long-range correlations between the following proton and carbon pairs (H-10 and C-8; H-100 and C-30; H-1000 and C-60 ) as shown in Figure 2. Enzymatic deacylation of echinacoside (4), whose absolute stereostructure was reported previously,3 with tannase13,14 gave 1. On the basis of the abovementioned evidence, the structure of kankanoside F was determined to be 2-(3,4-dihydroxy phenyl)ethyl O-a-L-rhamnopyranosyl(1-3)[b-D-glucopyranosyl(1-6)]-b- D-glucopyranoside (1).
Kankanoside G (2) was also obtained as a white powder with negative optical rotation (½a-26 D-35.0-in MeOH). The IR spectrum of 2 showed absorption bands at 3432, 1709, 1638, 1518, 1447, and 1040 cm-1, ascribable to hydroxyl, conjugated ester carbonyl, and ether functions and aromatic ring. The molecular formula, C29H36O14, of 2 was determined from the positive- and negative-ion FAB-MS [m/z 631 (M+Na)+ and 607 (M-H)-] and by high-resolution FAB-MS. Enzymatic deacylation of 2 with tannase yielded cistanoside G (12), whose absolute stereostructure was reported previously.16 On alkaline hydrolysis of 2 with 10% aqueous potassium hydroxide (KOH)—50% aqueous 1,4-dioxane (1:1, v/v), caffeic acid was identified by HPLC analysis. The 1 H and 13C NMR (CD3OD, Table 2) spectra19 of 2 indicated the presence of the following functions: 4-hydroxyphenylethyl alcohol part {two methylenes [d 2.82 (2H, m, H2-7), 3.70, 3.96 (1H each, both m, H2-8)] and ortho-coupled A2B2-type aromatic protons [d 6.65, 7.03 (2H each, both d, J = 8.6 Hz, H-3,5 and H-2,6)]} and two glucopyranose moieties [d 4.33 (1H, d, J = 7.9 Hz, H-10 ), 5.19 (1H, br s, H-100)] together with a caffeoyl moiety {an trans-olefin [d 6.29, 7.56 (1H each, both d, J = 15.9 Hz, H-8 and H-7)] and pyrocatechol-type dihydroxybenzene [d 6.78 (1H, d, J = 8.3 Hz, H-5), 6.89 (1H, dd, J = 1.9, 8.3 Hz, H-6), 7.04 (1H, d, J = 1.9 Hz, H-2)]}. In the HMBC experiment on 2, a long-range correlation was observed between the 60 -proton [d 4.36 (1H, dd, J = 6.1, 11.6 Hz), 4.50 (dd, J = 2.8, 11.6 Hz)] and the caffeoyl carbonyl carbon (dC 169.1). Consequently, the structure of kankanoside G was determined to be 4-hydroxyphenyl-O-a-L-rhamnopyranosyl(1 ! 3)-(6-O-caffeoyl)-b-D-glucopyranoside (2).
2.3. Structure of kankanose (3)
Kankanose (3), ½a- 27 D-35.3- (MeOH), was also obtained as a white powder. The IR spectrum of 3 showed absorption bands at 3430, 1702, 1639, 1611, 1458, and 1071 cm-1, ascribable to hydroxyl, conjugated ester carbonyl, and ether functions and aromatic ring. The positive- and negative-ion FAB-MS of 3 showed quasimolecular ion peaks at m/z 673 (M+Na)+ and m/z 649 (MH), respectively. The high-resolution FAB-MS of 3 revealed the molecular formula to be C27H38O18. Alkaline hydrolysis of 3 with 10% KOH– 50% aqueous 1,4-dioxane liberated caffeic acid, which was identified by HPLC analysis, together with an oligosugar. The acid hydrolysis of this oligo sugar with 1.0 M hydrochloric acid (HCl) liberated L-rhamnose and D-glucose, which were identified by HPLC analysis using an optical rotation detector.1,13,14 The 1 H and 13C NMR (CD3OD, Table 3) spectra19 of 3 showed signals assignable to the trisaccharide moiety {d [5.11 (d, J=3.4 Hz), 4.57 (d, J=7.9 Hz), a-and b-form mixture of H-10 (ca. 1:1)], 4.25, 4.27 (both d, J=7.7 Hz, H-1000), 5.13, 5.18 (both br s, H-100)} together with a caffeoyl group [d 6.28, 6.29 (both d, J=15.9 Hz, H-9), 6.78 (d, J=8.0 Hz, H-5), 6.96 (dd, J=2.0, 8.0 Hz, H-6), 7.06 (d, J = 2.0 Hz, H-2), 7.60 (d, J = 15.9 Hz, H-7)]. In the HMBC experiments on 3, long-range correlations were observed between the 40-proton [d 5.04, 4.99 (both dd, J=9.5, 9.8 Hz)] and the caffeoyl carbonyl carbon (dC 168.53, 168.65). On the basis of the above-mentioned evidence, the structure of kankanose was elucidated to be a-L-rhamnopyranosyl(1-3)[b-D-glucopyranosyl(1-6)]- O-(4-O-caffeoyl)-D-glucopyranose (3).
2.4. Vasorelaxant effect of principal constituents (1, 3–6, 10, 11) from Cistanche tubulosa on the contraction induced by noradrenaline and high K+ in isolated rat thoracic aorta
As shown in Table 4 and Figure 3, inhibitory effects of the principal constituents, kankanoside F (1) and kankanose (3), echinacoside (4), acteoside (5), isoacteoside (6), cistanoside F (10), and salidroside (11) on noradrenaline-induced contractions in isolated rat thoracic aorta were examined. As a result, compounds 3–5, and 10 having a caffeoyl group in the 40 -position significantly inhibited the contractions, time- and/or concentration-dependently (10–100 lM), while 6 having the 60 -O-caffeoyl moiety showed weak activity than 4 and 5. On the other hand, compound 1 with a catechol group showed significant inhibition, but 11 with a phenol group did not show such effect. These results led us to presume that the 40 -O-caffeoyl moiety and the 10 -O-dihydroxyphenethyl moiety are essential for strong vasorelaxant activity. On the other hand, compounds 1, 3–6, and 10 (100 lM) did not show any effects on contractions induced by a high concentration of potassium cation (high K+) different from nifedipine, a voltage-dependent calcium channel blocker, in the same tissues (data not shown). This finding suggested that these active constituents inhibited the contractions via receptor-operated calcium channel, but not via voltage-dependent calcium channel. Recently, Iizuka et al. reported that a phenylethanoid, forsythiaside, from the fruit of Forsythia suspensa inhibited the noradrenaline-induced contractions in rat aortic ring preparations and suggested that the inhibition by forsythiaside of noradrenaline-induced contraction is partly due to a decrease in noradrenaline-induced calcium influx.20 However, in the present study, the relaxant effects of the active constituents were observed ca. 30 min after addition of noradrenaline in a different way from that of prazosin, an adrenaline a1-receptor antagonist. The mechanisms of action of active constituents need to be studied further.
3. Experimental
3.1. General experimental procedure
The following instruments were used to obtain physical data: specific rotations, Horiba SEPA-300 digital polarimeter (l = 5 cm); UV spectra, Shimadzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrometer; 1 H NMR spectra, JEOL JNM-LA500 (500 MHz) spectrometer; 13C NMR spectra, JEOL JNM-LA500 (125 MHz) spectrometer with tetramethylsilane as an internal standard; FABMS and HR-FABMS, JEOL JMS-SX 102A mass spectrometer; HPLC detector, Shimadzu RID-6A refractive index and SPD10A UV–vis detectors. HPLC column, YMC-Pack ODS-A (250 · 4.6 mm id), and (250 · 20 mm id) columns were used for analytical and preparative purposes, respectively
The following experimental conditions were used for chromatography: normal-phase silica gel column chromatography, silica gel BW-200 (Fuji Silysia Chemical, Ltd, 150–350 mesh); reversed-phase silica gel column chromatography, Chromatorex ODS DM1020T (Fuji Silesia Chemical, Ltd, 100–200 mesh); TLC, pre-coated TLC plates with silica gel 60F254 (Merck, 0.25 mm) (normal-phase) and silica gel RP-18 F254S (Merck, 0.25 mm) (reversed-phase); reversed-phase HPTLC, pre-coated TLC plates with silica gel RP-18 WF254S (Merck, 0.25 mm); detection was achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4, followed by heating.
Cistanche tubulosa
3.2. Plant material
Dried stems of Cistanche tubulosa were purchased at Urumqi, Xinjiang Province, China, in January 2005 via Eishin Trading Co., Ltd, Osaka, Japan, as described previously.1
3.3. Extraction and isolation
Fractions 1-2 (590 mg), 2-1 (466 mg), 2-3 (3931 mg), 3-3 (3610 mg), 3-4 (190 mg), 4-5 [=echinacoside (4, 2595 mg, 0.44%)], 4-6 (1635 mg), 5-2 (1114 mg), 5-3 (306 mg), 5-4 (347 mg), 5-5 (=4, 1620 mg, 2.03%), and 5-6 (1453 mg) were obtained from the methanolic extract of the stems of Cistanche tubulosa as reported previously.1 Fraction 1–2 (590 mg) was purified by HPLC [MeOH/H2O (35:65, v/v)] to give 3-methylbutanol b-D-glucopyranoside (18 mg, 0.0030%), (+)-pinoresinol 40 -O-b-D-glucopyranoside (96 mg, 0.016%), and (+)-syringaresinol 40 -Ob-D-glucopyranoside (129 mg, 0.022%) together cistanochlorin (21 mg, 0.0035%). Fraction 2-1 (466 mg) was separated by HPLC [MeOH/H2O (5:95, v/v)] to give (2R,3R)-butane-2,3-diol 2-O-b-D-glucopyranoside (12 mg, 0.0087%), (2R,3S)-butane-2,3-diol 2-O-b-Dglucopyranoside (9 mg, 0.0065%), and ethyl b-D-glucopyranoside (76 mg, 0.055%) together with uridine (0.0069%). Fraction 2-2 (535 mg) was purified by HPLC [MeOH/H2O (5:95, v/v)] to afford salidroside (11, 41 mg, 0.022%) together with antirrhide (15 mg, 0.0079%) and 6-deoxycatalpol (214 mg, 0.11%). Fraction 2-3 (535 mg) was separated by HPLC [MeOH/H2O (5:95, v/v)] to furnish 11 (17 mg, 0.022%) together with gluroside (110 mg, 0.14%) and bartsioside (164 mg, 0.21%). Fraction 3-3 (510 mg) was subjected to HPLC [MeOH/ H2O (40:60, v/v)] to give acteoside (5, 257 mg, 0.31%), isoacteoside (6, 79 mg, 0.096%), and 20 -acetylacteoside (7, 14 mg, 0.017%). Fraction 3-4 (190 mg) was further purified by HPLC [MeOH/H2O (40:60, v/v)] to give kankanoside G (2, 17 mg, 0.0029%) and tubuloside B (9, 21 mg, 0.0036%). Fraction 4-6 (75 mg) was purified by HPLC [MeOH/H2O (35:65, v/v)] to afford 5 (18 mg, 0.065%). Fraction 5-2 (1114 mg) was subjected to HPLC [MeOH/H2O (10:90, v/v)] to give 2 (24 mg, 0.030%). Fraction 5-3 (306 mg) was purified by HPLC [MeOH/ H2O (15:85, v/v)] to give kankanoside F (1, 13 mg, 0.016%). Fraction 5-4 (347 mg) was subjected to HPLC [MeOH/H2O (10:90, v/v)] to give kankanose (3, 30 mg, 0.038%) and cistanoside F (10, 20 mg, 0.025%). Fraction 5-6 (530 mg) was purified by HPLC [MeOH/H2O (35:65, v/v)] to afford 4 (185 mg, 0.62%), 5 (91 mg, 0.30%), and tubuloside A (8, 16 mg, 0.052%).
3.3.1. Kankanoside F (1).
A white powder, ½a- 28 D -44.8- (c 0.50, MeOH). High-resolution positive-ion FAB-MS m/z: Calcd for C26H40O17Na (M+Na)+, 647.2163. Found: 647.2160. UV [MeOH, nm (log e)]: 224 (3.83), 283 (3.37). IR (KBr): 3432, 2940, 1560, 1508, 1458, 1075, 1044 cm-1 . 1 H NMR (500 MHz, CD3OD) d: given in Table 2. 13C NMR data (125 MHz, CD3OD) dC: given in Table 2. Positive-ion FAB-MS m/z: 647 (M+Na)+. Negative-ion FAB-MS m/z: 623 (MH), 477 (M-C6H11O4) -, 623 (M-C6H11O5)-, 623 (M-C12H21O9)-.
3.3.2. Kankanoside G (2).
A white powder, ½a- 26 D -35.0- (c 0.80, MeOH). High-resolution positive-ion FAB-MS m/z: Calcd for C29H36O14Na (M+Na)+, 631.2003. Found: 631.1996. UV [MeOH, nm (log e)]: 329 (4.10). IR (KBr): 3432, 2924, 1709, 1638, 1518, 1447, 1264, 1040 cm-1 . 1 H NMR (500 MHz, CD3OD) d: given in Table 2. 13C NMR data (125 MHz, CD3OD) dC: given in Table 2. Positive-ion FAB-MS m/z: 631 (M+Na)+. Negative-ion FAB-MS m/z: 607 (M-H)-, 461 (M-C6H11O4).
3.3.3. Kankanose (3).
A white powder, ½a- 27 D -35.3- (c 0.50, MeOH). High-resolution positive-ion FAB-MS m/z: Calcd for C27H38O18Na (M+Na)+, 673.1956. Found: 673.1959. UV [MeOH, nm (log e)]: 219 (4.10), 247 (3.96), 333 (4.21). IR (KBr): 3430, 2962, 1702, 1639, 1611, 1458, 1264, 1071 cm-1 . 1 H NMR (500 MHz, CD3OD) d: given in Table 3. 13C NMR data (125 MHz, CD3OD) dC: given in Table 3. Positive-ion FAB-MS m/z: 673 (M+Na)+. Negative-ion FABM-S m/z: 649 (MH), 487 (M-C9H8O3) -, 325 (M-C15H18O8) -.
Cistanche tubulosa
3.4. Enzymatic deacylation of 2 and 4 with tannase
A solution of 2 (4.8 mg, 0.008 mmol) in H2O (1.0 mL) was treated with tannase (3.7 mg, from Aspergillus oryzae, Wako Pure Chemical Ind., Ltd, Osaka, Japan) and the solution was stirred at 37℃ for 24 h. After EtOH was added to the reaction mixture, the solvent was removed under reduced pressure and the residue was purified by HPLC [MeOH/H2O (35:65, v/v)] to furnish cistanoside G (12, 2.8 mg, 80%).16 Through a similar procedure, a solution of 4 (9.2 mg, 0.012 mmol) in H2O (2.0 mL) was treated with tannase (7.5 mg) and the solution was stirred at 37 ℃ for 24 h. Work-up of the reaction mixture as described above gave a residue, which was purified by HPLC [MeOH/H2O (15:85, v/v)] to furnish 1 (4.7 mg, 62%).
3.5. Alkaline and acid hydrolysis of 2 and 3
A solution of 2 and 3 (each 3.5 mg) in 50% aqueous 1,4- dioxane (1.0 mL) was treated with 10% aqueous KOH (1.0 mL) and the whole was stirred at 37 C for 1 h, respectively. Removal of the solvent under reduced pressure gave a reaction mixture, which was subjected to HPLC analysis [column: YMC-Pack ODS-A, 250 · 4.6 mm id; mobile phase: MeOH/H2O (30:70, v/ v); detection: UV (254 nm); flow rate: 1.0 mL/min] to identify caffeic acid (tR: 13.1 min). Then the reaction mixture from 3 was dissolved in 1 M HCl (2.0 mL) and heated under reflux for 3 h. After cooling, the reaction mixture was extracted with EtOAc. The aqueous layer was subjected to HPLC analysis under the following conditions, respectively: HPLC column, Kaseisorb LC NH2-60-5, 250 · 4.6 mm id (Tokyo Kasei Co., Ltd, Tokyo, Japan); detection, optical rotation [Shodex OR-2 (Showa Denko Co., Ltd, Tokyo, Japan); mobile phase, CH3CN/H2O (85:15, v/v); flow rate 0.8 mL/ min]. Identification of L-rhamnose and D-glucose present in the aqueous layer was carried out by comparison of their retention time and optical rotation with those of authentic samples, tR: 9.9 min (L-rhamnose, negative optical rotation) and 17.9 min (D-glucose, positive optical rotation).
3.6. Vasorelaxant activity
3.6.1. Tissue preparation.
Male Sprague–Dawley rats weighing 250–350 g were sacrificed by severing both carotid arteries under anesthesia, and the thoracic aorta was isolated and cut into helical strips (2–3 mm · 15–20 mm). Physiological salt solution contained NaCl (118.0 mM), KCl (4.7 mM), KH2PO4 (1.2 mM), MgSO4 (1.2 mM), CaCl2 (2.5 mM), NaHCO3 (25.0 mM), and D-glucose (10.0 mM). The solution was aerated with a 95% O2–5% CO2 gas mixture and kept at 37℃. To investigate the mechanical response, each preparation was suspended in an organ bath (6 ml) and subjected to an initial load of about 1 g. One hour equilibration period was allowed before initiation of the experiments. Contractions were measured isometrically via a force-displacement transducer (AD Instruments) and recorded on a polygraph.
3.6.2. Inhibitory effect on the contraction induced by dl-noradrenaline or high K+.
After equilibration, dl-nor-adrenaline (final concentration: 1 lM) or 3 M KCl (0.1 mL, final concentration of K+: 54 mM) was added to the bath. The tissues were washed three times and re-equilibrated after the contraction had reached the maximum level. The sustained contraction was induced again by the addition of dl-noradrenaline or KCl, and then the test compound was applied at 10–100 lM (noradrenaline) or 100 lM (high K+). The contractile response prior to the application of the test sample was taken to be 100%. Prazosin hydrochloride and nifedipine were used as reference compounds.
3.6.3. Statistical analysis.
Values are expressed as means ± SEM. For statistical analysis, a one-way analysis of variance followed by Dunnett’s test for multiple comparison analysis was used. Probability (p) values less than 0.05 were considered significant.
Acknowledgments
This research was supported by the 21st COE Program, Academic Frontier Project, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References and notes
1. This paper is number 19 in the series ‘Bioactive Constituents from Chinese Natural Medicines.’ For paper number 18, see Xie, H.; Morikawa, T.; Matsuda, H.; Nakamura, S.; Muraoka, O.; Yoshikawa, M. Chem. Pharm. Bull. 2006, 54, 669–675.
2. Namba, T. In The Encyclopedia of Wakan-Yaku (Traditional Sino-Japanese Medicines) with Color Pictures; Hokuryusha: Osaka, 1994; Vol II.
3. Kobayashi, H.; Oguchi, H.; Takizawa, N.; Miyase, T.; Ueno, A.; Usmanghani, K.; Ahmad, M. Chem. Pharm. Bull. 1987, 35, 3309–3314.
4. Matsuda, H.; Morikawa, T.; Tao, J.; Ueda, K.; Yoshikawa, M. Chem. Pharm. Bull. 2002, 50, 208–215.
5. Morikawa, T.; Matsuda, H.; Toguchida, I.; Ueda, K.; Yoshikawa, M. J. Nat. Prod. 2002, 65, 1468–1474.
6. Tao, J.; Morikawa, T.; Toguchida, I.; Ando, S.; Matsuda, H.; Yoshikawa, M. Bioorg. Med. Chem. 2002, 10, 4005–4012.
7. Morikawa, T.; Tao, J.; Ando, S.; Matsuda, H.; Yoshikawa, M. J. Nat. Prod. 2003, 66, 638–645.
8. Tao, J.; Morikawa, T.; Ando, S.; Matsuda, H.; Yoshikawa, M. Chem. Pharm. Bull. 2003, 51, 654–662.
9. Matsuda, H.; Morikawa, T.; Xie, H.; Yoshikawa, M. Planta Med. 2004, 70, 847–855.
10. Sun, B.; Morikawa, T.; Matsuda, H.; Tewtrakul, H.; Wu, L. J.; Harima, S.; Yoshikawa, M. J. Nat. Prod. 2004, 67, 1464–1469.
11. Morikawa, T.; Sun, B.; Matsuda, H.; Wu, L. J.; Harima, S.; Yoshikawa, M. Chem. Pharm. Bull. 2004, 52, 1194–1199.
12. Xie, H.; Wang, T.; Matsuda, H.; Morikawa, T.; Yoshikawa, M.; Tani, T. Chem. Pharm. Bull. 2005, 53, 1416–1422.
13. Morikawa, T.; Xie, H.; Matsuda, H.; Yoshikawa, M. J. Nat. Prod. 2006, 69, 881–886.
14. Morikawa, T.; Xie, H.; Matsuda, H.; Wang, T.; Yoshikawa, M. Chem. Pharm. Bull. 2006, 54, 506–513.
15. Kobayashi, H.; Karasawa, H.; Miyase, T.; Fukushima, S. Chem. Pharm. Bull. 1985, 33, 1452–1457.
16. Karasawa, H.; Kobayashi, H.; Takizawa, N.; Miyase, T.; Fukushima, S. Yakugaku Zasshi 1986, 106, 721–724.
17. Kitajima, J.; Ishikawa, T.; Tanaka, Y. Chem. Pharm. Bull. 1998, 46, 1643–1646.
18. Chiba, M.; Hisada, S.; Nishibe, S.; Thieme, H. Phytochemistry 1980, 19, 335–336.
19. The 1H and 13C NMR spectra of 1–3 were assigned with the aid of distortionless enhancement by polarization transfer (DEPT), double quantum filter correlation spectroscopy (DQF COSY), and heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond connectivity (HMBC) experiments.
20. Iizuka, T.; Nagai, M. Yakugaku Zasshi 2005, 125, 219–224.
