Bioactive Constituents From Chinese Natural Medicines. XXXVI.1) Four New Acylated Phenylethanoid Oligoglycosides, Kankanosides J1, J2, K1, And K2, From Stems Of Cistanche Tubulosa

Yingni PAN, Toshio MORIKAWA, Kiyofumi NINOMIYA, Katsuya IMURA, Dan YUAN, Masayuki YOSHIKAWA, and Osamu MURAOKA

Pharmaceutical Research and Technology Institute, Kinki University; 3–4–1 Kowakae, Higashi-Osaka, Osaka 577–8502, Japan: School of Traditional Chinese Medicine, Shenyang Pharmaceutical University; 103 Wenhua Rd., Shenyang 110016, People’s Republic of China: and Kyoto Pharmaceutical University; Misasagi, Yamashina-Ku, Kyoto 607–8412, Japan. Received November 28, 2009; accepted February 2, 2010; published online February 4, 2010

Contact: joanna.jia@wecistanche.com

Abstract

Four new acylated phenylethanoid aminoglycosides, kankanosides J1 (1), J2 (2), K1 (3), and K2 (4), were isolated from stems of Cistanche tubulosa (Orobanchaceae) together with isocampneoside I (5). Their structures were elucidated based on chemical and physicochemical evidence. Among them, 3—5 were found to inhibit D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes.

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Keywords: Cistanche tubulosa; Lankan side; phenylethanoid glycoside; Orobanchaceae; hepatoprotective activity.

During our studies on bioactive constituents from Chinese natural medicines,1—3) we found that methanolic extract of dried stems of Cistanche tubulosa (SCHRENK) R. WIGHT (Orobanchaceae) showed vasorelaxant4) and hepatoprotective activities.1) From the dried stems of C. tubulosa, five iridoids, kankanosides A—D and kankanol, a monoterpene glycoside, kankanoside E, two phenylethanoid aminoglycosides, kankanosides F and G, and an acylated oligo sugar, kankanose, were isolated together with 30 known constituents.4,5) Recently, we additionally isolated 19 phenylethanoid aminoglycosides including kankanosides H1, H2, and I 1) and two acylated oligosugars from fresh stems of C. tubulosa. 1) Furthermore, principal phenylethanoid glycosides, echinacoside, acteoside, and isoacteoside, were found to inhibit the increase in serum aspartate aminotransferase (sAST) and alanine aminotransferase (sALT) levels in liver injured mice induced by D-galactosamine (D-GalN)/lipopolysaccharide at doses of 25—100 mg/kg per os (p.o.), and structural requirements of phenylethanoid glycosides for the hepatoprotective activity were elucidated.1) As a continuing study on constituents from the fresh stems of C. tubulosa, we further isolated four new acylated phenylethanoid oligoglycosides, kankanosides J1 (1), J2 (2), K1 (3), and K2 (4). This paper deals with isolation and structure elucidation of 1—4.

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 was described previously.1) By the intensive chromatographies on the MeOH-eluted fraction, four new phenylethanoid oligoglycosides, kankanosides J1 (1, 0.0002%), J2 (2, 0.0002%), K1 (3, 0.0002%), and K2 (4, 0.0005%) together with isocampneoside I6) (5, 0.0006%) were isolated.

Structures of Kankanosides J1 (1) and J2 (2) Kankanoside J1 (1) was obtained as a white powder with negative optical rotation ([a]D 25 - 6.5° in MeOH). The IR spectrum of 1 showed absorption bands at 3414, 1734, 1719, 1701, 1638, 1508, 1159, 1067, and 1046 cm- 1 ascribable to hydroxyls, ester carbonyls, ether functions, and aromatic rings. The positive- and negative-ion FAB-MS spectra of 1 showed quasimolecular ion peaks at m/z 719 (M- Na) and m/z 695 (M H) , and the molecular formula was determined as C32H40O17 by high-resolution positive-ion FAB-MS measurement. The 1 H- and 13C-NMR spectra of 1 (CD3OD, Tables 1, 2), which were assigned by various NMR experiments,7) showed signals assignable to a methoxy group [d 3.21 (3H, s, 7-OCH3)], a methylene and a methine bearing an oxygen function [d 3.58, 4.00 (1H each, both m, 8-H2), 4.18 (1H, dd-like, J ca. 4, 8 Hz, 7-H)], ortho- and meta-coupled ABC-type aromatic protons [d 6.63 (1H, dd, J 1.8, 8.2 Hz, 6-H), 6.74 (1H, d, J 1.8 Hz, 2-H), 6.74 (1H, d, J 8.2 Hz, 5- H)], a b-D-glucopyranosyl moiety [d 4.54 (1H, d, J 7.8 Hz, Glc-1-H)], and an a-L-rhamnopyranosyl moiety [d 1.07 (3H, d, J 6.4 Hz, Rha-6-H3), 4.80 (1H, br s, Rha-1-H)] together with an acetyl group [d 2.00 (3H, s)] and a trans-caffeoyl group {an trans-olefifin [d 6.26, 7.59 (1H each, both d, J- 16.0 Hz, 8-, 7-H)] and ortho- and meta-coupled ABC-type aromatic protons [d 6.77 (1H, d, J 8.2 Hz, 5-H), 6.95 (1H, dd, J- 1.8, 8.2 Hz, 6-H), 7.04 (1H, d, J 1.8 Hz, 2-H)]}. The 1 H- and 13C-NMR spectra of 1 were superimposable on those of campneoside I1,8—10) (6), except for the signals due to the acetyl group. Connectivities of the aminoglycoside and acyl moieties in 1 were confirmed by the heteronuclear multiple bond correlation (HMBC) experiment, which showed long-range correlations between the following proton and carbon pairs: 7-OCH3 and 7-C (dC 83.3); Glc-1-H and 8-C (dC 74.1); Glc-2-H [d 4.91 (1H, dd, J- 7.8, 9.2 Hz)] and the acetyl carbonyl carbon (dC 171.4); Glc-4-H [d 4.99 (1H, dd, J 9.2, 9.6 Hz)] and the trans-caffeoyl carbonyl carbon (dC 168.1); and Rha-1-H and Glc-3-C (dC 80.5) (Fig. 1). Finally, alkaline hydrolysis of 1 with 5% potassium hydroxide (KOH) liberated trans-caffeic acid, which was identified by HPLC analysis, together with a deacylated product. The deacylated product was successively treated with 1.0 M hydrochloric acid (HCl) to liberate L-rhamnose and D-glucose, which were identified by HPLC analysis using an optical rotation detector.1—5) Thus, the structure of kankanoside J1 was elucidated to be 2-methoxy-2-(3,4-dihydroxy phenyl)ethyl O-a-L-rhamnopyranosyl-(1→3)-2-O-acetyl-4-O-trans-caffeoyl-b-D-glucopyranoside (1).

Kankanoside J2 (2) was isolated as a white powder with negative optical rotation ([a]D 25 - 18.1° in MeOH). By high-resolution, positive-ion FAB-MS measurement, the molecular formula of 2 was found to be the same as that of 1. The 1 H- and 13C-NMR data of 2 (CD3OD, Tables 1, 2) were very similar to those of 1, except for the signals due to the ethyl bridge of the aglycone moiety {a methoxy group [d 3.24 (3H, s, 7-OCH3)], a methylene [d 3.63 (1H, m), 3.83 (1H, dd, J- 3.2, 11.0 Hz), 8-H2] and a methine bearing an oxygen function [d 4.22 (1H, dd, J- 3.2, 8.2 Hz, 7-H)]}. Alkaline hydrolysis of 2 with 5% KOH liberated trans-caffeic acid together with a deacylated product, and the deacylated product was successively treated with 1.0 M HCl to liberate L-rhamnose and D-glucose. As shown in Fig. 1, the same long-range correlations as in the case of 1 were observed in the HMBC experiment. Consequently, the planar structure of kankanoside J2 (2) was revealed to be the same as that of 1, and was elucidated to be 7-isomer of 1. 11)

Structures of Kankanosides K1 (3) and K2 (4) Kankanosides K1 (3) and K2 (4), C36H48O21, were also obtained as white powders with negative optical rotations (3: [a]D 25 - 75.3°; 4: [a]D 25 - 7.4° both in MeOH). The 1 H- and 13C-NMR spectra of 3 and 4 (CD3OD, Tables 1, 2) showed signals assignable to a methoxy group [3: d 3.23 (3H, s, 7- OCH3); 4: d 3.25 (3H, s, 7-OCH3)], a methylene and methine bearing an oxygen function {3: d [3.62 (1H, dd, J- 3.4,11.0 Hz), 4.02 (1H, dd, J- 8.1, 11.0 Hz), 8-H2], 4.34 (1H, dd, J- 3.4, 8.1 Hz, 7-H); 4: d [3.72 (1H, dd, J- 9.1, 11.0 Hz), 3.84 (1H, dd, J- 3.1, 11.0 Hz), 8-H2], 4.37 (1H, dd, J- 3.1, 9.1 Hz, 7-H)}, ortho- and meta-coupled ABC-type aromatic protons [3: d 6.68 (1H, dd, J- 2.0, 8.1 Hz, 6-H), 6.76 (1H, d, J- 8.1 Hz, 5-H), 6.80 (1H, d, J- 2.0 Hz, 2-H); 4: d 6.67 (1H, dd, J- 1.9, 8.2 Hz, 6-H), 6.76 (1H, d, J- 8.2 Hz, 5-H), 6.78 (1H, d, J- 1.9 Hz, 2-H)], two b-D-glucopyranosyl moieties [3: d 4.31 (1H, d, J- 7.9 Hz, terminal-Glc-1-H), 4.40 (1H, d, J- 7.9 Hz, inner-Glc-1-H); 4: d 4.26 (1H, d, J- 7.7 Hz, terminal-Glc-1-H), 4.44 (1H, d, J- 7.9 Hz, inner-Glc-1-H)], and an a-L-rhamnopyranosyl moiety [3: d 1.08 (3H, d, J 6.4 Hz, Rha-6-H3), 5.19 (1H, d, J- 1.7 Hz, Rha-1-H); 4: d 1.08 (3H, d, J- 6.2 Hz, Rha-6-H3), 5.20 (1H, d, J- 1.6 Hz, Rha-1-H)] together with a trans-caffeoyl group {an trans-olefifin [3: d 6.27, 7.60 (1H each, both d, J- 15.8 Hz, 8-, 7-H); 4: d 6.28, 7.60 (1H each, both d, J- 15.8 Hz, 8-, 7-H)] and ortho- and meta-coupled ABC-type aromatic protons [3: d 6.78 (1H, d, J- 8.3 Hz, 5-H), 6.96 (1H, dd, J- 1.9, 8.3 Hz, 6-H), 7.05 (1H, d, J- 1.9 Hz, 2-H)]; 4: d 6.78 (1H, d, J- 8.4 Hz, 5-H), 6.96 (1H, dd, J- 1.9, 8.4 Hz, 6-H), 7.05 (1H, d, J- 1.9 Hz, 2-H)}. The proton and carbon signals in the 1 H- and 13C-NMR spectra of 3 and 4 were superimposable on those of echinacoside,1,4,12) except for the signals due to the 7-methoxy group. The connectivities of the trans-caffeoyl group and the glycosyl moieties in 3 and 4 were elucidated on the basis of HMBC experiments as shown in Fig. 1. Finally, alkaline hydrolysis of 3 and 4 with 5% KOH gave the deacylated products together with trans-caffeic acid. Those deacylated products were successively treated with 1.0 M HCl to liberate Lrhamnose and D-glucose, respectively. Consequently, the structure of kankanosides K1 and K2 were determined to be 2-methoxy-2-(3,4-dihydroxy phenyl)ethyl O-a-L-rhamnopy-ranosyl-(1→3)-[b-D-glucopyranosyl-(1→6)]-4-O-trans-caf-feel-b-D-glucopyranoside (3 and 4).11,13)

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Previously, methanolic extract from stems of C. tubulosa and several phenylethanoid constituents such as echinacoside, acteoside, and isoacteoside were found to show hepatoprotective effects on D-galactosamine (D-GalN)/lipopolysaccharide-induced liver injury in mice and inhibitory effect on D-GalN-induced cytotoxicity in primary cultured mouse hepatocytes.1) We further examined inhibitory effects of kankanosides K1 (3) and K2 (4), and isocampneoside I (5) on D-GalN-induced cytotoxicity in primary cultured hepatocytes. Although their activities were weaker than those of echinacoside (IC50 10.2 mM), acteoside (4.6 mM), and isoacteoside (5.3 mM), the principle phenylethanoid constituents from stems of C. tubulosa, 1) 3—5 showed moderate activity.14)

Experimental

The following instruments were used to obtain spectral and 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- and 13C-NMR spectra, JEOL JNM-ECA600 (600, 150 MHz) and JEOL JNM-ECS400 (400, 100 MHz) spectrometers with tetramethylsilane as an internal standard; FAB-MS and high-resolution FAB-MS, JEOL JMS-SX 102A mass spectrometer; HPLC detector, Shimadzu RID-10A refractive index, Shimadzu SPD-10A UV–VIS, and Shodex OR-2 optical rotation detectors. HPLC column, Cosmosil 5C18-MS-II, and pNAP (Nacalai Tesque Inc., 250 4.6 mm i.d.) and (250 20 mm i.d.) columns were used for analytical and preparative purposes, respectively.

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The following experimental conditions were used for chromatography: normal-phase silica gel column chromatography (CC), silica gel 60N (Kanto Chemical Co., Ltd., 63—210 mesh, spherical, neutral); reversed-phase silica gel CC, Diaion HP-20 (Nippon Rensui), and Chromatorex ODS DM1020T (Fuji Silesia Chemical, Ltd., 100—200 mesh); normal-phase TLC, pre-coated TLC plates with silica gel 60F254 (Merck, 0.25 mm); reversed-phase TLC, pre-coated TLC plates with silica gel RP-18 F254S (Merck, 0.25 mm); reversed-phase HPTLC, pre-coated TLC plates with silica gel RP-18 WF254S (Merck, 0.25 mm), the detection was achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4, followed by heating.

Plant Material

This item was described in a previous report.1)

Extraction and Isolation

Fresh stems of C. tubulosa (2.98 kg) were fifinely cut and extracted three times with methanol under reflflux for 3 h. Evaporation of the solvent under reduced pressure provided a methanolic extract (249.1 g, 8.36%). The methanolic extract was subjected to Diaion HP- 20 CC (5.0 kg, H2O→MeOH) to give H2O- and MeOH-eluted fractions (167.84 g, 5.63% and 81.21 g, 2.73%, respectively). The MeOH-eluted fraction (61.00 g) was subjected to normal-phase silica gel CC [1.8 kg, CHCl3– MeOH–H2O (15 : 3 : 0.4→10 : 3 : 0.5→6 : 4 : 1, v/v/v)→MeOH] to give seven fractions [Fr. 1 (1.12 g), 2 (9.56 g), 3 (0.89 g), 4 (10.69 g), 5 (8.84 g), 6 (12.52 g), and 7 (4.60 g)], as was described previously.1) The fraction 4 (10.69 g) was separated by reversed-phase silica gel CC [500 g, MeOH–H2O (30 : 70, v/v)→MeOH→acetone] to give four fractions [Fr. 4-1 (878.2 mg), 4-2 (7.06 g), 4-3 (1.57 g), and 4-4 (792.8 mg)]. The fraction 4-3 (1.57 g) was purifified by HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (20 : 80, v/v)] to give 11 fractions {Fr. 4-3-1 (30.4 mg), 4-3-2 (55.2 mg), 4-3- 3 [ campneoside I (6, 22.1 mg, 0.0010%)], 4-3-4 [ acteoside (224.6 mg, 0.010%)], 4-3-5 (27.4 mg), 4-3-6 (43.6 mg), 4-3-7 [ isoacteoside (825.0 mg, 0.037%)], 4-3-8 [ syringalide A 3 -O-a-L-rhamnopyranoside (37.6 mg, 0.0017%)], 4-3-9 (39.8 mg), 4-3-10 [ 2 -acetylacteoside (85.4 mg, 0.0038%)], and 4-3-11 (64.6 mg)}, as was described previously.1) The fraction 4-3-5 (27.4 mg) was further purifified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (18 : 82, v/v)] to give isocampneoside I (5, 8.5 mg, 0.0004%). The fraction 4-3-6 (43.6 mg) was further purifified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (18 : 82, v/v)] to give 5 (3.7 mg, 0.0002%) together with kankanosides H1 1) (17.0 mg, 0.0008%) and H2 1) (3.3 mg, 0.0001%). The fraction 4-3-9 (39.8 mg) was further puri- fified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (18 : 82, v/v)] to give kankanosides J1 (1, 3.7 mg, 0.0002%) and J2 (2, 3.5 mg, 0.0002%) together with kankanoside I1) (15.4 mg, 0.0007%) and isoacteoside1) (3.1 mg, 0.0001%). The fraction 5 (8.84 g) was separated by reversed-phase silica gel CC [400 g, MeOH–H2O (20 : 80→30 : 70, v/v)→MeOH→acetone] to give seven fractions [Fr. 5-1 (870.2 mg), 5-2 (478.9 mg), 5-3 (3.72 g), 5-4 (979.9 mg), 5-5 (1.19 g), 5-6 (1.27 g), and 5-7 (130.1 mg)]. The fraction 5-3- 4 (72.3 mg) was further purifified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (10 : 90, v/v)] to give kankanosides K1 (3, 5.1 mg, 0.0002%) and K2 (4, 10.6 mg, 0.0005%) together with campneoside II1) (7, 10.6 mg, 0.0005%).

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Alkaline and Acid Hydrolysis of Kankanosides J1 (1), J2 (2), K1 (3), and K2 (4)

Solutions of 1—4 (each 1.0 mg) in 5% aqueous potassium hydroxide (KOH, 0.5 ml) was stirred at 40 °C for 1 h. Each solution was neutralized with Dowex HCR W2 (H- form), and the resin was removed by filtration. Evaporation of the solvent from the filtrates under reduced pressure yielded the corresponding deacylated products, which were subjected to HPLC analysis [column: Cosmosil pNAP, 250-4.6 mm i.d.; mobile phase: CH3CN–1% aqueous AcOH (15: 85, v/v); detection: UV (254 nm); fellow rate: 1.0 ml/min] to give trans-caffeic acid (tR 9.9 min from 1—4). Then each was dissolved in 1.0 M HCl (1.0 ml) and heated at 80 °C for 3 h. After being cooled, the reaction mixture was neutralized with Amberlite IRA-400 (OH- form), and the resins were removed by filtration. After removal of the solvent under reduced pressure, the residue was separated by the Sep-Pak C18 cartridge column (H2O→MeOH). The H2O-eluted fraction was subjected to HPLC analysis under the following conditions: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d.- 250 mm (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); fellow rate 0.8 ml/min]. Identification of L-rhamnose (i) and D-glucose (ii) from 1—4 present in the H2O-eluted fractions were carried out by comparison of their retention times and optical rotation with those of authentic samples [i, tR 9.9 min (negative)] and [ii, tR 17.9 min (positive)].

Acknowledgments

T. M., K. N., and O. M. were supported by ‘Hightech Research Center’ Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, 2007—2011 and also supported by a Grant-in-Aid for Scientific Research from MEXT. M. Y. and H. M. were supported by the 21st COE Program, Academic Frontier Project, and a Grant-in-Aid for Scientific Research from MEXT.

References and Notes

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6) Si C.-L., Liu Z., Kim J.-K., Bae Y.-S., Holzforschung, 62, 197—200 (2008).

7) The 1 H- and 13C-NMR spectra of 1—4 were assigned with the aid of distortionless enhancement by polarization transfer (DEPT), double quantum filter correlation spectroscopy (DQF COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond correlation (HMBC) experiments.

8) Imakura Y., Kobayashi S., Mima A., Phytochemistry, 24, 139—146 (1985).

9) Wu J., Huang J., Xiao Q., Zhang S., Xiao Z., Li Q., Long L., Huang L., Magn. Res. Chem., 42, 659—662 (2004).

10) Kitagawa S., Tsukamoto H., Hisada S., Nishibe S., Chem. Pharm. Bull., 32, 1209—1213 (1984).

11) Stereochemistries of 7-position in 1—4 have not been determined.

12) Kobayashi H., Oguchi H., Takizawa N., Miyase T., Ueno A., Usmanghani K., Ahmad M., Chem. Pharm. Bull., 35, 3309—3314 (1987).