Acylated Phenylethanoid Oligoglycosides With Hepatoprotective Activity From The Desert Plant Cistanche Tubulosa

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Toshio Morikawa et al

Abstract

The methanolic extract from fresh stems of Cistanche tubulosa (Orobanchaceae) was found to show hepatoprotective effects against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. From the extract, three new phenylethanoid oligoglycosides, kankanosides H1 (1), H2 (2), and I (3), were isolated together with 16 phenylethanoid glycosides (4–19) and two acylated oligosugars (20, 21). The structures of 1–3 were determined on the basis of spectroscopic properties as well as chemical evidence. Among the isolates, echinacoside (4, IC50 = 10.2 lM), acteoside (5, 4.6 lM), isoacteoside (6, 5.3 lM), 20 -acetylacteoside (8, 4.8 lM), and tubuloside A (10, 8.6 lM) inhibited D-GalN-induced death of hepatocytes. These five isolates, 4 (31.1 lM), 5 (17.8 lM), 6 (22.7 lM), 8 (25.7 lM), and 10 (23.2 lM), and cistantubuloside B1 (11, 21.4 lM) also reduced TNF-a-induced cytotoxicity in L929 cells. Moreover, principal constituents (4–6) exhibited in vivo hepatoprotective effects at doses of 25–100 mg/kg, PO.

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1. Introduction

Cistanche tubulosa (SCHRENK) R. WIGHT (Orobanchaceae) is a perennial parasitic plant growing on roots of Salvadora or Calotropis species, and distributed in North Africa, Arabia, and Asian countries.2 Stems of Cistanche tubulosa as well assistance salsa andCistanche deserticola have traditionally been used for the treatment of impotence, sterility, lumbago, and body weakness as well as a promoting agent of blood circulation.2,3 Previously, several phenylethanoids, iridoids, monoterpenes, and lignans were isolated from Chinese and PakistanCistanche tubulosa. 2,4–9 In the course of our characterization studies on bioactive constituents in this natural medicine, we previously reported that five iridoids, kankanosides A–D and kankanol, a monoterpene glycoside, kankanoside E, two phenylethanoid oligoglycosides, kankanosides F and G (23), and an acylated oligo sugar, kankanose (21), were isolated from the methanolic extract of dried stems ofCistanche tubulosa. 10,11 In addition, the methanolic extract and several isolates such as echinacoside (4), acteoside (5), cistanoside F (20), 21, and kankanoside F were found to show vasorelaxant effects, that is, inhibitory effects against contractions induced by noradrenaline in isolated rat thoracic aorta.11 In our continuing studies on constituents in the plant, a methanolic extract of fresh stems of Cistanche tubules was found to show a protective effect on liver injury induced by D-galactosamine (D-GalN)/lipopolysaccharide (LPS) in mice. From the methanolic extract, we have isolated three new phenylethanoid oligoglycosides named kankanosides H1 (1), H2 (2), and I (3) together with 16 phenylethanoid glycosides (4–19) and two acylated oligosugars (20, 21). This paper deals with the isolation and structural elucidation of these new phenylethanoid oligoglycosides (1–3). Structural requirements of the phenylethanoid glycosides for the hepatoprotective activity are also discussed (Fig. 1)

2. Results and discussion

2.1. Protective effect of methanolic extract from stems ofCistanche tubulosa on liver injury induced by D-GalN/LPS in mice

Fresh stems of Cistanche tubulosa (cultivated in Urumqi, Xinjiang Province, China) were extracted with methanol under reflux to yield a methanolic extract (8.36% from the fresh stems). As shown in Table 1, at doses of 250–500 mg/kg, PO, the methanolic extract showed an inhibitory effect on the increase in serum aspartate aminotransaminase (sAST) and alanine aminotransaminase (sALT), markers of liver injury, induced by D-GalN/LPS in mice.

2.2. Chemical constituents from stems of Cistanche tubulosa

A methanolic extract from the fresh stems of Cistanche tubulosa was subjected to Diaion HP-20 column chromatography (H2O?MeOH) to give H2O- and MeOH-eluted fractions (5.63% and 2.73%, respectively). The MeOH-eluted fraction was subjected to SiO2 and ODS column chromatographies and finally HPLC to furnish three newphenylethanoid oligoglycosides, kankanosides H1 (1, 0.0008%), H2 (2, 0.0001%), and I (3, 0.0007%) together with 16 phenylethanoid glycosides, echinacoside2,11 (4, 0.45%), acteoside2,11 (5, 0.28%), isoacteoside2,11 (6, 0.041%), cis-acteoside12,13 (7, 0.0007%), 20 -acetylac teoside2,11 (8, 0.0038%), decaffeoylacteoside14 (9, 0.0002%), tubuloside A2,11 (10, 0.013%), cistantubulosides B1 9 (11, 0.0020%), B2 9 (12, 0.0003%), arenarioside15 (13, 0.0006%), wiedemanninoside C16 (14, 0.0008%), cistantubuloside A9 (15, 0.0009%), syringalide A 30 -O-a-L rhamnopyranoside4 (16, 0.0017%), campneosides I17,18 (17, 0.015%) and II17,18 (18, 0.0005%), and salidroside11 (19, 0.0027%), and two acylated oligosugars, cistanoside F11 (20, 0.0004%) and kankanose11 (21, 0.0004%).

2.3. Structures of kankanosides H1 (1), H2 (2), and I (3)

Kankanoside H1 (1) was obtained as a white powder with negative optical rotation (½a-24 D-45.3 in MeOH). Its IR spectrum showed absorption bands at 3416, 1736, 1638, 1605, 1518, 1070, and 1040 cm1 ascribable to hydroxyls, ester carbonyls, ether functions, and aromatic rings. The positive- and negative-ion FABMS spectra of 1 showed quasimolecular ion peaks at m/z 835 (M+Na)+ and m/z 811 (MH), respectively, and the molecular formula was determined as C37H48O20 by high-resolution positive-ion FABMS measurement. The 1 H and 13C NMR spectra of 1 (CD3OD, Tables 2 and 3), which were assigned by various NMR experiments,19 showed signals assignable to two methylenes [d 2.70 (2H, m, H2-7), 3.66, 4.07 (1H each, both m, H2-8)], ortho- and meta-coupled ABC-type aromatic protons [d 6.52 (1H, dd, J = 1.8, 7.8 Hz, H-6), 6.65 (1H, d, J = 1.8 Hz, H-2), 6.67 (1H, d, J = 7.8 Hz, H-5)], two b-D-glucopyranosyl moieties [d 4.29 (1H, d, J = 7.8 Hz, terminal-Glc-H-1), 4.54 (1H, d, J = 8.2 Hz, inner-Glc-H-1)], and a a-L-rhamnopyranosyl moiety [d 1.06 (3H, d, J = 6.0 Hz, Rha-H3-6), 4.80 (1H, br s, Rha-H-1)] together with an acetyl group [d 1.98 (3H, s)] and a trans-p-coumaroyl group {an trans-olefin [d 6.34, 7.67 (1H each, both d, J = 16.0 Hz, H-8 and 7)] and ortho-coupled A2B2-type aromatic protons [d 6.80, 7.46 (2H each, both d, J = 8.7 Hz, H-3,5 and 2,6)]}. Connectivities between the oligoglycoside and acyl moieties in 1 were characterized by an HMBC experiment, which showed long-range correlations between the following proton and carbon pairs: inner-Glc-H-1 and C-8 (dC 71.9); inner-Glc-H-2 [d 4.88 (1H, dd-like)] and the acetyl carbonyl carbon (dC 171.4); inner-Glc-H-4 [d 5.08 (1H, dd, J = 9.6, 9.6 Hz)] and the p-coumaroyl carbonyl carbon (dC 168.2); Rha-H-1 and inner-Glc-C-3 (dC 80.5); and terminal-Glc-H-1 and inner-Glc-C-6 (dC 69.3) (Fig. 2). Finally, alkaline hydrolysis of 1 with 5% potassium hydroxide (KOH) liberated trans-p-coumaric 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.10,11 Thus, the structure of kankanoside H1 was elucidated to be 2-(3,4-dihydroxy phenyl)ethyl O-a-L-rhamnopyranosyl-(1- 3)- [b-D-glucopyranosyl-(1-6)]-2-O-acetyl-4-O-trans-p-coumaroyl-b-D-glucopyranoside (1).

Kankanoside H2 (2) was isolated as a white powder with negative optical rotation (½a 25 D 52.4 in MeOH). Its molecular formula C37H48O20 was found to be the same as that of 1 by high-resolution positive-ion FABMS measurement. The spectroscopic properties of 2 were very similar to those of 1, except for signals due to the cis-p-coumaroyl group [d 5.79, 6.95 (1H each, both d, J = 12.8 Hz, H-8 and 7), 6.76, 7.73 (2H each, both d, J = 8.7 Hz, H-3,5 and 2,6)]. Furthermore, HMBC experiment on 2 also revealed similar modes of long-range correlations as those detected for 1 between the following proton and carbon pairs: inner-Glc-H-1 [d 4.51 (1H, d, J = 7.8 Hz)] and C-8 (dC 72.0); inner-Glc-H-2 [d 4.88 (1H, m)] and the acetyl carbonyl carbon (dC 171.4); inner-Glc-H-4 [d 4.98 (1H, dd, J = 9.6, 9.7 Hz)] and the p-coumaroyl carbonyl carbon (dC 166.7); Rha-H-1 [d 4.77 (1H, d, J = 1.4 Hz, Rha-H-1)] and inner-Glc-C-3 (dC 80.7); and terminal-Glc-H-1 [d 4.28 (1H, d, J = 7.8 Hz)] and inner-Glc-C-6 (dC 69.4) (Fig. 2). By the degradation study, 2 gave cis-p-coumaric acid and two same sugars as those obtained in the case of 1. Consequently, the structure of kankanoside H2 was elucidated to be 2-(3,4-dihydroxy phenyl)ethyl O-a-L-rhamnopyranosyl-(1-3)- [b-D-glucopyranosyl-(1-6)]-2-O-acetyl-4-O-cis-p-coumaroyl-b-D-glucopyranoside (2).

Kankanoside I (3) was also isolated as a white powder with negative optical rotation (½a- 25 D-62.2 in MeOH). Its molecular formula, C35H46O18, was determined by positive- and negative-FABMS and HRFABMS measurements. The spectroscopic properties of 3 (CD3OD, Tables 2 and 3) were superimposable on those of echinacoside (4), except for signals due to the aglycone part {{two methylenes {d 2.95 (2H, dd, J = 7.3, 7.8 Hz, H2-7), [3.79 (1H, m), 4.10 (1H, dt, J = 16.9, 7.3 Hz), H2-8]}, monosubstituted aromatic protons [d 7.18 (1H, m, H-4), 7.26–7.28 (4H, m, H-2,6, 3,5)]}}. Alkaline hydrolysis of 3 with 5% KOH liberated trans-caffeic acid, and the deacylated product was successively treated with 1.0 M HCl to liberate L-rhamnose and D-glucose. Finally, connectivities of the acyl group and the sugar moieties in 3 were clarified by the HMBC experiment, as shown in Figure 2. On the basis of the above-mentioned evidence, the structure of kankanoside I was elucidated to be 2-phenylethyl O-a-L-rhamnopyranosyl-(1-3)-[b-D-glucopyrano-syl-(1-6)]-4-O-trans-caffeoyl-b-D-glucopyranoside (3)

2.4. Effects of chemical constituents from the stems of Cistanche tubulosa on D-GalN-induced cytotoxicity in primary cultured mouse hepatocytes

D-GalN/LPS-induced liver injury is recognized to develop via immunological responses.19 This type of liver injury is reported to occur in two forms. First, sensitivity to TNF-a by depletion of uridine triphosphate in hepatocytes is increased by D-GalN. Second, pro-inflammatory mediators, such as NO and TNF-a, are released from LPS-activated macrophages (Kupffer’s cells). Apoptosis of hepatocytes by TNF-a is reported to have an important role in D-GalN/LPS-induced liver injury.20

In our previous studies on hepatoprotective compounds from natural medicines, we reported that several constituents from Hovenia Dulcis, 21 Bupleurum scorzonerifolium, 22,23 Curcuma zedoaria, 24–26 Angelica furcijuga, 27,28 Betula sativum, 30 Salacia reticulata, 31 Tilia argentea, 32 Anastatica hierochuntica, 33 Panax notoginseng, 34 Cyperus longus, 35 Erycibe expansa, 36 Camellia sinensis, 37 Sedum sarmentosum, 38,39 Sinocrassula indica, 40 Hedychium coronarium, 41 and Piper chaba42,43 showed hepatoprotective effects on the liver injury induced by D-GalN/LPS in mice and/or platyphylla var. japonica, 29 Pisum inhibitory effect on D-GalN-induced cytotoxicity in primary cultured hepatocytes. Since the methanolic extract from the stems of Cistanche tubulosa showed hepatoprotective effects on D-GalN/LPS-induced liver injury in mice (vide ante), the inhibitory effect of the constituents on D-GalN-induced cytotoxicity in primary cultured mouse hepatocytes was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. As shown in Table 4, echinacoside (4, IC50 = 10.2 lM), acteoside (5, 4.6 lM), isoaceteoside (6, 5.3 lM), 20 -acetylacteoside (8, 4.8 lM), tubulosides A (10, 8.6 lM) and B11 (22, 14.6 lM), and kankanoside G11 (23, 14.8 lM) showed strong activity. Their activities were greater than that of commercial silybin (38.8 lM),33–43 as a positive control.44,45 The structural requirements of the phenylethanoid glycosides for the activity were as follows; (1) the aglycone part was essential for the activity [4 >> kankanose (21, >100 lM), 5 >> cistanoside F (20, >100 lM)]; (2) the aglycone having the 3,4-dihydroxy group showed stronger activity than that having the 4-hydroxy group (6 > 23); (3) the 60 -O-b-D-glucopyranosyl moiety reduced the activity (4 < 5, 10 < 8); (4) the 8-O-b-D-glucopyranosyl part having the 40 -O-caffeoyl group showed stronger activity than that having the 60 -O-caffeoyl group (5 = 6, 8 > 22); and (5) introduction of the 20 -O-acetyl moiety reduced the activity (8 5 5, 22 < 6).

Next, effects of the principal constituents (4–6) on NO and TNF productions, as markers of macrophage activation in LPS-activated mouse peritoneal macrophages were examined.43,46,47 As a result, these constituents (4–6) showed neither NO nor TNF production inhibitory activities (IC50 >100 lM, data not shown). Thus, these compounds were found not to affect the overproductions of NO and TNF-a from LPS-activated macrophages.

2.5. Effects of the chemical constituents on TNF-a-induced cytotoxicity in L929 cells

In order to clarify the effects of the constituents on the sensitivity of hepatocytes to TNF-a, TNF-a-induced decrease in viability of L929 cells, a TNF-a-sensitive cell line,48 was examined using the MTT assay. Without test samples, the cell viability was reduced to ca. 60% after incubation of the cells with 20 ng/mL TNF-a for 44 h compared to the case without TNF-a. As shown in Table 5, echinacoside (4, IC50 = 31.1 lM), acteoside (5, 17.8 lM), isoaceteoside (6, 22.7 lM), 20 -acetylacteoside (8, 25.7 lM), tubuloside A (10, 23.2 lM), and cistantubuloside B1 (11, 21.4 lM) inhibited the decrease of the cell viability, and their activities were found greater than those of piperine42,43 and silybin. The structural requirements of the phenylethanoid glycosides for the activity were as follows; (1) the aglycone part was essential for the activity [4 >> kankanose (21, >100 lM)]; (2) the aglycone having the 3,4-dihydroxy group showed stronger activity than that having the 4-hydroxy group [4 > cistantubuloside A (15, >100 lM), 5 > syringalide A 30 -O-a-L-rhamnopyranoside (16, >100 lM), 6 > kankanoside G (23, >100 lM)]; (3) the 60 -O-b-D-glucopyranosyl moiety reduced the activity (4 < 5); (4) the 8-O-b-D-glucopyranosyl part having the 40 -O-caffeoyl group showed stronger activity than that having the 60 -O-caffeoyl group [5 > 6, 8 > tubuloside B (22, >100 lM)]; and (5) introduction of the 20 -O-acetyl moiety reduced the activity (8 < 5, 22 < 6). These requirements were found similar to those observed in the previous chapter concerning the inhibitory effects on D-GalN-induced cytotoxicity in primary cultured mouse hepatocytes.

These in vitro findings suggest the following possible mechanisms of action for the hepatoprotective effect of the constituents of stems of Cistanche tubulosa: (i) decreasing of D-GalN-induced cytotoxicity (4–6, 8, 10, 22, and 23), and (ii) decreasing of TNF-a-induced cytotoxicity (4–6, 8, 10, and 11).

2.6. Protective effects of echinacoside (4), acteoside (5), and isoacteoside (6) against liver injury induced by D-GalN/LPS in mice and their mode of action

Finally, we examined the effect of principal phenylethanoid glycosides, echinacoside (4), acteoside (5), and isoacteoside (6) on the D-GalN/LPS-induced liver injury in mice. As shown in Tables 6 and 4–6 significantly inhibited the increase of sAST and sALT induced by D-GalN/LPS in mice at doses of 25–100 mg/kg, PO. On the other hand, 4–6 did not affect the overproduction of TNF-a from LPS-activated macrophages. Furthermore, 4–6 significantly inhibited the death of L929 cells caused by TNF-a, indicating a decrease in the sensitivity of L929 cells to TNF-a (vide ante). These findings suggest that 4–6 reduce the sensitivity of these cells to the TNF-a-induced cytotoxicity. Many compounds that inhibit cell death induced by D-GalN and production of TNF-a have been reported,24,26,32 but there reported few compounds that selectively reduce the sensitivity of hepatocytes to TNF-a. 42,43,49,50

In conclusion, from fresh stems of Cistanche tubulosa, three new phenylethanoid glycosides, kankanosides H1 (1), H2 (2), and I (3) together with 16 known phenylethanoid glycosides (4–19) and two acylated oligosugars (20, 21) were isolated. Structural requirements for their protective effects on hepatocyte injury induced by D-GalN, and cytoprotective effects on L929 cells caused by TNF-a were as follows; (1) the aglycone part was essential for the activity; (2) the aglycone having the 3,4-dihydroxy group showed stronger activity than that having the 4-hydroxy group; (3) the 60 -O-b-D-glucopyranosyl moiety reduced the activity; (4) the 8-O-b-D-glucopyranosyl part having the 40 -O-caffeoyl group showed stronger activity than that having the 60 -O-caffeoyl group; and (5) introduction of the 20 -O-acetyl moiety reduced the activity. Furthermore, principal phenylethanoid glycosides, echinacoside (4), acteoside (5), and isoacteoside (6), inhibited the increase in sAST and sALT at doses of 25–100 mg/kg PO in D-GalN/LPS-treated mice, and those inhibitory effects were suggested to be dependent on the reduced sensitivity of hepatocytes to TNF-a. The detailed mechanisms of action of the phenylethanoid glycosides should be studied further.

3. Experimental

3.1. General

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 and 150 MHz) and JEOL JNM-ECS400 (400 and 100 HMz) spectrometers with tetramethylsilane as an internal standard; FABMS and high-resolution FABMS, 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 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 (CC), silica gel 60 N (Kanto Chemical Co., Ltd, 63–210 mesh, spherical, neutral); reversed-phase silica gel CC, Diaion HP-20 (Nippon Rensui) and Chromatorex ODS DM1020T (Fuji Silysia 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.

3.2. Plant material

Fresh stems of Cistanche tubulosa cultivated at Urumqi, Xinjiang Province, China were collected in September 2007. The plant material was identified by one of the authors (M.Y.). A voucher for the plant material is on file in our laboratory.

3.3. Extraction and isolation

The fresh stems of Cistanche tubulosa (2.98 kg) were finely cut and extracted three times with methanol under reflux 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 HP20 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)?M eOH] 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)]. The fraction 2 (9.56 g) was separated by reversed-phase silica gel CC [400 g, MeOH–H2O (10:90, v/v)?MeOH?acetone] to give five fractions [Fr. 2–1 (55.9 mg), 2–2 (4.48 g), 2–3 (3.42 g), 2–4 (1.16 g), and 2–5 (31.9 mg)]. The fraction 2–3 (1.00 g) was subjected to HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (7:93, v/v)] to give seven fractions [Fr. 2–3–1 (66.5 mg), 2–3–2 (20.4 mg), 2–3–3 (26.0 mg), 2–3–4 (136.6 mg), 2–3–5 (380.6 mg), 2–3–7 (84.9 mg)]. The fraction 2–3–4 (106.6 mg) was further purified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (5:95, v/v)] to give salidroside (19, 13.7 mg, 0.0027%). 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 4–2 (1.50 g) was subjected to HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (20:80, v/v)] to give five fractions {Fr. 4–2–1 (42.9 mg), 4–2–2 [= campneoside I (17, 67.0 mg, 0.014%)], 4–2–3 [= acteoside (5, 1.21 g, 0.26%)], 4–2–4 (41.6 mg), and 4–2–5 (181.3 mg)}. The fraction 4–2–4 (41.6 mg) was further purified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (18:82, v/v)] to give isoacteoside (6, 19.1 mg, 0.0040%) and cis-acteoside (7, 3.4 mg, 0.0007%). The fraction 4–3 (1.57 g) was purified 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 (= 17, 22.1 mg, 0.0010%), 4–3–4 (= 5, 224.6 mg, 0.010%), 4–3–5 (27.4 mg), 4–3–6 (43.6 mg), 4–3–7 (= 6, 825.0 mg, 0.037%), 4–3–8 [= syringalide A 30 -O-a-L-rhamnopyranoside (16, 37.6 mg, 0.0017%)], 4–3–9 (39.8 mg), 4–3–10 [= 20 -acetylacteoside (8, 85.4 mg, 0.0038%)], and 4–3–11 (64.6 mg)]. The fraction 4–3–6 (43.6 mg) was further purified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (18:82, v/v)] to give kankanosides H1 (1, 17.0 mg, 0.0008%) and H2 (2, 3.3 mg, 0.0001%). The fraction 4–3–9 (39.8 mg) was further purified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (18:82, v/v)] to give kankanoside I (3, 15.4 mg, 0.0007%) and 6 (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 5–1 (870.2 mg) was purified by HPLC [CosmosilpNAP, CH3CN–1% aqueous AcOH (5:95, v/v)] to give decaffeoylacteoside (9, 5.1 mg, 0.0002%) and cistanoside F (20, 8.1 mg, 0.0004%). The fraction 5–3 (3.72 g) was purified by HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (15:85, v/v)] to give six fractions [Fr. 5–3–1 (128.6 mg), 5–3–2 [= kankanose (21, 9.4 mg, 0.0004%)], 5–3–3 (64.6 mg), 5–3–4 (72.3 mg), 5–3–5 [= echinacoside (4, 2.54 g, 0.11%), and 5–3–6 (318.5 mg)]. The fraction 5–3–4 (72.3 mg) was further purified by HPLC [Cosmosil pNAP, CH3CN– 1% aqueous AcOH (10:90, v/v)] to give campneoside II (18, 10.6 mg, 0.0005%). The fraction 5–4 (979.9 mg) was purified by HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (15:85, v/v)] to give nine fractions [Fr. 5–4–1 (28.6 mg), 5–4–2 (90.5 mg), 5–4–3 (99.7 mg), 5–4–4 (= 4, 25.7 mg, 0.0012%), 5–4–5 (16.4 mg), 5–4–6 (318.5 mg), 5–4–7 (21.6 mg), 5–4–8 (= 5, 204.4 mg, 0.0091%), and 5–4–9 (134.7 mg)]. The fraction 5–4–6 (318.5 mg) was purified by HPLC [Cosmosil pNAP, CH3CN–1% aqueous AcOH (15:85, v/v)] to give cistantubulosides B1 (11, 44.9 mg, 0.0020%), B2 (12, 7.6 mg, 0.0003%), and A (15, 21.1 mg, 0.0009%) and wiedemanninoside C (14, 17.2 mg, 0.0008%). The fraction 5–5 (1.19 g) was purified by HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (18:82, v/v)] to give tubuloside A (10, 300.3 mg, 0.013%) and arenarioside (13, 5.8 mg, 0.0006%). The fraction 6 (12.52 g) was separated by reversed-phase silica gel CC [600 g, MeOH–H2O (20:80?30:70, v/v)?MeOH?acetone] to give four fractions [Fr. 6–1 (553.8 mg), 6–2 (10.69 g), 6–3 (1.40 g), and 6–4 (17.8 mg)]. The fraction 6–2 (500.0 mg) was purified by HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (25:75, v/v)] to give 4 (353.1 mg, 0.34%).

3.3.1. Kankanoside H1 (1)

A white powder, ½a- 24 D -45.3 (c 1.06, MeOH). High-resolution positive-ion FABMS: Calcd for C37H48O20Na (M+Na)+ : 835.2637. Found: 835.2633. UV [MeOH, nm (log e)]: 226 (4.33), 293 (sh, 4.36), 317 (4.52). IR (KBr): 3416, 1736, 1638, 1605, 1518, 1070, 1044 cm-1 . 1 H NMR (600 MHz, CD3OD) d: given in Table 2. 13C NMR (150 MHz, CD3OD) dC: given in Table 3. Positive-ion FABMS: m/z 835 (M+Na)+ . Negative-ion FABMS: m/z 811 (M-H)-.

3.3.2. Kankanoside

H2 (2) A white powder, ½a- 25 D-52.4 (c 0.27, MeOH). High-resolution positive-ion FABMS: Calcd for C37H48O20Na (M+Na)+ : 835.2637. Found: 835.2644. UV [MeOH, nm (log e)]: 228 (4.26), 290 (sh, 4.22), 316 (4.37). IR (KBr): 3420, 1717, 1638, 1605, 1508, 1159, 1067 cm-1 . 1 H NMR (600 MHz, CD3OD) d: given in Table 2. 13C NMR (150 MHz,CD3OD) dC: given in Table 3. Positive-ion FABMS: m/z 835 (M+Na)+ . Negative-ion FABMS: m/z 811 (M-H)-.

3.3.3. Kankanoside I (3)

A white powder, ½a-25 D-62.2 (c 1.00, MeOH). High-resolution positive-ion FABMS: Calcd for C35H46O18Na (M+Na)+ : 777.2581. Found: 777.2585. UV [MeOH, nm (log e)]: 246 (3.97), 295 (sh, 4.04), 334 (4.23). IR (KBr): 3415, 1717, 1647, 1603, 1508, 1070, 1046 cm-1 . 1 H NMR (600 MHz, CD3OD) d: given in Table 2. 13C NMR (150 MHz, CD3OD) dC: given in Table 3. Positive-ion FABMS: m/z 777 (M+Na)+ . Negative-ion FABMS: m/z 753 (MH)-.

3.4. Alkaline and acid hydrolysis of 1–3

Solutions of 1, 2, and 3 (each 1.5 mg) in 5% aqueous potassium hydroxide (KOH, 0.5 mL) were stirred at 40℃ for 1 h. Each solution was neutralized with Dowex HCR W2 (H+ form), and the resins were removed by filtration. Evaporation of the solvent under reduced pressure yielded the corresponding deacylated products, which were subjected to HPLC analysis [column: Cosmosil pNAP, 250 - 4.6 mm id; mobile phase: CH3CN–1% aqueous AcOH (15:85, v/v); detection: UV (254 nm); flow rate: 1.0 mL/min] to be identified as trans-caffeic acid (tR 9.9 min from 3), trans-p-coumaric acid (tR 17.1 min from 1), and cis-p-coumaric acid (tR 17.8 min from 2), respectively. 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 id - 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); flow rate 0.8 mL/min]. Identification of L-rhamnose (i) and D-glucose (ii) liberated from 1, 2, or 3 in the H2O-eluted fraction 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)].

3.5. Bioassay

3.5.1. Reagents

LPS (from Salmonella enteritidis), minimum essential medium (MEM), and William’s E medium were purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA); fetal bovine serum (FBS) was from Invitrogen (Carlsbad, CA, USA); and other chemicals were from Wako Pure Chemical Industries, Co., Ltd (Osaka, Japan). 96- Well microplates were purchased from Sumitomo Bakelite Co., Ltd (Tokyo, Japan).

3.5.2. Animals

Male ddY mice were purchased from Kiwa Laboratory Animal Co., Ltd, (Wakayama, Japan). The animals were housed at a constant temperature of 23 ± 2 C and were fed a standard laboratory chow (MF, Oriental Yeast Co., Ltd, Tokyo, Japan). All of the experiments were performed with conscious mice unless otherwise noted. The experimental protocol was approved by the Experimental Animal Research Committee of Kinki University.

3.5.3. Protective effects on D-GalN/LPS-induced liver injury in mice

The method described by Tiegs et al.51 was modified and used for this experiment. Briefly, male ddY mice weighing about 25–30 g fasted for 20 h before the experiment. D-GalN (350 mg/kg) and LPS (10 lg/kg) dissolved in saline were injected intraperitoneally to produce liver injury. Each test sample was given orally 1 h before the D-GalN/LPS injection. Blood samples were collected from the infraorbital venous plexus 10 h after D-GalN/LPS injection. sAST and sALT levels were determined using the Reitman–Frankel method (commercial kit, Transaminase CII-Test Wako, Wako Pure Chemical Industries, Co., Ltd). Hydrocortisone was used as a reference compound. Test samples were suspended with 5% Arabic gum solution, and the solution was administered orally at 10 mL/kg in each experiment, while the vehicle was given orally at 10 mL/kg in the corresponding control group.

3.5.4. Protective effects on cytotoxicity induced by D-GalN in primary cultured mouse hepatocytes

The hepatoprotective effect of the constituents was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay using primary cultured mouse hepatocytes.33–43 Hepatocytes were isolated from male ddY mice (30–35 g) by the collagenase perfusion method. A cell suspension at 4 - 104 cells in 100 lL William’s E medium containing FBS (10%), penicillin G (100 units/mL), and streptomycin (100 lg/mL) was inoculated in a 96-well microplate and pre-cultured for 4 h at 37 ℃ under a 5% CO2 atmosphere. The medium was added with 100 lL of the fresh medium containing D-GalN (2 mM) with or without the test sample and the hepatocytes were cultured for 44 h. The medium was exchanged with 100 lL of the fresh medium, and 10 lL of MTT [5 mg/mL in phosphate-buffered saline (PBS)] solution was added to the medium. After 4 h of incubation, the medium was removed, and 100 lL of isopropanol containing 0.04 M HCl was then added to dissolve the formazan produced in the cells. The optical density (O.D.) of the formazan solution was measured by a microplate reader at 570 nm (reference: 655 nm). Inhibition (%) was obtained by the following formula.

Inhibition (%)= [(O.D.(sample) - O.D.(control))/(O.D.(normal)-O.D.(control))] x 100

3.5.5. Effects on the production of NO in LPS-stimulated mouse peritoneal macrophages

A screening test for NO production using TGC-induced mouse peritoneal macrophages was performed as described previously.43,46,

3.5.6. Effects on the production of TNF-a in LPS-stimulated mouse peritoneal macrophages

TNF-a released in the medium was determined as described previously28 with a slight modification. Briefly, TGC-induced mouse peritoneal macrophages (5 - 105 cells/well) were collected from the peritoneal cavities of male ddY mice, suspended in 100 lL of RPMI 1640 supplemented with 5% FBS, penicillin (100 units/mL), and streptomycin (100 lg/mL), and pre-cultured in 96-well microplates at 37℃ in 5% CO2 in the air for 1 h. Nonadherent cells were removed by washing with PBS, and 100 lL of fresh medium containing various concentrations of test compound was added. After 10 min, 100 lL of the medium containing 10 lg/mL LPS was added and incubated for 4 h. TNF-a production in each well was determined using an ELISA kit (Mouse TNF-a ELISA kit, Invitrogen). Each test compound was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration 0.5%)

3.5.7. Inhibitory effects on TNF-a-induced cell death in L929 cells

L929 cells (Dainippon Pharmaceuticals, Osaka, Japan) were maintained in Minimum Essential Medium Eagle (MEM, Sigma–Aldrich) containing 10% FBS, 1% MEM Non-Essential Amino acids (Invitrogen), penicillin G (100 units/mL), and streptomycin (100 lg/mL) at 37 C under a 5% CO2 atmosphere. The cells were inoculated in a 96-well tissue culture plate [3 -104 cells/well in 100 lL/well in MEM]. After 44 h of incubation in the medium TNF-a (20 ng/mL) with or without the test sample, the viability of the cells was assessed by the MTT colorimetric assay (vide ante).49,50 Each test compound was dissolved in DMSO, and the solution was added to the medium (final concentration in DMSO 0.5%).

3.6. Statistics

Values are expressed as means ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used for statistical analysis. Probability (p) values less than 0.05 were considered significant.

Acknowledgments

T.M., K.N., and O.M. were supported by a Grant-in-Aid for Scientific Research from ‘High-tech Research Center’ Project for Private Universities: matching fund subsidy from a Grant-in-Aid for Scientific Research from the 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 MEX

From: ' Acylated phenylethanoid oligoglycosides with hepatoprotective activity from the desert plant Cistanche tubulosa' by Toshio Morikawa et al

---Bioorganic & Medicinal Chemistry 18 (2010) 1882–1890.