Monoterpene Constituents From Cistanche Tubulosa—Chemical Structures Of Kankanosides A—E And Kankanol—

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Haihui XIE, Toshio MORIKAWA, Hisashi MATSUDA, Seikou NAKAMURA, Osamu MURAOKA, andMasayuki YOSHIKAWA

Abstract

Four new iridoid glycosides, kankanosides A (1), B (2), C (3), and D (4), a chlorinated iridoid, kankanol (5), and an acyclic monoterpene glycoside, kankanoside E (6), were isolated from the methanolic extract of dried stems of Cistanche tubulosa (SCHRENK) R. WIGHT (Orobanchaceae) together with 16 known compounds. The structures of these new compounds (1—6) were determined based on chemical and physicochemical evidence.

Key words: Cistanche tubulosa; kankanoside; kankanol; iridoid; monoterpene; Orobanchaceae

Cistanche tubulosa (SCHRENK) R. WIGHT (Orobanchaceae) is a perennial parasitic plant growing on the roots of Salvadora or Calotropis species, and distributed in North Africa, Arabia, and Asian countries.1) The stems of this plant (Kanka-nikujuyou in Japanese) have been used traditionally for treating impotence, sterility, lumbago, and body weakness.2) Previously, several iridoids, monoterpenoids, phenylethanoids, and lignans were isolated from Chinese and Pakistan C. tubulosa. 1,3–7) In the course of our serial studies on bioactive constituents from Chinese natural medicines,8–18) four new iridoid glycosides, kankanosides A (1), B (2), C (3), and D (4), a chlorinated iridoid, kankanol (5), and an acyclic monoterpene glycoside, kankanoside E (6), were isolated from the methanolic extract of this herbal medicine together with 16 known compounds including 12 monoterpenes (7—18). This paper deals with the isolation and structure elucidation of new monoterpene constituents (1—6).

Cistanche tubulosa

The methanolic extract from dried stems of Cistanche tubulosa (26.8% from this herbal medicine) was subjected to normalphase and reversed-phase silica gel column chromatography and repeated HPLC to give kankanosides A (1, 0.0054%), B(2, 0.030%), C (3, 0.0027%), and D (4, 0.0015%), kankanol (5, 0.0034%), and kankanoside E (6, 0.027%) together with mussaenosidic acid19) (7, 0.020%), geniposidic acid19) (8, 0.030%), 8-epiloganic acid7) (9, 0.033%), gluroside19) (10, 0.14%), antirrhide20) (11, 0.0079%), ajugol19) (12, 0.011%), bartsioside19) (13, 0.21%), 6-deoxycatalpol19) (14, 0.11%), argyol21) (15, 0.0030%), cistanin22,23) (16, 0.0040%), cistachlorin22) (17, 0.0035%), (2E,6Z)-8-b-D-glucopyranosyloxy-2,6-dimethyl-2,6-octadienoic acid24) (18, 0.0028%), Dmannitol25) (4.19%), uridine25) (0.0069%), (3R)-3-hydroxy-2- pyrrolidinone26,27) (0.0020%), and (3R)-3-hydroxy-1-methyl- 2-pyrrolidinone27) (0.0059%).

Structure of Kankanoside A (1) Kankanoside A (1) was obtained as an amorphous powder and exhibited a negative optical rotation ([a]D 25
107.4° in MeOH). The IR spectrum of 1 showed an absorption band at 1647 cm
1 assignable to olefin function in addition to strong absorption bands at 3410 and 1076 cm
1 suggestive of a glycoside moiety. In the positive- and negative-ion fast atom bombardment (FAB)-MS of 1, quasimolecular ion peaks were observed at m/z 369 (M Na) and 345 (M
H)
, and high-resolution FAB-MS analysis revealed the molecular formula of 1 to be C16H26O8. 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.8,10—12,15—18) The 1 H- (CD3OD, Table 1) and 13C-NMR (Table 2) spectra of 1, which were assigned by various NMR experiments,28) showed signals assignable to two methyls [d 1.31 (s, 10-H3), 1.51 (br s, 11-H3)], two methylenes [d 1.49, 2.02 (both m, 6a- and 6b-H), 1.64, 1.67 (both m, 7b- and 7a-H)], two methines [d 2.21 (dd, J 2.7, 9.5 Hz, 9-H), 2.71 (m, 5-H)], and an a,b-unsaturated acetal group [d 5.33 (d, J 2.7 Hz, 1-H), 5.95 (br s, 3-H)] together with a b-glucopyranosyl moiety [d 4.62 (d, J 7.9 Hz, 1 -H)]. As shown in Fig. 1, the 1 H–1H 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 correlations (HMBC) experiment on 1, long-range correlations were observed between the following protons and carbons (3-H, 1 -H and 1-C; 11-H3 and 3-C; 3-H, 5-H, 6-H2, 9-H, 11-H3 and 4- C; 11-H3 and 5-C; 10-H3 and 7-C; 1-H, 7-H2, 9-H, 10-H3, and 8-C; 10-H3 and 9-C; 7-H2 and 10-C) as shown in Fig. 1. Enzymatic hydrolysis of 1 with b-glucosidase gave an aglycon, kankagenin a (1a) as shown in Fig. 3. Comparison of the 13CNMR spectrum for 1 with those for 1a revealed the glycosylation shift around the 1-position in 1 [1: dC 94.1 (1-C), 134.6 (3-C), 53.3 (9-C); 1a: dC 92.9 (1-C), 135.3 (3-C), 54.7 (9-C)]. Thus, the connectivity of the b-D-glucopyranosyl moiety in 1 was also clarified to be at the 1-position of 1a. Next, the relative stereostructure of 1 was characterized by nuclear Overhauser enhancement spectroscopy (NOESY) experiment, which showed NOE correlations between the following proton pairs (1-H and 10-H3; 3-H and 11-H3; 5-H and 6b-H, 9-H; 6b-H and 7b-H; 7a-H and 10-H3; 7b-H and 9-H) as shown in Fig. 2. Finally, the absolute configuration of the 1-position in 1 was determined by application of the 13CNMR glycosylation shift rule of 1,1 -disaccharide,29) which was found to apply to the hemiacetal compounds.30,31) The stereostructure of the 1-position in 1 was confirmed to be maintained in 1a by comparison of the 1 H- NMR analysis including the NOESY experiment. The glycosylation shift values [Dd 1.2 ppm (1 -C) and 0.9 ppm (1- C), in pyridine-d5] were found to be characteristic of the R, Rhemiacetal combination, which corresponded to the absolute stereostructure of 1 as shown in Fig. 3. Consequently, the absolute configuration at the 1-position of 1 was determined to be S configuration, and the absolute stereostructure of 1 was elucidated as shown.

Structures of Kankanosides B (2) and C (3) Kankanoside B (2) were also isolated as an amorphous powder with negative optical rotation ([a]D 26
118.7° in MeOH). The IR spectrum of 2 showed absorption bands at 3410, 1647, and 1085 cm
1 ascribable to hydroxyl, olefin, and ether functions. The molecular formula C15H24O10 of 2 was determined by the quasimolecular ion peaks in positive-ion FAB-MS and by high-resolution FAB-MS. Acid hydrolysis of 2 with 1.0 M HCl liberated D-glucose, which was identified by HPLC analysis using an optical rotation detector.8,10—12,15—18) The 1 H- (CD3OD, Table 1) and 13C-NMR (Table 2) spectra28) of 2 showed signals assignable to two methylenes [d 1.40 (DDD, J 5.2, 7.3, 13.5 Hz, 6a-H), 2.52 (DDD, J 7.0, 9.2, 13.5 Hz, 6b-H), 3.85, 3.99 (both d, J 11.9 Hz, 10-H2)], four methines [d 2.21 (dd, J 6.4, 8.6 Hz, 9-H), 2.83 (m, 5-H), 4.02 (dd, J 5.2, 7.0 Hz, 7-H), 5.49 (d, J 6.4 Hz, 1-H)], and a cisolefifin pair [d 4.95 (dd, J 4.0, 6.1 Hz, 4-H), 6.22 (dd, J 1.8, 6.1 Hz, 3-H)], together with a b-glucopyranosyl moiety [d 4.72 (d, J 7.9 Hz, 1 -H)]. The proton and carbon signals in the 1 H- and 13C-NMR data of 2 were similar to those of 6-deoxycatalpol (14), except for the signals due to the 7- and 8-positions. 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, 1 -H and 1-C; 1-H and 3-C; 10-H2 and 7- C; 1-H, 7-H, 9-H, 10-H2, and 8-C; 7-H, 10-H2 and 9-C; 7-H and 10-C). The relative stereostructure of 2 was characterized by the NOESY experiment, which showed NOE correlations between the following proton pairs (1-H and 10-H2; 3- H and 4-H; 5-H and 6b-H, 9-H; 6b-H and 7-H; 7-H and 9-H) as shown in Fig. 2. Finally, alkaline treatment of 14 with 5% aqueous potassium hydroxide (KOH) yielded 2 and 19, 32) so that the stereostructure of 2 was clarified.

Kankanoside C (3) was isolated as an amorphous powder with negative optical rotation ([a]D 26
34.0° in MeOH). In the negative-ion FAB-MS of 3, a pair of isotope quasimolecular ion peaks were observed at m/z 399 and 401 (M
H)
. The molecular formula of 3 was determined to be C15H25ClO10 by high-resolution FAB-MS measurement. Acid hydrolysis of 3 with 1.0 M HCl liberated D-glucose, which was identified by HPLC analysis using an optical rotation detector.8,10—12,15—18) The 1 H- (CD3OD, Table 1) and 13C-NMR (Table 2) spectra28) of 3 showed signals assignable to three methylenes [d 1.70 (br dd, J ca. 3, 13 Hz, 4a-H), 2.70 (br dd, J ca. 6, 13 Hz, 4b-H), 1.68 (br d, J ca. 13 Hz, 6aH), 2.44 (m, 6b-H), 4.01, 4.04 (both d, J 11.3 Hz, 10-H2)], five methines [d 2.47 (dd, J 2.5, 7.9 Hz, 9-H), 2.61 (m, 5- H), 3.94 (br s, 7-H), 5.10 (br d, J ca. 3 Hz, 3-H), 5.48 (d, J 2.5 Hz, 1-H)], and a b-glucopyranosyl moiety [d 4.60 (d, J 8.0 Hz, 1 -H)]. The proton and carbon signals in the 1 H- and 13C-NMR spectra of 3 were superimposable on those of 2, except for the signals due to the 3- and 4-positions. The positions of b-D-glucopyranosyl and chlorine function in 3 were elucidated from the H–H COSY and HMBC experiments as shown in Fig. 1. Consequently, the planar structure of 3 was constructed to be as shown. The relative stereostructure of 3 was determined by a NOESY experiment, in which NOE correlations were observed between the following proton pairs (1-H and 3-H, 10-H2; 3-H and 4a-H; 4b-H and 5- H; 5-H and 6b-H, 9-H; 6b-H and 7-H; 7-H and 9-H) as shown in Fig. 2.

Structures of Kankanoside D (4) and Kankanol (5) Kankanoside D (4) was isolated as an amorphous powder with negative optical rotation ([a]D 25
30.6° in MeOH). The IR spectrum of 4 showed an absorption band at 1655 cm
1 ascribable to olefin function and strong absorption bands at 3410 and 1078 cm
1 suggestive of its glycosidic structure. In the positive-ion FAB-MS of 4, a quasimolecular ion peak was observed at m/z 341 (M Na) . The molecular formula C15H26O7 of 4 was determined by high-resolution FAB-MS measurement. Acid hydrolysis of 4 with 1.0 M HCl liberated D-glucose,8,10—12,15—18) whereas (R)-rotundity (4a) 33,34) was obtained by enzymatic hydrolysis of 4 with b-glucosidase. The 1 H-NMR (Table 3, CD3OD) and 13C-NMR (Table 4) spectra28) of 4 showed signals assignable to a methyl [d 1.69 (s, 10-H3)], three methylenes [d 1.41, 2.06 (both m, 4-H2), 1.51, 2.01 (both m, 6a- and 6b-H), 2.23 (br dd, J ca. 8, 15 Hz, 7a-H), 2.37 (br dd, J ca. 8, 15 Hz, 7b-H)], a methine [d 2.90 (m, 5-H)], and two methylenes bearing an oxygen function {d [3.56 (DDD, J 2.8, 7.4, 13.2 Hz), 3.97 (DDD, J 4.9, 8.0, 13.2 Hz), 3-H2], 4.04, 4.18 (both d, J 12.2 Hz, 1-H2)} together with a b-glucopyranosyl part [d 4.25 (d, J 7.7 Hz, 1 -H)]. The position of the b-D-glucopyranosyl part in 4 was clarified by the HMBC experiment to be 3-position (Fig. 1). Based on this evidence, the absolute stereostructure of 4 was determined to be as shown.

Kankanol (5) was obtained as an amorphous powder with positive optical rotation ([a]D 25  11.1°). The chemical ionization (CI)-MS of 5 showed a pair of isotope ion peaks at m/z 221 and 223 due to a quasimolecular ion (M H) . The high-resolution CI-MS measurement of 5 revealed the molecular formula to be C9H13ClO4. The 1 H-NMR (Table 1, CD3OD) and 13C-NMR (Table 2) spectra28) of 5 showed the presence of the following functions: three methylenes [d 1.60 (DDD, J 2.5, 5.5, 13.1 Hz, 4a-H), 1.80 (br dd, J ca. 3, 13 Hz, 4bH), 1.83 (br d, J ca. 12 Hz, 6a-H), 2.57 (m, 6b-H), 3.75, 3.88 (both d, J 9.2 Hz, 10-H2)], five methines [d 2.76 (dd, J 4.3, 8.0 Hz, 9-H), 2.50 (m, 5-H), 3.80 (br s, 7-H), 5.17 (br d, J ca. 3 Hz, 3-H), 5.26 (d, J 4.3 Hz, 1-H)]. The planar structure of 5 was confirmed by 1 H–1 H COSY and HMBC experiments. That is, the 1 H–1 H COSY experiment on 5 indicated the presence of the partial structures written in bold lines, and in the HMBC experiment, long-range correlations were observed as shown in Fig. 1. The relative stereostructure of 5 was determined by the NOESY experiment, in which NOE correlations were observed between the following proton pairs (1-H and 9-H; 3-H and 4a-H; 4b-H and 5- H; 5-H and 6b-H, 9-H; 6b-H and 7-H; 7-H and 9-H) as shown in Fig. 2. By comparison of the 1 H- and 13C-NMR data for 5 with those for 14a, which was obtained by the treatment of 14 with 5% aqueous HCl as shown in Chart 1,35) the position of the chlorine group in 5 was supported to be the 3-position. Furthermore, acetylation of 5 with acetic anhydride (Ac2O) and pyridine yielded the 3,7-oxide (5a), while 14a gave the diacetate (14b) under the same acetylation condition. This evidence also led us to confirm the position of chlorine function to be the 3b-position (Chart 3). Consequently, the stereostructure of 5 was determined to be as shown.

Structure of Kankanoside E (6) Kankanoside E (6) was isolated as an amorphous powder with negative optical rotation ([a]D 25
20.0° in MeOH). The IR spectrum of 6 showed absorption bands at 3410, 1647, 1085 cm
1 ascribable to glycosidic and carbonyl functions, while its UV spectrum showed absorption maximum at 211 nm (log e 4.63), indicating the presence of a b-unsaturated carboxylic acid. The molecular formula C16H28O8 of 6 was characterized by the positive- and negative-ion FAB-MS and by high-resolution MS measurement. Acid hydrolysis of 6 liberated D-glucose,8,10—12,15—18) whereas (2E,6R)-8-hydroxy-2,6-dimethyl- 2-octanoic acid (6a) 36) was obtained by enzymatic hydrolysis of 6 with b-glucosidase. The 1 H-NMR (Table 3, CD3OD) and 13C-NMR (Table 4) spectra28) of 6 indicated the presence of a (2E,6R)-8-hydroxy-2,6-dimethyl-2-octanoic acid moiety [d 0.95 (d, J 6.4 Hz, 10-H3), 1.30, 1.48 (both m, 5-H2), 1.45, 1.70 (both m, 7-H2), 1.65 (m, 6-H), 1.81 (s, 9-H3), 2.22 (2H, m, 4-H2), 3.61, 3.94 (both m, 8-H2), 6.78 (dd, J 1.2, 7.3 Hz, 3-H)] together with a b-D-glucopyranosyl part [d 4.26 (d, J 7.6 Hz, 1 -H)]. By comparison of the carbon signals in the 13C-NMR spectrum of 6 with those of 6a, the glycosylation shift was observed around the 8-position of 6. The position of the glucoside linkage was also confirmed by HMBC experiments as shown in Fig. 1. Consequently, the absolute stereostructure of 6 was clarified to be (2E,6R)-8-b- D-glucopyranosyloxy-2,6-dimethyl-2-octanoic acid.

cistanche extract benefit

Experimental

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; EI-MS, CI-MS, and high-resolution CI-MS, JEOL JMS-GCMATE mass spectrometer; FAB-MS and high-resolution MS, JEOL JMS-SX 102A mass spectrometer; 1 H-NMR spectrum, JEOL EX-270 (270 MHz) and JNM-LA500 (500 MHz) spectrometers; 13C-NMR spectra, JEOL EX-270 (68 MHz) and JNM-LA500 (125 MHz) spectrometers with tetramethylsilane as an internal standard; and HPLC detector, Shimadzu RID-6A refractive index and SPD- 10Avp UV-VIS detectors. HPLC column, YMC-Pack ODS-A (250 4.6 mm i.d.), and (250 20 mm i.d.) columns were used for analytical and preparative purposes, respectively.

The following experimental conditions were used for chromatography: ordinary-phase silica gel column chromatography, Silica gel BW-200 (Fuji Silysia Chemical, Ltd., Aichi, Japan, 150—350 mesh); reverse-phase silica gel column chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., Aichi, Japan, 100—200 mesh); TLC, precoated TLC plates with Silica gel 60F254 (Merck, 0.25 mm) (ordinary phase) and Silica gel RP- 18 F254S (Merck, 0.25 mm) (reverse phase); reverse-phase HPTLC, precoated TLC plates with Silica gel RP-18 WF254S (Merck, 0.25 mm); and detection was achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4 followed by heating.

Plant Material

Dried stems of Cistanche tubulosa (SCHRENK) R. WIGHT were purchased at Urumqi, Xinjiang Province, China in January 2005 via Eishin Trading Co., Ltd. Osaka, Japan, and botanical identification was undertaken by professor Jia Xiaoguang in Xinjiang Institute of Traditional Chinese and Ethnologic Medicines. A voucher specimen (2005.01. Xinjiang-01) of this plant is on file in our laboratory.

Extraction and Isolation

Dried stems of C. tubulosa (5.0 kg) was powdered and extracted three times with methanol under reflflux for 3 h. Evaporation of the solvent under reduced pressure provided the methanolic extract (1340 g, 26.8% from this herbal medicine). The methanolic extract (160 g) was subjected to normal-phase silica gel column chromatography [3.2 kg, CHCl3–MeOH–H2O (10 : 3 : 1→7 : 3 : 1, lower layer→6 : 4 : 1)MeOH] to give six fractions [Fr. 1 (5.04 g), Fr. 2 (9.84 g), Fr. 3 (7.80 g), Fr. 4 (13.28 g), Fr. 5 (113.60 g), and Fr. 6 (7.61 g)]. Fraction 1 (5.00 g) was separated by reversedphase silica gel column chromatography [150 g, MeOH–H2O (40 6: 60→ 50 : 50→60 : 40, v/v)→MeOH] to afford fifive fractions [Fr. 1-1 (830 mg), Fr. 1-2 (590 mg), Fr. 1-3 (180 mg), Fr. 1-4 (124 mg), and Fr. 1-5 (3200 mg)]. Fr. 1-1 (830 mg) was further separated by HPLC [MeOH–H2O (10 : 90, v/v)] to give kankanol (5, 20 mg, 0.0034%), argyol (15, 18 mg, 0.0030%), cistanin (16, 24 mg, 0.0040%), and Fr. 1-1-2 (62 mg), which was further separated by HPLC [MeOH–H2O (2 : 98, v/v)] to give (3R)-3-hydroxy-2-pyrrolidinone (0.0020%) and (3R)-3-hydroxy-1-methyl-2-pyrrolidinone (0.0059%). Fr. 1-2 (590 mg) was purifified by HPLC [MeOH–H2O (35 : 65, v/v) and CH3CN– H2O (20 : 80, v/v)] to give cistanchlorin (17, 21 mg, 0.0035%). Fraction 2 (9.72 g) was subjected to reversed-phase silica gel column chromatography [290 g, MeOH–H2O (20 : 80→30 : 70→40 : 60→60 : 40, v/v)→MeOH] to afford seven fractions [Fr. 2-1 (1986 mg), Fr. 2-2 (1563 mg), Fr. 2-3 (3931 mg), Fr. 2-4 (375 mg), Fr. 2-5 (486 mg), Fr. 2-6 (460 mg), and Fr. 2-7 (336 mg)]. Fr. 2-1 (466 mg) was separated by HPLC [MeOH–H2O (5 : 95, v/v)] to give uridine (0.0069%). Fr. 2-2 (535 mg) was separated by HPLC [MeOH–H2O (10 : 90, v/v)] to give antirrhide (11, 15 mg, 0.0079%) and 6-deoxycatalpol (14, 214 mg, 0.11%). Fr. 2-3 (535 mg) was separated by HPLC [MeOH–H2O (20 : 80, v/v)] to furnish gluroside (10, 110 mg, 0.14%) and bartsioside (13, 164 mg, 0.21%). Fr. 2-4 (375 mg) was separated by HPLC [MeOH–H2O (30 : 70, v/v)] to give kankanosides A (1, 32 mg, 0.0054%) and D (4, 9 mg, 0.0015%). Fr. 2-6 (460 mg) was further separated by HPLC [MeOH–H2O (45 : 55, v/v)] to provide kankanoside E (6, 161 mg, 0.027%) and (2E,6Z)- 8-b-D-glucopyranosyloxy-2,6-dimethyl-2,6-octadienoic acid (18, 17 mg, 0.0028%). Fraction 3 (7.60 g) was subjected to reversed-phase silica gel column chromatography [230 g, MeOH–H2O (20 : 80→40 : 60→50 : 50→ 60 : 40, v/v)→MeOH] to give fifive fractions [Fr. 3-1 (2652 mg), Fr. 3-2 (593 mg), Fr. 3-3 (3610 mg), Fr. 3-4 (190 mg), and Fr. 3-5 (336 mg)]. Fr. 3-1 (480 mg) was purifified by HPLC [MeOH–H2O (10 : 90, v/v)] to give kankanoside B (2, 19 mg, 0.018%), geniposidic acid (8, 32 mg, 0.030%), and ajugol (12, 12 mg, 0.011%). Fraction 4 (13.10 g) was subjected to reversed-phase silica gel column chromatography [390 g, MeOH–H2O (10 : 90→20 : 80→30 : 70→40 : 60→50 : 50, v/v)→MeOH] to give seven fractions [Fr. 4-1 (6114 mg), Fr. 4-2 (430 mg), Fr. 4-3 (1058 mg), Fr. 4-4 (170 mg), Fr. 4-5 (2595 mg), Fr. 4-6 (1635 mg), and Fr. 4-7 (1064 mg)]. Fr. 4-2 (430 mg) was further separated by HPLC [MeOH–H2O (5 : 95, v/v)] to afford 2 (70 mg, 0.012%) and kankanoside C (3, 16 mg, 0.0027%). Fr. 4-6 (1058 mg) was also separated by HPLC [MeOH–H2O (15 : 85, v/v)] to furnish mussaenosidic acid (7, 116 mg, 0.020%) and 8-epiloganic acid (9, 193 mg, 0.033%). Fraction 5 (15.15 g) was subjected to reversed-phase silica gel column chromatography [455 g, MeOH–H2O (0 : 100→10 : 90→ 20 : 80→40 : 60→50 : 50, v/v)→MeOH] to give seven fractions [Fr. 5-1 (9311 mg), Fr. 5-2 (1114 mg), Fr. 5-3 (306 mg), Fr. 5-4 (347 mg), Fr. 5-5 (1620 mg), Fr. 5-6 (1453 mg), and Fr. 5-7 (1106 mg)]. Fr. 5-1 (9311 mg) was crystallized from MeOH to give D-mannitol (3337 mg, 4.19%).

The known compounds (7—18) were identified by comparison of their physical data ([a]D, IR, 1 H-NMR, 13C-NMR, MS) with reported values1,7,19—24,26,27) or those of commercial samples.25) Kankanoside A (1): An amorphous powder, [a]D 25
107.4° (c 1.50, MeOH). High-resolution positive-ion FAB-MS: Calcd for C16H26O8Na (M Na) 369.1525; Found 369.1522. IR (KBr): 3410, 2940, 1647, 1076 cm
1 . 1 H-NMR (500 MHz, CD3OD and pyridine-d5) d: given in Table 1. 13C-NMR (125 MHz, CD3OD and pyridine-d5) d C: given in Table 2. Positive-ion FAB-MS: m/z 369 (M Na) . Negative-ion FAB-MS: m/z 345 (M
H)
.

Kankanoside B (2): An amorphous powder, [a]D 26
118.7° (c 0.10, MeOH). High-resolution positive-ion FAB-MS: Calcd for C15H24O10Na (M Na) 387.1267; Found 387.1261. IR (KBr): 3410, 2940, 1647, 1085 cm 1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 1. 13C-NMR (125 MHz, CD3OD) d C: given in Table 2. Positive-ion FAB-MS: m/z 387 (M Na) . Negative-ion FAB-MS: m/z 363 (M
H)
.

Kankanoside C (3): An amorphous powder, [a]D 26
34.0° (c 1.00, MeOH). High-resolution negative-ion FAB-MS: Calcd for C15H24ClO10 (M
H)
399.1058; Found 399.1077. IR (KBr): 3410, 2964, 1159, 1078, 1048, 949 cm
1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 1. 13CNMR (125 MHz, CD3OD) d C: given in Table 2. Negative-ion FAB-MS: m/z 399, 401 (M
H)
.

Kankanoside D (4): An amorphous powder, [a]D 25
30.6° (c 0.50, MeOH). High-resolution positive-ion FAB-MS: Calcd for C15H26O7Na (M Na) 341.1204; Found 341.1210. IR (KBr): 3410, 2940, 1655, 1078, 1040 cm
1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 3. 13C-NMR (125 MHz, CD3OD) d C: given in Table 4. Positive-ion FAB-MS: m/z 341 (M Na) . Negative-ion FAB-MS: m/z 317 (M
H)
.

Kankanol (5): An amorphous powder, [a]D 25  11.1° (c 1.40, MeOH). High-resolution CI-MS: Calcd for C9H14ClO4 (M H) 221.0580; Found 221.0582. IR (KBr): 3399, 3004, 1165, 1096, 1059, 1048, 955 cm
1 . 1 H- NMR (500 MHz, CD3OD) d: given in Table 1. 13C-NMR (125 MHz, CD3OD) d C: given in Table 2. CI-MS m/z (%): 221 (M H) (5), 223 (M H) (2), 185 (88), 167 (100), 149 (71), and 57 (49).

Kankanoside E (6): An amorphous powder, [a]D 25
20.0° (c 2.00, MeOH). High-resolution positive-ion FAB-MS: Calcd for C16H28O8Na (M Na) 371.1682; Found 371.1690. UV [MeOH, nm (log e)]: 215 (4.16). IR (KBr): 3410, 2940, 1647, 1085, 1043 cm
1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 3. 13C-NMR (125 MHz, CD3OD) d C: given in Table 4. Positive-ion FAB-MS: m/z 371 (M Na) . Negative-ion FAB-MS: m/z 347 (M
H).

cistanche extract benefit

Acid Hydrolysis of 1—4 and 6 with 1 M HCl

A solution of 1—4 or 6 (each 1.5 mg) in 1 M HCl (0.5 ml) was heated under reflux for 3 h. After cooling, the reaction mixture was poured into ice water and neutralized with Amberlite IRA-400 (OH
form), and the resin was removed by filtration. Then, the filtrate was extracted with EtOAc. The aqueous layer was subjected to HPLC analysis under the following conditions: HPLC column, Kasensor 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 (75: 25, v/v); fellow rate 0.8 ml/min; column temperature, room temperature. Identification of D-glucose present in the aqueous layer was carried out by comparison of its retention time and optical rotation with those of an authentic sample: tR 12.3 min (positive optical rotation)

Enzymatic Hydrolysis of 1, 4, and 6 with b-Glucosidase

A solution of 1 (7.7 mg) in H2O (1.5 ml) was treated with b-glucosidase (5.0 mg, from almond, Oriental Yeast Co., Tokyo, Japan) and the solution was stirred at 37 °C for 3 d. After EtOH was added to the reaction mixture, the solvent was removed under reduced pressure and the residue was purified by HPLC [MeOH–H2O (55: 45, v/v)] to furnish kankagenin a (1a, 2.3 mg, 56%). Through a similar procedure, (R)-rotundiol33,34) (4a, 1.2 mg, 69%) and (2E,6R)-8-hydroxy-2,6-dimethyl-2-octanoic acid36) (6a, 6.8 mg, 62%) were obtained from 4 (3.5 mg) and 6 (20.4 mg), respectively.

Kankagenin a (1a): A white powder, [a]D 25
18.4° (c 0.20, MeOH). High-resolution EI-MS: Calcd for C10H16O3 (M ) 184.1099; Found 184.1106. IR (KBr): 3410, 2940, 1684 cm
1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 1. 13C-NMR (125 MHz, CD3OD) d C: given in Table 2. EIMS: m/z (%): 184 (M , 37), 95 (100).

Alkaline Treatment of 14 with 5% aqueous KOH

A solution of 14 (23.0 mg) in 5% aqueous KOH (1.0 ml) was stirred at 80 °C for 2 h. The reaction mixture was neutralized with Amberlite HCR-W2 (H form). Removal of the solvent from the filtrate under reduced pressure furnished a residue, which was purified by HPLC [MeOH–H2O (5: 95, v/v)] to give 2 (4.0 mg, 16%) and 1932) (10.5 mg, 43%).

Acid Treatment of 14 with 5% aqueous HCl

A solution of 14 (25.0 mg) in 5% aqueous HCl (2.0 ml) was stirred at room temperature for 3 h. The reaction mixture was poured into ice water and the whole reaction mixture was extracted with EtOAc. The EtOAc extract was successively washed with saturated aqueous NaHCO3 and brine, then dried over anhydrous MgSO4 powder and filtered. Removal of the solvent from the filtrate under reduced pressure furnished a residue, which was separated by HPLC [MeOH–H2O (20: 80, v/v)] to give 14a (4.0 mg, 25%).

14a

A white powder, [a]D 20  21.5° (c 0.30, MeOH). High-resolution CI-MS: Calcd for C9H14ClO4 (M H) 221.0580; Found 221.0587. IR (KBr): 3410, 2962, 1365, 1152, 1055, 945 cm
1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 1. 13C-NMR (125 MHz, CD3OD) d C: given in Table 2. CI-MS: m/z (%): 221 (M H) (7), 223 (M H) (3), 203 (M
H2O) (97), 205 (M
H2O) (33), 185 (7), 167 (12), 159 (32), 121(27), 110 (48), 95 (64), 85 (100), 67 (56), and 57 (65).

Acetylation of 5 and 14a

A solution of 5 (2.5 mg) in pyridine (0.5 ml) was treated with acetic anhydride (Ac2O, 0.4 ml) and the mixture was stirred at room temperature for 12 h. The reaction mixture was poured into icewater and the whole reaction mixture was extracted with EtOAc. The EtOAc extract was successively washed with 5% aqueous HCl, saturated aqueous NaHCO3, and brine then dried over anhydrous MgSO4 powder and filtered. Removal of the solvent from the filtrate under reduced pressure furnished a residue, which was purified by HPLC [MeOH–H2O (35: 65, v/v)] to give 5a (2.3 mg, 77%). Through a similar procedure, 14b (2.7 mg, 87%) was also prepared and purified by HPLC [MeOH–H2O (55: 45, v/v)] from 14a (2.1 mg).

5a

A white powder, [a]D 20  1.8° (c 0.18, MeOH). High-resolution CIMS: Calcd for C11H15O5 (M H) 227.0919; Found 227.0925. IR (KBr): 2962, 1734, 1374, 1258, 1237, 1169, 1103, 1053, 947 cm
1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 1. 13C-NMR (125 MHz, CD3OD) d C: given in Table 2. CI-MS m/z (%): 227 (M H) (28), 209 (M
H2O) (4), 184 (3), 166 (45), 149 (22), 138 (38), 122 (44), 94 (31), 85 (100), and 57 (34).

14b

A white powder, [a]D 20  23.7° (c 0.06, MeOH). High-resolution CI-MS: Calcd for C13H18ClO6 (M H) 305.0792; Found 305.0790. IR (KBr): 1744, 1376, 1243, 1231, 1001, 941 cm
1 . 1 H-NMR (500 MHz, CD3OD) d: given in Table 1. 13C-NMR (125 MHz, CD3OD) d C: given in Table 2. CI-MS: m/z (%): 305 (M H) (2), 307 (M H) (1), 263 (M H
C2H3O) (6), 265 (M H
C2H3O) (3), 245 (30), 203 (8), 185 (100), 167 (7), 149 (33), 121 (26), 95 (15), 85 (37), and 57 (93).

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. The authors thank Professor Xiaoguang Jia in Xinjiang Institute of Traditional Chinese and Ethnologic Medicines in Urumqi, China, for the identification of plant material.

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