354 Vol. 60, No. 3

© 2012 The Pharmaceutical Society of Japan

Chem. Pharm. Bull. 60(3) 354—362 (2012)

New Triterpenoid Saponins from Fruits Specimens of Panax japonicus Collected in Kumamoto and Miyazaki Prefectures (1)Kouichi Yoshizaki and Shoji Yahara*Graduate School of Pharmaceutical Sciences, Kumamoto University; 5–1 Oe-honmachi, Kumamoto, Kumamoto 862–0973, Japan.Received November 17, 2011; accepted December 20, 2011; published online December 26, 2011

Seven new dammarane-type triterpenoid saponins, chikusetsusaponin FK1 (1), chikusetsusaponin FK2 (2), chikusetsusaponin FK3 (3), chikusetsusaponin FK4 (4), chikusetsusaponin FK5 (5), chikusetsusaponin FK6 (6), and chikusetsusaponin FK7 (7), and eleven known triterpenoid saponins, ginsenoside Rb3 (9), ginsenoside Rc (10), chikusetsusaponin VI (11), ginsenoside Re (12), ginsenoside Rg1 (13), pseudo-ginsenoside RS1 (14), notoginsenoside R1 (15), chikusetsusaponin L5 (17), chikusetsusaponin L10 (18), chikusetsusaponin IVa (19), and chikusetsusaponin V (20), were isolated from the fruits of Panax japonicus C. A. MEYER, collected in Kumamoto prefecture, Japan, and two new dammarane-type triterpenoid saponin, chikusetsusaponin FK5 (5) and chikusetsusaponin FM1 (8), and five known triterpenoid saponins, ginsenoside Rb3 (9), ginsenoside Rc (10), ginsenoside Re (12), ginsenoside Rg1 (13), and floralquinquenoside E (16), were isolated from the fruits of P. japonicus C. A. MEYER, collected in Miyazaki prefecture, Japan. The structures of new chikusetsusapo-nins were elucidated on the basis of chemical and physicochemical evidences.

Key words Panax japonicus; fruit; chikusetsusaponin; triterpenoid saponin; dammarane type saponin

In continuation of chemical constituents studies on aerial parts of Panax spp., main dammarane-type triterpenoid sa-ponins from the leaves of P. japonicus C. A. MEYER (=P. pseudo-ginseng subsp. japonicus HARA) collected at 19 points in Japan, were reported.1,2) It was also reported that saponin composition of rhizome of this plant grown in southern area of Miyazaki prefecture is different from that of other area in Japan.3) But aerial parts of this plant grown in southern area of Miyazaki prefecture had not been investigated. Recently, we had opportunities to collect the fruits of P. japonicus C. A. MEYER grown in Kumamoto prefecture and southern area of Miyazaki prefecture, and investigated on saponins in these resources. In this paper, we report the isolation and structure elucidation of seven new dammarane-type triterpe-noid saponins, chikusetsusaponin FK1—FK7 (1—7), from the fruits of P. japonicus C. A. MEYER, collected in Kumamoto prefecture, Japan, together with eleven known triterpenoid saponins, ginsenoside Rb3

4) (9), ginsenoside Rc5,6) (10), chi-kusetsusaponin VI7) (11), ginsenoside Re8,9) (12), ginsenoside Rg1

10,11) (13), pseudo-ginsenoside RS19) (14), notoginsenoside

R112) (15), chikusetsusaponin L51) (17), chikusetsusaponin L10

1) (18), chikusetsusaponin IVa13) (19), and chikusetsusaponin V14) (20). In addition, we also report two new dammarane-type triterpenoid saponin, chikusetsusaponin FK5 (5) and chiku-setsusaponin FM1 (8), from the fruits of P. japonicus C. A. MEYER, collected in Miyazaki prefecture, Japan, together with five known triterpenoid saponins, ginsenoside Rb3 (9), ginsen-oside Rc (10), ginsenoside Re (12), ginsenoside Rg1 (13), and floralquinquenoside E15) (16).

Results and DiscussionThe aqueous MeOH extracts from the fruits of P. japonicus

C. A. MEYER, collected in Kumamoto and Miyazaki prefec-tures, were each subjected to reverse-phase polystyrene gel and ordinary-phase and reverse-phase silica gel column chro-matography to afford compounds 1—20.

Chikusetsusaponin FK1 (1) was a white amorphous powder with negative optical rotation ([α]D

30 −15.6° in MeOH), and

its molecular formula C48H82O18 was determined from the quasimolecular ion peak observed in the negative-ion FAB-MS and by high-resolution (HR)-FAB-MS measurement. The 1H-NMR (pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 1 showed signals assignable to be a dammarane-type tri-terpenoid part [δ 0.85, 0.87, 1.03, 1.31, 1.35, 2.11 (3H-each, all s, H3-30, 19, 18, 21, 29, 28), 1.65 (6H, s, H3-26, 27), 2.04 (1H, t, J=10.7 Hz, H-13), 3.48 (1H, dd, J=4.9, 11.0 Hz, H-3), 5.35 (1H, m, H-24)], two β-glucopyranosyl [δ 5.24 (1H, d, J=6.7 Hz, 3Glc-H-1′), 5.28 (1H, d, J=7.9 Hz, 12Glc-H-1‴)], a α-rhamnopyranosyl [δ 1.77 (3H, d, J=6.3 Hz, Rha-H-6″) 6.48 (1H, br s, Rha-H-1″)] moieties. The proton and carbon signals of 1 in the 1H- and 13C-NMR spectra were resembled to those of ginsenoside Re (12), except for the signals due to the C-12 and C-20 positions, which were similar to those of chikuset-susaponin L10 (18). This evidence indicated that 1 should be a 6,12-O-bisdesmoside of 20(S)-protopanaxatriol. The structure of 1 was characterized using 1H–1H correlation spectroscopy (1H–1H COSY), 1H–13C heteronuclear multiple-quantum co-herence (HMQC), and 1H-13C heteronuclear multiple bond correlation (HMBC) experiments. The HMBC experiment showed long-range correlations, as shown in Fig. 1. In com-parison with 1, the carbon signals of 20(S)-protopanaxatriol11) in the 13C-NMR spectra due to C-12 (δ 70.9) was displaced by +7.5 ppm (at δ 78.4), and the signals assignable to C-11 (δ 31.9) and C-13 (δ 48.1) of 20(S)-protopanaxatriol were each shielded by −4.1 ppm (at δ 27.8) and −1.7 ppm (at δ 46.4) by the β-D-glucosylation shift effects.16) The difference of molec-ular optical rotations between 1 ([M]D −147.6° in MeOH) and chikusetsusaponin L10 ([M]D +268.0° in MeOH) is −415.6°, which reveals the α-L-rhamnopyranoside ([M]D of methyl-α-L-rhamnopyranoside is −111°)17) and β-D-glucopyranoside ([M]D of methyl-β-D-glucopyranoside is −66°)17) in 1. Consequently, the structure of chikusetsusaponin FK1 (1) was confirmed as 6-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol.

Chikusetsusaponin FK2 (2) was a white amorphous pow-der with positive optical rotation ([α]D

30 +10.0° in MeOH),

Regular Article

* To whom correspondence should be addressed. e-mail: yaharas1@gpo.kumamoto-u.ac.jp

March 2012 355

and its molecular formula C48H80O18 was determined from the quasimolecular ion peak observed in the negative-ion FAB-MS and by HR-FAB-MS measurement. The 1H-NMR

(pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 2 showed signals assignable to be a dammarane-type triterpenoid part [δ 0.67 (1H, m, H-5), 0.80, 0.91, 1.10, 1.28, 1.29, 1.57, 1.62,

Fig. 1. Significant HMBC Correlations for 1—8

Chart 1. Structures of Saponins from the Fruits of P. japonicus C. A. MEYER

356 Vol. 60, No. 3

1.64 (3H-each, all s, H3-19, 30, 29, 28, 18, 21, 26, 27), 2.95 (1H, m, H-17), 3.25 (1H, dd J=4.3, 11.6 Hz, H-3), 3.61 (1H, d, J=9.2 Hz, H-13), 5.22 (1H, m, H-24)], three β-glucopyranosyl [δ 4.92 (1H, d, J=7.3 Hz, 3Glc-H-1′), 5.11 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.38 (1H, d, J=7.3 Hz, Glc-H-1″)] moieties. The proton and carbon signals of 2 in the 1H- and 13C-NMR spectra resembled those of ginsenoside Rd,5,18) except for the signals due to the C-12 position, which was similar to that of chikusetsusaponin LT8.2) This evidence indicated that 2 should be a 3, 20-O-bisdesmoside of dammar-24-ene-3β, 20(S)-diol-12-one. The structure of 2 was characterized using 1H–1H COSY, HMQC, and HMBC experiments. The HMBC experiment showed long-range correlations, as shown in Fig. 1. In comparison with 2, the carbon signals of dammar-24-ene-3β, 20(S)-diol-12-one2) in the 13C-NMR spectra due to C-3 (δ 77.9), C-4 (δ 38.0), C-20 (δ 73.3), and C-22 (δ 39.9) were each displaced by +10.8 ppm (at δ 88.7), +1.7 ppm (at δ 39.7), +8.1 ppm (at δ 81.4), and +0.7 ppm (at δ 40.6), and the signals assignable to C-2 (δ 27.9), C-17 (δ 44.4), and C-21 (δ 26.6) of dammar-24-ene-3β, 20(S)-diol-12-one were each shielded by −1.3 ppm (at δ 26.6), −1.8 ppm (at δ 42.6), and −4.1 ppm (at δ 22.5), by the β-D-glucosylation shift effects. The carbon signal of ginsenoside Rh2

19) in the 13C-NMR spectra due to C-2′ (δ 75.8) was displaced by +7.7 ppm (at δ 83.5), and the signal assignable to C-1′ (δ 106.7) of ginsenoside Rh2 was shielded by −1.6 ppm (at δ 105.1) by the β-D-glucosylation shift ef-

fects. Consequently, the structure of chikusetsusaponin FK2 (2) was confirmed as dammar-24-ene-3β, 20(S)-diol-12-one-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside-20-O-β-D-glucopyranoside.

Chikusetsusaponin FK3 (3) was a white amorphous powder with negative optical rotation ([α]D

30 −7.7° in MeOH), and its molecular formula C53H88O22 was determined from the qua-simolecular ion peak observed in the negative-ion FAB-MS and by HR-FAB-MS measurement. The 1H-NMR (pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 3 showed signals as-signable to be a dammarane-type triterpenoid part [δ 0.63 (1H, m, H-5), 0.81, 0.88, 1.10, 1.25, 1.28, 1.57, 1.63, 1.65 (3H-each, all s, H3-19, 30, 29, 28, 18, 21, 26, 27), 2.94 (1H, m, H-17), 3.20 (1H, dd J=4.3, 11.6 Hz, H-3), 3.59 (1H, d, J=9.8 Hz, H-13), 5.23 (1H, m, H-24)], three β-glucopyranosyl [δ 4.87 (1H, d, J=7.9 Hz, 3Glc-H-1′), 5.11 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.32 (1H, d, J=7.9 Hz, Glc-H-1″)], a β-xylopyranosyl [δ 4.97 (1H, d, J=7.3 Hz, Xyl-H-1″″)] moieties. The proton and carbon signals of 3 in the 1H- and 13C-NMR spectra resembled to those of chikusetsusaponin FK2 (2), except for the additional β-xylopyranosyl moiety in 3. This evidence indicated that 3 should be a 3, 20-O-bisdesmoside of dammar-24-ene-3β, 20(S)-diol-12-one. The structure of 3 was characterized using 1H–1H COSY, HMQC, HMBC, and 1H–1H totally correlated spectroscopy (1H–1H TOCSY) experiments. The HMBC ex-periment showed long-range correlations, as shown in Fig.

Table 1. 13C-NMR Data of the Aglycones of 1—8 and 4a (in C5D5N)

1 2 3 4 4a (S) 4a (R) 5 6 7 8

C-1 39.1 38.9 38.8 39.2 39.2 39.1 39.2 38.9 39.2C-2 27.9 26.6 26.7 26.7 26.7 26.6 26.7 26.8 26.7C-3 78.7 88.7 88.9 89.2 89.1 89.1 89.1 88.9 89.1C-4 39.7 39.7 39.7 39.7 39.7 39.7 39.7 39.7 39.7C-5 60.8 56.2 56.4 56.4 56.4 56.4 56.4 56.4 56.4C-6 74.5 18.5 18.6 18.5 18.5 18.5 18.5 18.4 18.5C-7 45.7 34.8 34.8 35.1 35.2 35.1 35.1 35.0 35.2C-8 41.1 40.9 40.9 40.1 40.0 (40.1) 40.0 40.1 39.9 40.0C-9 49.6 54.9 54.9 50.2 50.4 50.2 50.2 50.2 50.2C-10 40.0 37.5 37.5 36.9 37.0 36.9 37.0 37.0 36.9C-11 27.8 40.1 40.1 30.8 32.1 (32.2) 30.7 30.8 27.9 30.8C-12 78.4 211.3 211.5 70.3 71.0 (70.9) 70.2 70.2 78.5 70.5C-13 46.4 56.5 56.4 49.4 48.5 (49.2) 49.4 49.5 46.8 49.5C-14 52.2 56.3 56.3 51.5 51.7 (51.8) 51.4 51.5 52.2 51.5C-15 31.3 32.3 32.3 30.8 31.4 (31.4) 30.8 31.0 31.3 31.0C-16 27.2 24.0 24.0 26.8 26.9 26.8 26.9 27.2 26.8C-17 54.1 42.6 42.6 51.8 54.8 (50.7) 51.7 51.9 54.1 51.9C-18 17.0 16.0 16.0 16.0 16.4 16.0 16.0 16.3 16.0C-19 17.5 16.3 16.3 16.3 15.9 16.2 16.3 15.7 16.3C-20 73.0 81.4 81.4 83.5 72.9 83.5 83.3 73.0 83.9C-21 26.8 22.5 22.5 22.4 27.1 (22.8) 22.3 22.5 26.9 22.8C-22 36.4 40.6 40.6 36.2 36.0 (43.3) 36.2 36.2 36.5 34.2C-23 23.0 24.6 24.6 23.3 23.0 (22.7) 23.2 23.3 23.0 27.0C-24 126.6 125.8 125.8 126.1 126.4 (126.2) 126.0 126.0 126.6 79.7C-25 130.6 130.9 130.9 131.0 130.8 131.0 130.9 130.9 73.0C-26 25.8 25.8 25.8 25.8 25.8 (25.9) 25.8 25.8 25.8 26.3a)

C-27 17.7 17.8 17.8 17.9 17.7 (17.1) 17.9 17.8 17.7 26.0a)

C-28 32.2 28.0 28.0 28.1 28.1 28.1 28.2 28.1 28.1C-29 17.6 16.5 16.5 16.6 16.6 16.6 16.7 16.6 16.6C-30 17.3 17.0 17.0 17.4 17.4 17.4 17.4 17.4 17.3

a) Interchangeable values.

March 2012 357

1. In comparison with 3, the carbon signals of dammar-24-ene-3β, 20(S)-diol-12-one in the 13C-NMR spectra due to C-3 (δ 77.9), C-4 (δ 38.0), C-20 (δ 73.3), and C-22 (δ 39.9) were each displaced by +11.0 ppm (at δ 88.9), +1.7 ppm (at δ 39.7), +8.1 ppm (at δ 81.4), and +0.7 ppm (at δ 40.6), and the signals assignable to C-2 (δ 27.9), C-17 (δ 44.4), and C-21 (δ 26.6) of dammar-24-ene-3β, 20(S)-diol-12-one were each shielded by −1.2 ppm (at δ 26.7), −1.8 ppm (at δ 42.6), and −4.1 ppm (at δ 22.5), by the β-D-glucosylation shift effects. The carbon signal of ginsenoside Rh2 in the 13C-NMR spectra due to C-2′ (δ 75.8) was displaced by +7.3 ppm (at δ 83.1), and the signal assignable to C-1′ (δ 106.7) of ginsenoside Rh2 was shielded by −1.7 ppm (at δ 105.0) by the β-D-glucosylation shift effects. The difference of molecular optical rotations between 3 ([M]D −82.9° in MeOH) and 2 ([M]D +94.4° in MeOH) is −177.3°, which reveals the β-D-xylopyranoside ([M]D of methyl-β-D-xylopyranoside is −108°)17) in 3. Consequently, the structure of chikusetsusaponin FK3 (3) was confirmed as dammar-24-ene-3β, 20(S)-diol-12-one-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranoside-20-O-β-D-gluco-pyranoside.

Chikusetsusaponin FK4 (4) was a white amorphous powder with negative optical rotation ([α]D

27 −14.2° in MeOH), and its molecular formula C58H98O26 was determined from the qua-simolecular ion peak observed in the negative-ion FAB-MS and by HR-FAB-MS measurement. The 1H-NMR (pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 4 showed signals assignable to be a dammarane-type triterpenoid part [δ 0.61

(1H, m, H-5), 0.80, 0.91, 0.94, 1.10, 1.24, 1.67 (3H-each, all s, H3-19, 30, 18, 29, 28, 27), 1.64 (6H, s, H3-21, 26), 2.59 (1H, m, H-17), 3.21 (1H, dd, J=4.3, 11.6 Hz, H-3), 5.33 (1H, m, H-24), 5.58 (1H, br s, 12-OH)], an α-arabinofuranosyl [δ 5.66 (1H, br s, Ara-H-1″‴)], three β-glucopyranosyl [δ 4.87 (1H, d, J=7.9 Hz, 3Glc-H-1′), 5.15 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.32 (1H, d, J=7.9 Hz, Glc-H-1″)], a β-xylopyranosyl [δ 4.97 (1H, d, J=7.3 Hz, Xyl-H-1″″)] moieties. The proton and carbon signals of 4 in the 1H- and 13C-NMR spectra resembled to those of ginsenoside Rc (10), except for the additional β-xylopyranosyl moiety of 4. This evidence indicated that 4 should be a 3,20-O-bisdesmoside of 20(S)-protopanaxadiol. The structure of 4 was characterized using 1H–1H COSY, HMQC, HMBC, and 1H–1H TOCSY experiments. The HMBC experiment showed long-range correlations, as shown in Fig. 1. In com-parison with 4, the carbon signals of 20(S)-protopanaxadiol11) in the 13C-NMR spectra due to C-3 (δ 77.9), C-4 (δ 39.5), C-20 (δ 72.9), and C-22 (δ 35.8) were each displaced by +11.3 ppm (at δ 89.2), +0.2 ppm (at δ 39.7), +10.6 ppm (at δ 83.5), and +0.4 ppm (at δ 36.2), and the signals assignable to C-2 (δ 28.2), C-17 (δ 54.7), and C-21 (δ 26.9) of 20(S)-protopanaxadiol were each shielded by −1.5 ppm (at δ 26.7), −2.9 ppm (at δ 51.8), and −4.5 ppm (at δ 22.4), by the β-D-glucosylation shift ef-fects. The carbon signal of ginsenoside Rh2 in the 13C-NMR spectra due to C-2′ (δ 75.8) was displaced by +7.4 ppm (at δ 83.2), and the signal assignable to C-1′ (δ 106.7) of ginsen-oside Rh2 was shielded by −1.6 ppm (at δ 105.1) by the β-D-glucosylation shift effects. The difference of molecular optical

Table 2. 13C-NMR Data of the Sugar Part of 1—8 and 4a (in C5D5N)

1 2 3 4 4a 5 6 7 8

3(6)Glc-C-1′ 101.8 105.1 105.0 105.1 105.1 105.0 105.1 105.1 105.13(6)Glc-C-2′ 79.4 83.5 83.1 83.2 83.2 83.1 83.3 83.5 83.33(6)Glc-C-3′ 78.3a) 78.4a) 78.2a) 78.3a) 78.2a) 78.2a) 78.3a) 78.4a) 78.4a)

3(6)Glc-C-4′ 72.3b) 71.7b) 71.3 71.3 71.4 71.3 71.4 71.7b) 71.7b)

3(6)Glc-C-5′ 78.6 78.0a) 76.5 76.6 76.6 76.4 76.5 78.2a) 78.1a)

3(6)Glc-C-6′ 63.1 62.9 70.0 70.1 70.0 70.0 70.1 62.8 62.9Glc(Rha)-C-1″ 101.9 106.1 105.9 106.0 106.0 105.9 106.1 106.1 106.0Glc(Rha)-C-2″ 72.6 77.1 77.0 77.0 77.0 77.0 77.1 77.2 77.1Glc(Rha)-C-3″ 72.3b) 78.0a) 78.3a) 78.3a) 78.3a) 78.2a) 78.3a) 78.0a) 77.9a)

Glc(Rha)-C-4″ 74.2 71.8b) 71.8b) 71.8b) 71.8 71.7b) 71.7b) 71.8b) 71.8b)

Glc(Rha)-C-5″ 69.5 78.3a) 78.0a) 78.0a) 78.0a) 77.9a) 78.1a) 78.3a) 78.3a)

Glc(Rha)-C-6″ 18.8 62.8 62.8 62.8 62.8 62.8 62.9 62.7 62.812(20)Glc-C-1‴ 100.4 98.5 98.5 98.1 98.1 98.4 100.5 98.112(20)Glc-C-2‴ 75.3 75.7 75.7 75.1 74.9 75.2 75.3 74.912(20)Glc-C-3‴ 78.5a) 79.3 79.3 79.2 79.2 79.3 78.6 79.012(20)Glc-C-4‴ 71.2 72.0b) 72.0b) 72.2b) 71.6b) 71.8b) 71.3 71.6b)

12(20)Glc-C-5‴ 77.5 78.2a) 78.2a) 76.5 76.9 78.0 77.7 76.812(20)Glc-C-6‴ 62.6 63.1 63.1 68.5 70.0 62.8 63.0 69.9Xyl(Ara)-C-1″″ 106.0 105.9 105.9 105.9 106.1 105.5Xyl(Ara)-C-2″″ 74.8 74.8 74.8 74.8c) 74.8 74.8Xyl(Ara)-C-3″″ 78.2a) 78.1a) 78.1a) 78.0a) 78.2a) 78.0a)

Xyl(Ara)-C-4″″ 71.2 71.2 71.2 71.1d) 71.2 71.1Xyl(Ara)-C-5″″ 67.1 67.1 67.1 67.0 67.1 66.9Ara(Xyl)-C-1″‴ 110.1 105.8Ara(Xyl)-C-2″‴ 83.3 74.7c)

Ara(Xyl)-C-3″‴ 78.9 78.0a)

Ara(Xyl)-C-4″‴ 86.1 71.1d)

Ara(Xyl)-C-5″‴ 62.7 66.9

a—d) Interchangeable values in each vertical column.

358 Vol. 60, No. 3

rotations between 4 ([M]D −171.8° in MeOH) and ginsenoside Rd ([M]D +183.5° in MeOH) is −355.3°, which reveals the α-L-arabinofuranoside ([M]D of methyl-α-L-arabinofuranoside is −226°)5) and β-D-xylopyranoside in 4. On mild acid hy-drolysis with aqueous acetic acid (Fig. 2), 13C-NMR (Tables 1, 2) spectra of reaction product (4a) showed signal assign-able to be a 20-epimeric pair of protopanaxadiol part, two β-glucopyranosyl and a β-xylopyranosyl moieties. Compared with 4, the carbon signals due to α-L-arabinofuranoside which connect to C-6‴ and β-D-glucopyranoside which connect to C-20 were not observed in 4a. And the carbon signals due to 3-O-glycoside moieties of 4a were exactly same as those of 4. This evidence indicated that 4a must be 20-epi-meric pair of chikusetsusaponin III.7,19,20) Consequently, the structure of chikusetsusaponin FK4 (4) was confirmed as 3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl-20-O-α-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl-20(S)-protopanaxadiol.

Chikusetsusaponin FK5 (5) was a white amorphous pow-der with negative optical rotation ([α]D

27 −6.9° in MeOH), and its molecular formula C58H98O26 was determined from the quasimolecular ion peak observed in the negative-ion FAB-MS and by HR-FAB-MS measurement. The 1H-NMR (pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 5 showed signal assignable to be a dammarane-type triterpenoid part [δ 0.61 (1H, m, H-5), 0.80, 0.93, 0.94, 1.10, 1.25, 1.62, 1.64, 1.66 (3H-each, all s, H3-19, 30, 18, 29, 28, 26, 21, 27), 2.56 (1H, m, H-17), 3.21 (1H, dd, J=4.0, 11.3 Hz, H-3), 5.33 (1H, m, H-24), 5.55 (1H, brs, 12-OH)], three β-glucopyranosyl [δ 4.87 (1H, d, J=7.3 Hz, 3Glc-H-1′), 5.13 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.33 (1H, d, J=7.3 Hz, Glc-H-1″)], two β-xylopyranosyl [δ 4.94 (1H, d, J=7.3 Hz, Xyl-H-1″″), 4.99 (1H, d, J=7.3 Hz, Xyl-H-1″‴)] moieties. The proton and carbon signals of 5 in the 1H- and 13C-NMR spectra resembled to those of ginsenoside Rb3 (9), except for the additional β-xylopyranosyl moiety of 5. This evidence indicated that 5 should be a 3,20-O-bisdesmoside of 20(S)-protopanaxadiol. The structure of 5 was character-ized using 1H–1H COSY, HMQC, HMBC, and 1H–1H TOCSY experiments. The HMBC experiment showed long-range correlations, as shown in Fig. 1. In comparison with 5, the carbon signals of 20(S)-protopanaxadiol in the 13C-NMR spectra due to C-3 (δ 77.9), C-4 (δ 39.5), C-20 (δ 72.9), and C-22 (δ 35.8) were each displaced by +11.2 ppm (at δ 89.1), +0.2 ppm (at δ 39.7), +10.6 ppm (at δ 83.5), and +0.4 ppm (at δ 36.2), and the signals assignable to C-2 (δ 28.2), C-17 (δ 54.7), and C-21 (δ 26.9) of 20(S)-protopanaxadiol were each shielded by −1.6 ppm (at δ 26.6), −3.0 ppm (at δ 51.7), and −4.6 ppm (at δ 22.3), by the β-D-glucosylation shift ef-fects. The carbon signal of ginsenoside Rh2 in the 13C-NMR

spectra due to C-2′ (δ 75.8) was displaced by +7.3 ppm (at δ 83.1), and the signal assignable to C-1′ (δ 106.7) of ginseno-side Rh2 was shielded by −1.7 ppm (at δ 105.0) by the β-D-glucosylation shift effects. The difference of molecular optical rotations between 5 ([M]D −83.5° in MeOH) and ginsenoside Rd ([M]D +183.5° in MeOH) is −267.0°, which reveals the β-D-xylopyranoside in 5. On mild acid hydrolysis of 5 with aqueous acetic acid, it gave 4a. Compared with 5, the carbon signals due to β-D-xylopyranoside which connect to C-6‴ and β-D-glucopyrano- side which connect to C-20 were not ob-serve in 4a. Consequently, the structure of chikusetsusaponin FK5 (5) was confirmed as 3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl-20-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-20(S)-protopanaxa-diol.

Chikusetsusaponin FK6 (6) was a white amorphous powder with positive optical rotation ([α]D

15 +2.8° in MeOH), and its molecular formula C53H88O22 was determined from the qua-simolecular ion peak observed in the negative-ion FAB-MS and by HR-FAB-MS measurement. The 1H-NMR (pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 6 showed signals assignable to be a dammarane-type triterpenoid part [δ 0.62 (1H, m, H-5), 0.81, 0.92, 0.94, 1.12, 1.26, 1.63 (3H-each, all s, H3-19, 30, 18, 29, 28, 21), 1.61 (3H, s, H3-26, 27), 2.54 (1H, m, H-17), 3.22 (1H, dd, J=4.3, 11.6 Hz, H-3), 5.26 (1H, m, H-24), 5.57 (1H, br s, 12-OH)], three β-glucopyranosyl [δ 4.88 (1H, d, J=7.3 Hz, 3Glc-H-1′), 5.21 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.34 (1H, d, J=7.9 Hz, Glc-H-1″)], a β-xylopyranosyl [δ 4.95 (1H, d, J=7.3 Hz, Xyl-H-1″″)] moieties. The proton and carbon signals of 6 in the 1H- and 13C-NMR spectra resembled to those of chikusetsusaponin FK3 (3), except for the signals due to the C-12 position, which was similar as that of 4 or 5. This evidence indicated that 6 should be a 3,20-O-bisdesmoside of 20(S)-protopanaxadiol. The structure of 6 was character-ized using 1H–1H COSY, HMQC, HMBC, and 1H–1H TOCSY experiments. The HMBC experiment showed long-range cor-relations, as shown in Fig. 1. In comparison with 6, the carbon signals of 20(S)-protopanaxadiol in the 13C-NMR spectra due to C-3 (δ 77.9), C-4 (δ 39.5), C-20 (δ 72.9), and C-22 (δ 35.8) were each displaced by +11.2 ppm (at δ 89.1), +0.2 ppm (at δ 39.7), +10.4 ppm (at δ 83.3), and +0.4 ppm (at δ 36.2), and the signals assignable to C-2 (δ 28.2), C-17 (δ 54.7), and C-21 (δ 26.9) of 20(S)-protopanaxadiol were each shielded by -1.5 ppm (at δ 26.7), −2.8 ppm (at δ 51.9), and −4.4 ppm (at δ 22.5), by the β-D-glucosylation shift effects. The carbon signal of ginsenoside Rh2 in the 13C-NMR spectra due to C-2′ (δ 75.8) was displaced by +7.5 ppm (at δ 83.3), and the signal assignable to C-1′ (δ 106.7) of ginsenoside Rh2 was shielded by −1.6 ppm (at δ 105.1) by the β-D-glucosylation shift ef-

Fig. 2. Partial Hydrolysis of 4 and 5

March 2012 359

fects. The difference of molecular optical rotations between 6 ([M]D +30.2° in MeOH) and ginsenoside Rd ([M]D +183.5° in MeOH) is −153.3°, which reveals the β-D-xylopyranoside in 6. Consequently, the structure of chikusetsusaponin FK6 (6) was confirmed as 3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl-20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol.

Chikusetsusaponin FK7 (7) was a white amorphous powder with positive optical rotation ([α]D

30 +2.3° in MeOH), and its molecular formula C48H82O18 was determined from the qua-simolecular ion peak observed in the negative-ion FAB-MS and by HR-FAB-MS measurement. The 1H-NMR (pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 7 showed signals assignable to be a dammarane-type triterpenoid part [δ 0.64 (1H, m, H-5), 0.69, 0.78, 0.85, 1.10, 1.29, 1.35 (3H-each, all s, H3-19, 18, 30, 29, 28, 21), 1.65 (6H, s, H3-26, 27), 2.06 (1H, t, J=10.7 Hz, H-13), 2.39 (1H, m, H-17), 3.28 (1H, J=4.3, 11.6 Hz, H-3)], three β-glucopyranosyl [δ 4.96 (1H, d, J=7.9 Hz, 3Glc-H-1′), 5.27 (1H, d, J=7.9 Hz, 12Glc-H-1‴), 5.40 (1H, d, J=7.3 Hz, Glc-H-1″)] moieties. The proton and carbon signals of 7 in the 1H- and 13C-NMR spectra resembled to those of ginsenoside Rd, except for the signals due to the C-12 and C-20 positions, which were similar to that of chikusetsusaponin FK1 (1). This evidence indicated that 7 should be a 3, 12-O-bisdesmoside of 20(S)-protopanaxadiol. The structure of 7 was characterized using 1H–1H COSY, HMQC, and HMBC experiments. The HMBC experiment showed long-range correlations, as shown in Fig. 1. In com-parison with 7, the carbon signals of 20(S)-protopanaxadiol in the 13C-NMR spectra due to C-3 (δ 77.9), C-4 (δ 39.5), and C-12 (δ 70.9) were each displaced by +11.0 ppm (at δ 88.9), +0.2 ppm (at δ 39.7), and +7.6 ppm (at δ 78.5), and the signals assignable to C-2 (δ 28.2), C-11 (δ 32.0), and C-13 (δ 48.5) of 20(S)-protopanaxadiol were each shielded by −1.4 ppm (at δ 26.8), −4.1 ppm (at δ 27.9), and −1.7 ppm (at δ 46.8), by the β-D-glucosylation shift effects. The carbon signal of ginseno-side Rh2 in the 13C-NMR spectra due to C-2′ (δ 75.8) was displaced by +7.7 ppm (at δ 83.5), and the signal assignable to C-1′ (δ 106.7) of ginsenoside Rh2 was shielded by −1.6 ppm (at δ 105.1) by the β-D-glucosylation shift effects. Consequently, the structure of chikusetsusaponin FK7 (7) was confirmed as 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol.

Chikusetsusaponin FM1 (8) was a white amorphous powder with negaitive optical rotation ([α]D

15 −2.5° in MeOH), and its molecular formula C53H92O24 was determined from the quasi-molecular ion peak observed in the negative-ion FAB-MS and by HR-FAB-MS measurement. The 1H-NMR (pyridine-d5) and 13C-NMR (Tables 1, 2) spectra of 8 showed signals as-signable to be a dammarane-type triterpenoid part [δ 0.67 (1H, m, H-5), 0.81, 0.93, 0.95, 1.10, 1.28, 1.64 (3H-each, all s, H3-19, 30, 18, 29, 28, 21), 1.55, 1.55 (3H each, both s, H3-26, 27), 2.51 (1H, m, H-17), 3.27 (1H, dd, J=4.9, 11.3 Hz, H-3), 3.82 (1H, m, H-24)], three β-glucopyranosyl [δ 4.91 (1H, d, J=7.9 Hz, 3Glc-H-1′), 5.11 (1H, d, J=7.9 Hz, 20Glc-H-1‴), 5.37 (1H, d, J=7.3 Hz, Glc-H-1″), an β-xylopyranosyl [δ 4.97 (1H, d, J=7.3 Hz, Xyl-H-1″″)] moieties. The proton and carbon signals of 8 in the 1H- and 13C-NMR spectra resembled to those of ginsenoside Rb3 (9), except for the signals due to the side chain part (C-24—C-27), which were similar to those of vina-ginsenoside R13.21) This evidence indicated that 8 should

be a 3,20-O-bisdesmoside of dammar-3β,12β,20(S),24,25-pentol. The structure of 8 was characterized using 1H–1H COSY, HMQC, and HMBC experiments. The HMBC ex-periment showed long-range correlations, as shown in Fig. 1. In comparison with 8, the carbon signals of dammar-3β,12β,20(S),24,25-pentol21) in the 13C-NMR spectra due to C-3 (δ 77.9), C-4 (δ 39.5), C-20 (δ 73.4), and C-22 (δ 33.5) were each displaced by +11.2 ppm (at δ 89.1), +0.2 ppm (at δ 39.7), +10.5 ppm (at δ 83.9), and +0.7 ppm (at δ 34.2), and the signals assignable to C-2 (δ 28.2), C-17 (δ 54.9), and C-21 (δ 27.3) of dammar-3β,12β,20(S),24,25-pentol were each shielded by −1.5 ppm (at δ 26.7), −3.0 ppm (at δ 51.9), and −4.5 ppm (at δ 22.8), by the β-D-glucosylation shift effects. The carbon signal of ginsenoside Rh2 in the 13C-NMR spectra due to C-2′ (δ 75.8) was displaced by +7.5 ppm (at δ 83.3), and the signal assignable to C-1′ (δ 106.7) of ginsenoside Rh2 was shielded by −1.6 ppm (at δ 105.1) by the β-D-glucosylation shift effects. The difference of molecular optical rotations between 8 ([M]D −27.8° in MeOH) and vina-ginsenoside R13 ([M]D +21.6° in MeOH) is −49.4°, which reveals the β-D-xylopyranoside in 8. Consequently, the structure of chikusetsusaponin FM1 (8) was confirmed as dammar-3β,12β,20(S),24,25-pentol-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside-20-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside.

At this time, we found that there are remarkable differences on saponin composition between fruits of P. japonicus C. A. MEYER collected at southern area of Miyazaki prefecture and that of Kumamoto prefecture. Main saponins in the fruits of P. japonicus C. A. MEYER collected in Kumamoto prefecture were chikusetsusaponins FK4 and FK5. On the other hand, ginsenoside Rb3 and Rc were found as main constituents in the sample from the southern area of Miyazaki prefecture. From a geographical point of view, further study on the sa-ponins in the fruits of P. japonicus C. A. MEYER grown other area in Japan is in progress.

ExperimentalThe following instruments were used to obtain physical

data: specific rotations, JASCO DIP-1000KUY digital po-larimeter (l=5 cm); FAB-MS and high-resolution MS, JEOL JMS-700 MStation spectrometer; 1H-NMR spectra, JEOL α-500 (500 MHz) spectrometer; 13C-NMR spectra, JEOL α-500 (125 MHz) spectrometer; and 1H–1H COSY, TOCSY, HMQC and HMBC spectra, JEOL α-500 spectrometer, with tetramethylsilane (TMS) as an internal standard.

The following experimental conditions were used for chromatography; reverse-phase polystyrene gel column chro-matography, MCI GEL CHP20P (Mitsubishi Kasai Co., 75—150 μm); ordinary-phase silica gel column chromatography, Silica gel 60 (Merck, 0.040—0.063 mm); reverse-phase silica gel column chromatography, Chromatrex ODS (Fuji Silysia Chemical, Ltd., 30—50 μm); pre-coated TLC plates with Silica gel 60 F254 (Merck, 0.2 mm)(ordinary phase); and detection was achieved by spraying with 10% aqueous H2SO4 followed by heating.

Plant Materials Fresh fruits of P. japonicus were col-lected in July 27, 2007 at Minamioguni-machi, Aso-gun, Kumamoto prefecture, Japan, and in July 16, 2006 at Kitagou, Naka-gun, Miyazaki prefecture, Japan. These voucher specimens have been deposited at Medicinal Plant Garden in Kumamoto University, Kumamoto, Japan.

360 Vol. 60, No. 3

Isolation of Saponins from the Dried Fruits of P. japoni-cus Collected in Kumamoto Prefecture The freeze-dried fruits of P. japonicus C. A. MEYER (17.6 g) were extracted with hot 50% aqueous MeOH and then with hot 90% aque-ous MeOH. Evaporation of the solvent under reduced pres-sure provided the methanolic extract (4.0 g). The methanolic extract (4.0 g) was subjected to reverse-phase polystyrene gel column chromatography [H2O→MeOH–H2O (30 : 70→40 : 60→50 : 50→60 : 40→70 : 30, v/v)→MeOH] to give fractions 1—8. Fraction 5 (615 mg) was separated by reverse-phase silica gel column chromatography [MeOH–H2O (50 : 50→55 : 45→60 : 40→65 : 35→70 : 30→75 : 25, v/v)→MeOH] to give 17 fractions. Fraction 5-2 (51 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (8 : 2.5 : 0.2, v/v)] to give notoginsenoside R1 (15, 3 mg) and chikusetsu-saponin V (20, 10 mg). Fraction 5-3 (96 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (8 : 2.5 : 0.2, v/v)] to give 7 fractions, include gin-senoside Rg1 (fr. 5-3-2, 13, 11 mg). Fraction 5-3-4 (63 mg) was separated by reverse-phase silica gel column chromatography [MeOH–H2O (51 : 49→52 : 48→53 : 47, v/v)] to give ginsenoside Re (12, 57 mg). Fraction 5-4 (66 mg) was separated by ordi-nary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.3, v/v)] to give chikusetsusaponin FK1 (1, 6 mg). Fraction 5-5 (48 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (8 : 2.5 : 0.2, v/v)] to give pseudo-ginsenoside RS1 (14, 19 mg). Fraction 5-7 (46 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give chikusetsusaponin L5 (17, 3 mg). Fraction 5-10 (12 mg) was separated by ordinary-phase silica gel column chromatogra-phy [CHCl3–MeOH–H2O (8 : 2.5 : 0.2, v/v)] to give chikuset-susaponin FK2 (2, 5 mg) and chikusetsusaponin FK3 (3, 2 mg). Fraction 5-12 (45 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give chikusetsusaponin FK4 (4, 33 mg) and chikuset-susaponin VI (11, 9 mg). Fraction 5-13 (82 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give ginsenoside Rc (10, 3 mg), chikusetsusaponin FK4 (4, 61 mg), and chikusetsusaponin VI (11, 10 mg). Fraction 5-15 (65 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give ginsenoside Rb3 (9, 6 mg) and chikuset-susaponin FK5 (5, 52 mg). Fraction 5-16 (8 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give chikusetsusaponin FK6 (6, 5 mg). Fraction 6 (143 mg) was separated by reverse-phase silica gel column chromatography [MeOH–H2O (50 : 50→55 : 45→60 : 40→65 : 35→70 : 30→75 : 25, v/v)→MeOH] to give 16 fractions, include chikusetsusaponin IVa (fr. 6-2, 19, 6 mg), chikusetsusaponin FK2 (fr. 6-7, 2, 3 mg), and chikusetsusapo-nin L10 (fr. 6-9, 18, 4 mg). Fraction 6-11 (30 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give ginsenoside Rc (10, 4 mg) and chikusetsusaponin FK4 (4, 20 mg). Fraction 6-13 (56 mg) was separated by ordinary-phase silica gel column chromatog-raphy [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give ginsenoside Rb3 (9, 7 mg) and chikusetsusaponin FK5 (5, 35 mg). Fraction 6-15 (15 mg) was separated by ordinary-phase silica gel col-umn chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give chikusetsusaponin FK6 (6, 12 mg). Fraction 7 (70 mg)

was separated by reverse-phase silica gel column chromatog-raphy [MeOH–H2O (60 : 40→65 : 35→70 : 30→75 : 25→80 : 20→85 : 15→90 : 10, v/v)→MeOH] to give 9 fractions. Fraction 7-4 (5 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give chikusetsusaponin FK6 (6, 1 mg). Fraction 7-6 (6 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (8 : 2.5 : 0.2, v/v)] to give chikusetsusapo-nin FK7 (7, 3 mg).

Isolation of Saponins from the Fresh Fruits of P. ja-ponicus Collected in Miyazaki Prefecture The fresh fruits of P. japonicus C. A. MEYER (34 g) were extracted with hot 60% aqueous MeOH and then with hot MeOH. Evaporation of the solvent under reduced pressure provided the methanolic extract (4.3 g). The methanolic extract (4.3 g) was subjected to reverse-phase polystyrene gel column chromatography [H2O→MeOH–H2O (30 : 70→40 : 60→70 : 30, v/v)→MeOH] to give fractions 1—7. Fraction 4 (235 mg) was separated by reverse-phase silica gel column chromatography [MeOH–H2O (70 : 30, v/v)] to give 4 fractions. Fraction 4-2 (143 mg) was separated by reverse-phase silica gel column chromatogra-phy [MeOH–H2O (60 : 40→63 : 37, v/v)→MeOH] to give 6 fractions. Fraction 4-2-1 (72 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.5→7 : 3.2 : 0.5, v/v)] to give ginsenoside Rg1 (13, 3 mg), ginsenoside Re (12, 25 mg), and floralquinquenoside E (16, 8 mg). Fraction 6 (413 mg) was separated by reverse-phase silica gel column chromatography [MeOH–H2O (50 : 50→60 : 40→65 : 35→70 : 30→75 : 25→85 : 15, v/v)→MeOH] to give 13 fractions, include ginsenoside Rb3 (fr. 6-10, 9, 114 mg). Fraction 6-8 (85 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (6 : 3.5 : 0.6, v/v)] to give ginsenoside Rc (10, 73 mg). Fraction 6-9 (59 mg) was separated by ordinary-phase silica gel column chromatog-raphy [CHCl3–MeOH–H2O (7 : 3 : 0.5, v/v)] to give 7 fractions. Fraction 6-9-4 (24 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give chikusetsusaponin FK5 (5, 12 mg).

The fresh fruits of P. japonicus C. A. MEYER (36 g) were extracted with hot 60% aqueous MeOH and then with hot MeOH. Evaporation of the solvent under reduced pressure provided the methanolic extract (3.9 g). The methanolic extract (3.9 g) was subjected to reverse-phase polystyrene gel column chromatography [H2O→MeOH–H2O (30 : 70→40 : 60→70 : 30, v/v)→MeOH] to give fractions 1—8. Fraction 6 (685 mg) was separated by reverse-phase silica gel column chromatography [MeOH–H2O (50 : 50→60 : 40→70 : 30→90 : 10, v/v)] to give 19 fractions. Fraction 6-7 (47 mg) was separated by ordinary-phase silica gel column chromatography [CHCl3–MeOH–H2O (7 : 3 : 0.4, v/v)] to give ginsenoside Re (12, 5 mg). Fraction 6-11 (47 mg) was separated by ordinary-phase silica gel col-umn chromatography [CHCl3–MeOH–H2O (7 : 3.5 : 0.5, v/v)] to give chikusetsusaponin FM1 (8, 24 mg).

Chikusetsusaponin FK1 (1): A white amorphous pow-der; [α]D

30 −15.6° (c=0.57, MeOH); 1H-NMR (pyridine-d5, 500 MHz) δ: 0.85, 0.87, 1.03, 1.31, 1.35, 2.11 (3H each, all s, H3-30, 19, 18, 21, 29, 28), 1.65 (6H, s, H3-26, 27), 1.77 (3H, d, J=6.3 Hz, Rha-H3-6″), 2.04 (1H, t, J=10.7 Hz, H-13), 3.48 (1H, dd, J=4.9, 11.0 Hz, H-3), 5.24 (1H, d, J=6.7 Hz, 3Glc-H-1′), 5.28 (1H, d, J=7.9 Hz, 12Glc-H-1‴), 5.35 (1H, m, H-24), 6.48 (1H, br s, Rha-H-1″); 13C-NMR data, see Tables 1, 2; negative-

March 2012 361

ion FAB-MS m/z 945 (M−H)−; negative-ion HR-FAB-MS: m/z 945.5469 (Calcd for C48H81O18 [M−H]−, 945.5423).

Chikusetsusaponin FK2 (2): A white amorphous pow-der; [α]D

30 +10.0° (c=0.87, MeOH); 1H-NMR (pyridine-d5, 500 MHz) δ: 0.67 (1H, m, H-5), 0.80, 0.91, 1.10, 1.28, 1.29, 1.57, 1.62, 1.64 (3H each, all s, H3-19, 30, 29, 28, 18, 21, 26, 27), 2.95 (1H, m, H-17), 3.25 (1H, dd J=4.3, 11.6 Hz, H-3), 3.61 (1H, d, J=9.2 Hz, H-13), 4.92 (1H, d, J=7.3 Hz, 3Glc-H-1′), 5.11 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.38 (1H, d, J=7.3 Hz, Glc-H-1″), 5.22 (1H, m, H-24); 13C-NMR data, see Tables 1, 2; negative-ion FAB-MS m/z 943 (M−H)−, 781 (M−C6H11O5)−; negative-ion HR-FAB-MS: m/z 943.5219 (Calcd for C48H79O18 [M−H]−, 943.5226).

Chikusetsusaponin FK3 (3): A white amorphous powder; [α]D

30 −7.7° (c=0.62, MeOH); 1H-NMR (pyridine-d5, 500 MHz) δ: 0.63 (1H, m, H-5), 0.81, 0.88, 1.10, 1.25, 1.28, 1.57, 1.63, 1.65 (3H each, all s, H3-19, 30, 29, 28, 18, 21, 26, 27), 2.94 (1H, m, H-17), 3.20 (1H, dd J=4.3, 11.6 Hz, H-3), 3.59 (1H, d, J= 9.8 Hz, H-13), 4.87 (1H, d, J=7.9 Hz, 3Glc-H-1′), 4.97 (1H, d, J=7.3 Hz, Xyl-H-1″″), 5.11 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.32 (1H, d, J=7.9 Hz, Glc-H-1‴), 5.23 (1H, m, H-24); 13C-NMR data, see Tables 1, 2; negative-ion FAB-MS m/z 1075 (M−H)−, 943 (M−C5H9O4)−, 913 (M−C6H11O5)−; negative-ion HR-FAB-MS: m/z 1075.5638 (Calcd for C53H87O22 [M−H]−, 1075.5689).

Chikusetsusaponin FK4 (4): A white amorphous pow-der; [α]D

27 −14.2° (c=0.92, MeOH); 1H-NMR (pyridine-d5, 500 MHz) δ: 0.61 (1H, m, H-5), 0.80, 0.91, 0.94, 1.10, 1.24, 1.67 (3H each, all s, H3-19, 30, 18, 29, 28, 27), 1.64 (6H, s, H3-21, 26), 2.59 (1H, m, H-17), 3.21 (1H, dd, J=4.3 Hz, 11.6 Hz, H-3), 4.87 (1H, d, J=7.3, 3Glc-H-1′), 4.95 (1H, d, J=7.3 Hz, Xyl-H-1″″), 5.15 (1H, d, J=7.9 Hz, 20Glc-H-1‴), 5.32 (1H, d, J=7.3 Hz, Glc-H-1″), 5.33 (1H, m, H-24), 5.58 (1H, br s, 12-OH), 5.66 (1H, br s, Ara-H-1″‴); 13C-NMR data, see Tables 1, 2; negative-ion FAB-MS m/z 1209 (M−H)−, 1077 (M−C5H9O4)−, 1047 (M−C6H11O5)−, 915 (M−C11H19O9)−; negative-ion HR-FAB-MS: m/z 1209.6237 (Calcd for C58H97O26 [M−H]−, 1209.6268).

Chikusetsusaponin FK5 (5): A white amorphous pow-der; [α]D

27 −6.9° (c=0.92, MeOH); 1H-NMR (pyridine-d5, 500 MHz) δ: 0.61 (1H, m, H-5), 0.80, 0.93, 0.94, 1.10, 1.25, 1.62, 1.64, 1.66 (3H each, all s, H3-19, 30, 18, 29, 28, 26, 21, 27), 2.56 (1H, m, H-17), 3.21 (1H, dd, J=4.0 Hz, 11.3 Hz, H-3), 4.87 (1H, d, J=7.3 Hz, 3Glc-H-1′), 4.94 (1H, d, J=7.3 Hz, Xyl-H-1″″), 4.99 (1H, d, J=7.3 Hz, Xyl-H-1″‴), 5.13 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.33 (1H, d, J=7.3 Hz, Glc-H-1″), 5.33 (1H, m, H-24), 5.55 (1H, br s, 12-OH); 13C-NMR data, see Table 1; negative-ion FAB-MS m/z 1209 (M−H)−, 1077 (M−C5H9O4)−, 1047 (M−C6H11O5)−, 915 (M−C11H19O9)−; negative-ion HR-FAB-MS: m/z 1209.6222 (Calcd for C58H97O26 [M−H]−, 1209.6268).

Chikusetsusaponin FK6 (6): A white amorphous powder; [α]D

15 +2.8° (c=0.45, MeOH); 1H-NMR (pyridine-d5, 500 MHz) δ: 0.62 (1H, m, H-5), 0.81, 0.92, 0.94, 1.12, 1.26, 1.63 (3H, s, H3-19, 30, 18, 29, 28, 21), 1.61 (6H, s, H3-26, 27), 2.54 (1H, m, H-17), 3.22 (1H, dd, J=4.3, 11.6 Hz, H-3), 4.88 (1H, d, J=7.3 Hz, 3Glc-H-1′), 4.95 (1H, d, J=7.3 Hz, Xyl-H-1″″), 5.21 (1H, d, J=7.3 Hz, 20Glc-H-1‴), 5.26 (1H, m, H-24), 5.34 (1H, d, J=7.9 Hz, Glc-H-1″), 5.57 (1H, br s, 12-OH); 13C-NMR data, see Tables 1, 2; negative-ion FAB-MS m/z 1077 (M−H)−, 945 (M−C5H9O4)−, 915 (M−C6H11O5)−; negative-ion HR-FAB-

MS: m/z 1077.5833 (Calcd for C53H89O22 [M−H]−, 1077.5846).Chikusetsusaponin FK7 (7): A white amorphous pow-

der; [α]D30 +2.3° (c=0.26, MeOH); 1H-NMR (pyridine-d5,

500 MHz) δ: 0.64 (1H, m, H-5), 0.69, 0.78, 0.85, 1.10, 1.29, 1.35 (3H each, all s, H3-19, 18, 30, 29, 28, 21), 1.65 (6H, s, H3-26, 27), 2.06 (1H, t, J=10.7 Hz, H-13), 2.39 (1H, m, H-17), 3.28 (1H, J=4.3, 11.6 Hz, H-3), 4.96 (1H, d, J=7.9 Hz, 3Glc-H-1′), 5.27 (1H, d, J=7.9 Hz, 12Glc-H-1‴), 5.33 (1H, m, H-24), 5.40 (1H, d, J=7.3 Hz, Glc-H-1″); 13C-NMR data, see Tables 1, 2; negative-ion FAB-MS m/z 945 (M−H)−, 783 (M−C6H11O5)−; negative-ion HR-FAB-MS: m/z 945.5667 (Calcd for C48H81O18 [M−H]−, 945.5423).

Chikusetsusaponin FM1 (8): A white amorphous powder; [α]D

15 −2.5° (c=0.71, MeOH); 1H-NMR (pyridine-d5, 500 MHz) δ: 0.67 (1H, m, H-5), 0.81, 0.93, 0.95, 1.10, 1.28, 1.64 (3H-each, all s, H3-19, 30, 18, 29, 28, 21), 1.55, 1.55 (3H each, both s, H3-26, 27), 2.51 (1H, m, H-17), 3.27 (1H, dd, J=4.9, 11.3 Hz, H-3), 3.82 (1H, m, H-24), 4.91 (1H, d, J=7.9 Hz, 3Glc-H-1′), 4.97 (1H, d, J=7.3 Hz, Xyl-H-1″″), 5.11 (1H, d, J=7.9 Hz, 20Glc-H-1‴), 5.37 (1H, d, J=7.3 Hz, Glc-H-1″); 13C-NMR data, see Tables 1, 2; negative-ion FAB-MS m/z 1111 (M−H)−, 979 (M−C5H9O4)−, 817 (M−C11H19O9)−; negative-ion HR-FAB-MS: m/z 1111.5928 (Calcd for C53H91O24 [M−H]−, 1111.5900).

Partial Hydrolysis of 4 and 5 A solution of 4 (33 mg) and 5 (35 mg) in 55% aqueous acetic acid (5 mL) was heated at 70°C for 1 h separately. Each reaction mixture was diluted with H2O and then subjected to reverse-phase polystyrene gel column chromatography [H2O→MeOH–H2O (70 : 30→80 : 20, v/v)] to give 12 mg and 10 mg of 4a, respectively. The 1H- and 13C-NMR (Table 1) spectra of 4a showed signals assignable to be a 20-epimeric pair of chikusetsusaponin III.

Acknowledgments We are grateful to Kaneyoshi Yamashita in Miyazaki for providing fresh fruits of P. japoni-cus, Katsushi Takeda and Teruo Tanaka, Institute of Resource Development and Analysis, Kumamoto University for the measurements of NMR spectra, and Toshiyuki Iriguchi, Institute of Resource Development and Analysis, Kumamoto University for the measurement of MS spectra.

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