Two new triterpenoid saponins from Polygala crotalarioides

Yan Hua a,*, Chang Xiang Chen b, Jun Zhou b

a Key Laboratory for Forest Resources Conservation and Use in the Southwest Mountains of China,

Ministry of Education, Southwest Forestry University, Kunming 650224, Chinab State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany,

Academia Sinica, Kunming 650204, China

Received 29 January 2010

Abstract

Two new oleanane-type saponins, crotalarioside A (1) and crotalarioside B (2), were isolated from the roots of Polygala

crotalarioides. Their structures were elucidated on the basis of spectroscopic and chemical evidence.

# 2010 Yan Hua. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Polygala crotalarioides; Chemical constituents; Oleanane-type saponins

Polygala crotalarioides Buch. Ham. (Polygalaceae) is known to be a folk tonic medicine in Yunnan Wa nationality

[1]. Its bioactivities attracted us to investigate its chemical constituents. In the previous paper, we reported structural

elucidation of five new xanthones [2,3]. Our continuing phytochemical investigation into the constituents of this plant

has resulted in the isolation of two new oleanane-type saponins, crotalarioside A and crotalarioside B. Their structures

were elucidated on the basis of spectral analysis.

1. Experimental

The dried roots (1 kg) of P. crotalarioides were extracted with 75% EtOH four times under reflux. After removal of

the solvent in vacuo, the aqueous solution was passed through a HPD-100 column and the absorbed materials were

eluted with water, 75% aqueous ethanol and ethanol, successively. The 75% ethanol eluate was concentrated in vacuo

to give a residue (96 g), which was chromatographed on a silica gel (200–300 mesh) column chromatography with

CHCl3/MeOH/H2O (7:4:1) to afford 10 fractions (fraction 1–10). Fractions 1–4 were chromatographed on Si gel with

CHCl3/MeOH/H2O (7:3:0.5) and then resubjected to Sephadex LH-20 with MeOH to give the total saponins (15 g).

The total saponins were passaged over RP-18 eluted with MeOH/H2O (5:5–7:3) to give 4 fractions (fraction A–D).

Fraction D was further purified by semi-pre HPLC eluted by CH3CN/0.1% AcOH–H2O solution (24:76) to afford

compounds 1 (24 mg) and 2 (10 mg).

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Chinese Chemical Letters 21 (2010) 1107–1110

* Corresponding author.

E-mail address: huayan@mail.kib.ac.cn (Y. Hua).

1001-8417/$ – see front matter # 2010 Yan Hua. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2010.05.005

2. Results and discussion

Compound 1 was obtained as white powder, and analyzed for C53H82O24 by negative-ion HRFABMS (m/z

1101.3976 [M�H]� (calcd. for C53H81O24, 1101.3965)). Its IR spectrum exhibited absorption bands for hydroxyl

(3424 cm�1), carbonyl (1718 and 1737 cm�1), and olefinic groups (1631 cm�1). The 1H and 13C NMR spectra showed

signals due to 30 aglycone carbon signals, including five singlet methyls [dC 33.1 (s), 24.0 (s), 18.4 (s), 17.1 (s), 13.8

(s)], one oxygenated methylene [dC 64.4 (t)], one oxygenated methine [dC 86.1 (d)], two olefinic carbons [dC 139.1 (s),

127.0 (d)], three carboxyl carbons [dC 208.0(s), 180.1 (s), 176.8 (s)]. Also observed were signals of four anomeric

carbons and their corresponding anomeric protons [dC 107.4 (d), 104.0 (d), 101.3 (d), 94.8 (d); dH 5.03 (d, 1H,

J = 6.8 Hz), 5.13 (d, 1H, J = 7.5 Hz), 6.47 (br s, 1H), 6.06 (d, 1H, J = 8.2 Hz)], indicating a triterpenoid saponin with

four sugar moieties. Comparison of the 1H and 13C NMR data of the aglycone unit with those of polygalasaponinXXIII

[4] showed that the structures of the two aglycones were very similar. The only difference was the replacement of a

methyl group in polygalasaponinXXIII by an oxygenated methine group in 1. Both of the two aglycones were

oleanane-type triterpenoids with a ketone quaternary carbon at C-2.

Acid hydrolysis of 1 with 1 mol/L HCl furnished four monosaccharides, which may be determined to be D-glucose

D-fucose, L-rhamnose and D-xylose, by TLC comparison with authentic samples. This was further confirmed by the13C and 1H NMR spectral data of these sugar moieties, which were consistent with those reported [4]. Sugar proton and

carbon signals in the spectra were assigned by HMQC, HMBC, and HMQC-TOCSY spectra. In the HMBC spectrum,

long-range couplings were observed for H-10 of the glucosyl unit to C-3 of the aglycone, H-100 of the fucosyl unit to C-

28 of the aglycone, H-1000 of the rhamnosyl unit to C-200 of the fucosyl unit, H � 10000 of the xylosyl unit to C-4000 of the

rhamnosyl unit. The anomeric configurations of D-glucosyl, D-fucosyl, L-rhamnosyl and D-xylosyl were determined to

be b, b, a and b, respectively, from the coupling constants of the anomeric proton signals. On the basis of the above

evidence, the structure of 1 was elucidated as 2-oxo-olean-12-ene-27-hydroxy-23, 28-dioic acid 3-O-b-D-

glucopyranosyl-28-O-b-D-xylopyranosyl-(1! 4)-a-L-rhamnopyranosyl-(1! 2)-b-D-fucopyranoside, and was

named crotalarioside A (Fig. 1).

Compound 2 was obtained as white powder, and analyzed for C59H92O29 by negative-ion HRFABMS (m/z

1263.4251 [M�H]� (calcd. for C59H91O29, 1263.4236)). Its IR spectrum exhibited absorption bands for hydroxyl

Y. Hua et al. / Chinese Chemical Letters 21 (2010) 1107–11101108

[(Fig._1)TD$FIG]

Fig. 1. Structures of compounds 1 and 2.

(3425 cm�1), carbonyl (1720 and 1737 cm�1), and olefinic groups (1630 cm�1). Comparison of the 1H and 13C NMR

spectral data of 2 with those of 1 showed that the two structures were very similar except that there was an additional

glucosyl moiety in compound 2. In the HMBC spectrum, long-range couplings were observed for H � 100000 of the

additional glucosyl unit to C-300 of the fucosyl unit. The binding sites of the other sugars are identical to compound 1.

Based on the above evidence, the structure of 2 can be elucidated as 2-oxo-olean- 12-ene-27-hydroxy-23, 28-dioic acid

3-O-b-D-glucopyranosyl-28-O-b-D-xylopyranosyl-(1! 4)-a-L-rhamno-pyranosyl-(1! 2)-[b-D-glucopyranosyl-

(1! 3)]-b-D-fucopyranoside, and was named crotalarioside B (Table 1).

Y. Hua et al. / Chinese Chemical Letters 21 (2010) 1107–1110 1109

Table 11H and 13C NMR spectral data of 1 and 2 (400 and 500 MHz, C5D5N, d).

Position 1 dC 1 dH 1 HMBC 2 dC 2 dH 2 HMBC

1 54.8 t 54.8 t

2 208.0 s 207.9 s

3 86.1 d 5.66 (s, 1H) Glc-C-10, C-1,

C-5, C-23, C-24

85.7 d 5.65 (s, 1H) Glc-C-10, C-1,

C-5, C-23, C-24

4 58.4 s 58.4 s

5 52.4 d 2.74 (br d,

1H, J = 10.7 Hz)

C-1, C-7, C-23,

C-24, C-25

52.4 d 2.75 (m, 1H) C-1, C-7, C-23,

C-24, C-25

6 21.0 t 21.0 t

7 33.8 t 33.9 t

8 40.7 s 40.7 s

9 48.5 d 48.5 d

10 43.6 s 43.7 s

11 23.5 t 23.6 t

12 127.0 d 5.74 (t-like, 1H) C-9, C-11, C-14, 127.0 d 5.73 (t-like, 1H) C-9, C-14,

13 139.1 s 139.1 s

14 47.0 s 47.0 s

15 24.3 t 24.3 t

16 24.0 t 24.0 t

17 48.0 s 48.1 s

18 42.0 d 3.20 (dd, 1H,

J = 14.0, 3.8 Hz)

C-12, C-16,

C-20, C-28

42.0 d 3.20 (dd, 1H,

J = 14.0,

4.0 Hz)

C-12, C-16,

C-20, C-28

19 45.4 t 45.5 t

20 30.8 s 30.8 s

21 33.3 t 33.5 t

22 32.3 t 32.3 t

23 180.1 s 180.4 s

24 13.8 q 1.51 (s, 3H) C-3, C-4,

C-5, C-23

14.0 q 1.50 (s, 3H) C-3, C-5, C-23

25 17.1 q 0.96 (s, 3H) C-1, C-5, C-9 17.1 q 0.96 (s, 3H) C-1, C-5, C-9

26 18.4 q 1.03 (s, 3H) C-7, C-9, C-14 18.4 q 1.05 (s, 3H) C-7, C-9, C-14

27 64.4 t 3.83 (d, 1H,

J = 12.0 Hz),

4.07 (d, 1H,

J = 12.0 Hz)

C-8, C-13,

C-14, C-15

64.4 t 3.82 (d, 1H,

J = 12.0 Hz),

4.06 (d, 1H,

J = 12.0 Hz)

C-8, C-13, C-15

28 176.8 s 176.8 s

29 33.1 q 0.76 (s, 3H) C-19, C-20,

C-21, C-30,

33.1 q 0.77 (s, 3H) C-19, C-20,

C-21, C-30,

30 24.0 q 0.89 (s, 3H) C-19, C-20,

C-21, C-29,

24.0 q 0.90 (s, 3H) C-19, C-20,

C-21, C-29,

Glc-10 104.0 d 5.13 (d, 1H,

J = 7.5 Hz)

C-3, Glc-C-30,Glc-C-50

103.9 d 5.12 (d, 1H,

7.5 Hz)

C-3, Glc-C-30,Glc-C-50

20 75.1 d 75.0 d

30 78.6 d 78.6 d

40 71.4 d 71.4 d

50 78.1 d 78.1 d

Acknowledgments

The authors are grateful to the Analytical Group of the Laboratory of Phytochemistry, Kunming Institute of Botany,

Chinese Academy of Sciences, for the spectral measurements.

References

[1] B.X. Xiang, P.F. Zhang, Y.H. Xiang, Guizhou Sci. 13 (1995) 24.

[2] Y. Hua, J. Zhou, C.X. Chen, Chin. Chem. Lett. 17 (2006) 773.

[3] Y. Hua, C.X. Chen, J. Zhou, J. Asian Nat. Prod. Res. 9 (2007) 273.

[4] D.M. Zhang, T. Miyase, M. Kuroyanagi, Chem. Pharm. Bull. 44 (1996) 173.

Y. Hua et al. / Chinese Chemical Letters 21 (2010) 1107–11101110

Table 1 (Continued )

Position 1 dC 1 dH 1 HMBC 2 dC 2 dH 2 HMBC

60 62.6 t 4.29 (dd, 1H,

J = 2.0, 5.0 Hz),

4.45a

Glc-C-40,Glc-C-50

62.6 t 4.30a, 4.47a Glc-C-40, Glc-C-50

Fuc-100 94.8 d 6.06 (1H, d,

J = 8.2 Hz)

C-28, Fuc-C-300,Fuc-C-500

94.8 d 6.05 (d, 1H,

J = 8.1 Hz)

C-28, Fuc-C-300,Fuc-C-500

200 74.0 d 72.7 d

300 76.8 d 85.5 d

400 73.3 d 72.4 d

500 72.6 d 72.1 d

600 17.0 q 1.49 (3H, d,

J = 6.2 Hz)

Fuc-C-400,Fuc-C-500

16.9 q 1.40 (d, 3H,

J = 6.2 Hz)

Fuc-C-400, Fuc-C-500

Rha-100 0 101.3 d 6.47 (br s, 1H) Fuc-C-200,Rha -C-300 0,Rha -C-500 0

101.2 d 6.42 (br s, 1H) Fuc-C-200, Rha -C-300 0,Rha -C-500 0

200 0 71.9 d 72.1 d

300 0 72.6 d 72.4 d

400 0 85.1 d 84.9 d

500 0 68.3 d 68.4 d

600 0 18.6 q 1.67 (d, 3H,

J = 6.0 Hz)

Rha -C-400 0,Rha -C-500 0,

18.7 q 1.68 (d, 3H,

J = 6.1 Hz)

Rha -C-400 0, Rha -C-500 0,

xyl-100 00 107.4 d 5.03 (d, 1H,

J = 6.8 Hz)

Rha -C-400 0,xyl-C-300 00,xyl-C-500 00

107.4 d 5.02 (d, 1H,

J = 6.8 Hz)

Rha -C-400 0,xyl-C-300 00,xyl-C-500 00

200 00 76.2 d 76.2 d

300 00 78.7 d 78.7 d

400 00 70.9 d 70.9 d

500 00 67.5 t 67.4 t

Glc-100 00 0 105.7 d 5.09 ((d, 1H,

J = 7.5 Hz))

Fuc-C-300, Glc-C-300 00 0,Glc-C-500 00 0

200 00 0 75.3 d

300 00 0 78.6 d

400 00 0 71.6 d

500 00 0 78.1 d

600 00 0 62.5 t 4.28a, 4.46a Glc-C-400 00 0

a Overlapping with other signals.