Original Papers

1647

Authors

Bao-Song Cui 1, Yong-Qi Qiao 1, Yi Yuan 1, Li Tang 2, Hui Chen 1, Yan Li 1, Shuai Li 1

Affiliations

1

2

Key words " Gentianaceae l " Comastoma pedunculatum l " saikosaponin homologs l " hepatoprotective activity l " cytotoxic activity l

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, Peopleʼs Republic of China College of Life and Environmental Science, Central University for Nationalities, Beijing, Peopleʼs Republic of China

Abstract !

Eight new triterpenoid saponins, the saikosaponin homologs comastomasaponins A–H (1–8), as well as a known triterpenoid (9) and eight known saponins (10–17) were isolated from the aerial portions of Comastoma pedunculatum. The structures of these compounds were elucidated spectroscopically, and their hepatoprotective activity

Introduction !

received revised accepted

April 22, 2014 July 20, 2014 Sept. 1, 2014

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383123 Published online September 24, 2014 Planta Med 2014; 80: 1647–1656 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Shuai Li Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College 1 Xian Nong Tan Street Beijing 100050 Peopleʼs Republic of China Phone: + 86 10 63 16 46 28 Fax: + 86 10 63 01 77 57 [email protected]

The herb Comastoma pedunculatum (Royle ex D. Don) Holub has been used to treat hepatitis, liver fibrosis, and cholecystitis in traditional Tibetan medicine [1]. The genus Comastoma is in the family Gentianaceae and comprises 17 species worldwide [2]. Phytochemical investigations of this genus have revealed a number of structurally diverse metabolites, such as xanthones, flavonoids and oleanolic acid, triterpenoids, steroids, lignins, coumarin, and long-chain alkanes [3–7]. In the course of our investigations on the constituents of C. pedunculatum, we have reported the isolation and characterization of xanthones [8, 9], which are characteristic chemical constituents of the Comastoma genus, and flavone glycosides [10]. Further investigation of the same source resulted in the isolation of triterpenoid saponins [10], which are saikosaponin homologs and represent the first identification of saikosaponins in the Gentianaceae family. The saikosaponins were first derived from Bupleurum falcatum L. (Umbelliferae) [11] and exhibit a broad range of biological activities, including analgesic, antiviral, antiinflammatory [12], immunoregulatory [13], hepatoprotective, and anticancer activities [14]. Herein, we report the isolation and structural characterization of eight new triterpenoid saponins, saikosaponin homologs from the aqueous ethanol extract of the aerial portion of C. pedun-

and cytotoxic activity were evaluated against five human tumor cell lines in vitro. Compounds 1, 5– 12, 14, 15, and 17 exhibited potent hepatoprotective activity, and compound 11 displayed cytotoxic activity against HCT-8, Bel-7402, BGC-825, A549, and A2780 human tumor cell lines. Supporting information available online at http://www.thieme-connect.de/products

culatum. Spectroscopic methods were used to elucidate the structures of these new compounds. In addition, the compounds were evaluated for hepatoprotective activity against D-galactosamine-induced toxicity and cytotoxic activity against five human tumor cell lines in vitro.

Results and Discussion !

The aqueous ethanol extract of the aerial portions of C. pedunculatum were suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. The n-BuOH extract was purified by multiple chromatographic steps over silica gel, reversed-phase C18, Sephadex LH-20, and semipreparative HPLC over C18 to yield eight new triterpenoid saponins, the comastomasaponins " Fig. 1). In addition, a known triterpeA–H (1–8, l noid (9) and eight known saponins (10–17) were identified as saikogenin F (9) [11], 3-O-β-D-fucopyranosyl saikogenin F (10) [11], clinoposaponin XV (11) [15], saikosaponin A (12) [11], 6′′-acetylsaikosaponin A (13) [11], clinoposaponin I (14) [16], bupleuroside I (15) [17], clinoposaponin XII (16) [15], and bupleuroside b3 (17) [18]. Compounds 1–8 were isolated as white amorphous powder. The 1H and 13C NMR spectra of 18 displayed characteristic signals of triterpene saponins. The monosaccharides obtained by acid hydrolysis of each compound were identified by

Cui B-S et al. Hepatoprotective Saikosaponin Homologs …

Planta Med 2014; 80: 1647–1656

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Hepatoprotective Saikosaponin Homologs from Comastoma pedunculatum

Original Papers

Fig. 1

The structural formulas of compounds 1–8 and 11.

comparison on TLC with authentic samples as glucose for 1, 2, 7, and glucose, and fucose for 3–6 and 8. The absolute configurations were determined by HPLC analysis to be D for all the sugars [19]. The JH‑1,H‑2 values in the 1H NMR spectrum of the glucose and fucose in their pyranose form (7.0–8.0 Hz) indicated their β anomeric configuration. Comastomasaponin A (1) has the molecular formula C42H68O14, as determined by HR‑ESI‑MS analysis (m/z 819.4507 [M + Na]+, calcd. for C42H68O14Na, 819.4501). Thirty of the 42 carbons were assigned to the triterpenoid skeleton and 12 to a disaccharide moiety. In the 1H NMR spectra, singlets corresponding to six tertiary methyl groups at δH 0.88, 0.91, 0.92, 0.95, 1.10, and 1.38 " Table 1) correlated with the signals in the 13C NMR spectra (l " Tafor six sp3 carbons at δC 13.0, 18.7, 20.0, 20.8, 23.8, and 33.6 (l ble 2), which were correlated in the HSQC spectrum. Two broad, doublet-like, vinyl signals at δH 5.64 (1H, br d, J = 10.0 Hz) and 5.95 (1H, d, J = 10.0 Hz) correlated with two sp2 carbons at δC 131.1 and 132.1. An AB system of methylene protons on a carbon bonded to an oxygen appearing at δH 3.33 (1H, d, J = 7.0 Hz) and 4.39 (1H, d, J = 7.0 Hz) correlated with the carbon signal at δC 73.0, two carbinol protons (δH 4.31, 1H, dd, J = 12.0, 4.0 Hz; δH 4.51, 1H, br d, J = 11.0 Hz; and δC 81.8 and 64.0), and a hydroxymethyl group (δH 3.70, 1H, d, J = 10.0 Hz; δH 4.36, 1H, d, J = 10.0 Hz; and δC 64.0). On the basis of these assignments, compound 1 was determined to be an oleanane-type triterpenoid. From detailed analyses of the molecular formula, NMR spectroscopic data, and literature comparisons, it was concluded that compound 1 was a saikosaponin homolog containing a double bond (Δ11,12) and six rings, one of which is an epoxide bridge between the quaternary carbon at C-13 (δC 84.0) and the secondary carbon at C-28 (δC 73.0). HMBC correlations were used to assign the glycosylated hydroxyl at C-3 (δH 4.31, δC 81.8), the unglycosylated hydroxyl at C-16 (δH 4.51, δC 64.0), and the hydroxymethyl group at C-23 " Fig. 2). The hydroxyl groups at posi(δH 3.70 and 4.36, δC 64.0) (l tions 3 and 16 must be equatorial because the coupling constants of H-3 (4.31, dd, J = 12.0, 4.0 Hz) and H-16 (4.51, br d, J = 11.0 Hz) were consistent with axial configurations for both protons [20, 21], and were further supported by the NOESY correlations between H-3 (4.31) and H-1α (1.00), H-5α (1.63); and between H16α (4.51) and H-15α (1.71), H-18α (1.97). Therefore, the triterpenoid skeleton of 1 was identified as 3β,16β,23-trihydroxy13,28-epoxyolean-11-ene, and this conclusion was further sup" Fig. 2). The two anomeric ported by HMBC correlation analysis (l

protons at δH 5.06 and 5.35 and the two corresponding carbons at δC 106.2 and 106.7, which were correlated in the HSQC spectrum, indicated a disaccharide moiety. The two sets of glucopyranosyl signals were assigned by the analysis of gCOSY, HSQC, and HMBC " Tables 1 and 2). The linkage between the glucopyranospectra (l syls was established by analysis of the HMBC correlations between δH 5.06 (H-1′ of Glc) and δC 81.8 (C-3 of the aglycone) and between δH 5.35 (H-1′′ of Glc) and δC 85.2 (C-3′ of Glc). Therefore, compound 1 was concluded to be 3β,16β,23-trihydroxy-13,28epoxyolean-11-ene-3-O-[β-D-glucopyranosyl-(1 → 3)]-β-D-glucopyranoside. Comastomasaponin B (2) has the molecular formula C48H78O19, as determined by HR‑ESI‑MS data (m/z 981.5037 [M + Na]+). De" Tables 1 and 2) tailed analysis of the NMR spectroscopic data (l revealed that the aglycone signals of 2 were nearly identical to those of 1. The saccharide signals, however, displayed significant differences. The three anomeric proton signals at δH 5.04, 5.06, and 5.31 and the three corresponding carbon signals at δC 103.5, 105.0, and 105.6, which were correlated in the HSQC spectrum, indicated a trisaccharide moiety. The three sets of glucopyranosyl signals were assigned by analysis of the gCOSY, HSQC, and HMBC " Tables 1 and 2). The linkage between the glucopyranospectra (l syls was established by analysis of the HMBC correlations between δH 5.04 (H-1′ of Glc) and δC 82.3 (C-3 of the aglycone); δH 5.31 (H-1′′ of Glc) and δC 83.5 (C-2′ of Glc); and δH 5.06 (H-1′′ of Glc) and δC 69.7 (C-6′′ of Glc). The trisaccharide was thus elucidated as a [β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl(1 → 2)-β-D-glucopyranosyl moiety, and compound 2 was determined to be 3β,16β,23-trihydroxy-13,28-epoxyolean-11-ene-3O-[β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl-(1 → 2)-βD-glucopyranoside. Comastomasaponin C (3) has the molecular formula C54H88O23, as determined by HR‑ESI‑MS analysis (m/z 1127.5611 [M + Na]+). " Tables 1 and 2) revealed Detailed analysis of the NMR data (l that the aglycone signals of 3 and 1 were nearly identical. The saccharide signals, however, displayed significant differences. The four anomeric proton signals at δH 4.92, 5.01, 5.02, and 5.10 and the four corresponding carbon signals at δC 104.3, 105.3, 105.4, and 107.1, which were correlated in the HSQC spectrum, indicated a tetrasaccharide moiety. The fucopyranosyl group was confirmed by analysis of the gCOSY, HSQC, and HMBC spectra and comparison with literature data [11]. The 1H signals at δH 4.92 (d, J = 8.0 Hz), 4.50, 3.86, 4.22, 3.75 (q, J = 6.5 Hz), and 1.54 (d, J = 6.5 Hz) and the 13C signals at δC 105.3, 71.0, 86.7, 71.6, 71.0, and 17.3 supported the assignment of the D-fucopyranosyl group. The linkage between the fucopyranosyl and the glucopyranosyls was established by analysis of the HMBC correlations between δH 4.92 (H-1′ of Fuc) and δC 82.0 (C-3 of the aglycone); δH 5.02 (H-1′′ of Glc) and δC 86.7 (C-3′ of Fuc); δH 5.10 (H-1′′′ of Glc) and δC 85.8 (C-2′′ of Glc); and δH 5.01 (H-1′′′′ of Glc) and δC 69.8 (C-6′′ of Glc). The tetrasaccharide was thus elucidated as a [β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranosyl moiety, and 3 was therefore determined to be 3β,16β,23-trihydroxy-13, 28-epoxyolean11-ene-3-O-[β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl(1 → 2)]- β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranoside. Comastomasaponin D (4) has the molecular formula C48H76O18, as determined by HR‑ESI‑MS analysis (m/z 963.4923 [M + Na]+). " Tables 1 and 2) revealed Detailed analysis of the NMR data (l that the aglycone signals of 4 and 1 were nearly identical, including six tertiary methyl signals at δH 0.88, 0.90, 0.95, 1.14, 1.28, and 1.32, the Δ11,12 olefinic protons at δH 5.68 and 5.93, and the

Cui B-S et al. Hepatoprotective Saikosaponin Homologs … Planta Med 2014; 80: 1647–1656

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1648

Original Papers

1

H NMR spectroscopic data of compounds 1–8 (500 MHz in pyridine-d5).

Proton

1

2

3

4

5

6

7

8

Aglycone H-1

1.00a

1.07a

0.98a

1.53 m

1.53a

1.77 m

2.05a

2.06a

1.89a

2.13a

2.06a

2.48 (br d, 13.0) 4.20 (dd, 9.0, 3.5) 1.60 (br d, 10.5) 1.51a 1.72 m

2.19 (br d, 14.0) 4.07a

2.06 (dt, 13.5, 2.0) 2.13 (br t, 13.5) 2.34 m

2.05a

H-2

1.85 (br d, 10.0) 2.10 (dd, 10.0, 4.0) 2.49a

1.53 (br t, 13.5) 2.03 (br t, 13.5) 2.06 (br t, 13.5) 2.28 (br d, 11.0) 4.20 (br d, 11.0) 1.72 (br d, 12.0) 1.36a 1.73a

1.53a

1.76 (br d, 12.0) 2.03a

1.04 (dd, 10.5, 3.5) 1.82 m

2.45 m

2.29 m

4.20a 1.63a

4.22 (dd, 9.0, 3.0) 1.71a

1.25a

1.38a 1.75 br, d (12.5) 1.26a

1.37a 1.71 (br d, 11.5) 1.26a

1.62a

1.63a

1.61 (t, 13.0)

1.95 (d, 8.0) 3.80 (dd, 8.0, 2.5) 5.52 (d, 2.5)

1.91 (d, 8.5) 3.80 (dd, 6.0, 2.5) 5.51 (d, 3.0)

1.95 (d, 6.5) 3.78a

1.66 (dd, 13.0, 4.0) 2.14 (t, 13.0)

1.69a

2.14 (t, 12.5)

1.67 (dd, 12.0, 4.0) 2.13 (t, 12.0)

4.62 m

4.61 m

4.62 m

4.62 m

2.52 (dd, 14.0, 4.0) 1.27a

2.51 (dd, 13.0, 2.0) 1.26a

2.51 (dd, 14.0, 4.0) 1.27a

2.53 (br d, 13.5) 1.27a

1.83 (t, 13.5)

1.83a

1.83 (t, 14.0)

1.84 (t, 13.0)

1.26a

1.29a

1.28a

1.26a

1.61a

1.62a

1.62a

1.62a

1.83 (br d, 13.5) 2.78 (br d, 13.5) 3.77a 4.40a 1.13 s 1.10 s 1.06 s 1.38 s 3.73a 4.38a) 0.88 s 0.99 s 3.22 s Fucose 4.94 (d, 8.0) 4.51 (dd, 9.0, 8.0) 4.06 (dd, 9.0, 3.0) 3.97 (d, 3.0) 3.68 (br q, 6.0)

1.83a

1.83 (t, 14.0)

1.83 (t, 13.0)

2.77 (br d, 13.5) 3.69 (d, 11.5) 4.33 (d, 11.5) 0.93 s 1.09 s 1.06 s 1.38 s 3.71 (d, 11.0) 4.38a 0.88 s 0.98 s 3.22 s Fucose 4.94 (d, 7.0) 4.46 (dd, 9.0, 7.0) 4.15 (br d, 9.0) 4.33 br s 3.87 (br q, 6.0)

2.80 (br d, 14.0) 3.80a 4.39a 1.15 s 1.11 s 1.06 s 1.34 s 3.72a 4.39a 0.88 s 0.98 s 3.27 s Glucose 5.02 (d, 7.5) 4.11a

2.78 (br d, 13.0) 3.69a 4.32a 1.04 s 1.09 s 1.06 s 1.38 s 3.72a 4.36a 0.88 s 0.99 s 3.22 s Fucose 4.92 (d, 7.0) 4.50a

3.89a

3.89a

4.19a 4.10a

4.22a 3.73 (q, 5.5) continued

H-3 H-5 H-6

H-7

2.33 (br d, 12.0) 4.31 (dd, 12.0, 4.0) 1.63 (br d, 12.0) 1.53a 1.76 (br d, 12.0) 1.26 (br d, 10.0) 1.54a

4.15 (dd, 12.0, 2.0) 1.46a 1.45a 1.77 m 1.21a

1.36 (br d, 13.0) – – 1.16a

1.46a

1.24 (br d, 9.5) 1.52a

–

H-9 H-11

2.01a 5.95 (d, 10.0)

1.96a 5.93 (d, 10.0)

2.01a 5.98 (d, 10.5)

2.00a 5.93 (d, 10.0)

H-12

5.64 (br d, 10.0) 1.71 (dd, 12.0, 4.5) 2.05 (d, 10.5)

5.59 (br d, 10.0) 1.68 (dd, 12.5, 5.5) 2.04 (dd, 12.5, 10.5) 4.51 (br d, 12.0) 1.96a

5.64 (dd, 10.5, 3.0) 1.67 (dd, 12.5, 5.5) 2.04a

5.68 (dd, 10.0, 2.5) 1.70 (dd, 12.5, 5.5) 1.99a 4.53 (dd, 10.0, 4.0) 1.94a

1.57 (t, 13.5)

4.49 (br d, 10.0) 1.96 (dd, 15.5, 3.0) 1.32 (dd, 14.5, 4.0) 1.83 (dd, 14.5, 14.5) 1.18 (br d, 13.5) 1.58a

1.31a

1.32a

1.85 (dd, 14.5, 12.0) 1.20 (br d, 14.0) 1.60 (dd, 14.0, 3.0) 1.34a

2.49 (br d, 14.0) 3.70 (d, 10.0) 4.36 (d, 10.0) 0.92 s 0.95 s 1.38 s 1.10 s 3.33 (d, 7.0) 4.39 (d, 7.0) 0.91 s 0.88 s

2.49a

2.29 m

2.50 m

3.77 (d, 11.0) 4.40 (d 11.0) 1.11 s 0.98 s 1.37 s 1.01 s 3.32 (d, 6.5) 4.39 (d, 6.5) 0.93 s 0.89 s

3.68 (d, 11.0) 4.39a 0.97 s 1.00 s 1.37 s 1.09 s 3.32 (d, 7.0) 4.40a 0.91 s 0.88 s

9.70 s 1.28 s 0.88 s 1.32 s 1.14 s 3.34 (d, 6.5) 4.38a 0.95 s 0.90 s

Glucose 5.06 (d, 7.5) 4.63 (dd, 10.0, 7.5) 4.11 (dd, 10.0, 3.0) 4.68 m 3.95 (dd, 6.0, 5.0)

Glucose 5.04 (d, 7.0) 4.09a

Fucose 4.92 (d, 8.0) 4.50a

Fucose 4.61 (d, 8.0) 4.49a

3.90a

3.86a

4.17a

4.19a 4.10a

4.22a 3.75 (q, 6.5)

4.38a 3.86 m

H-15

H-16 H-18 H-19

H-21

H-22

H-23 H-24 H-25 H-26 H-27 H-28 H-29 H-30 H–OCH3 H-1 H-2 H-3 H-4 H-5

4.51 (br d 11.0) 1.97 (dd, 14.0, 3.5) 1.32 (dd, 13.5, 3.5) 1.83 (dd, 14.0, 13.5) 1.18 (br d, 13.5) 1.56 (br d, 14.0) 1.33 m

1.25 (br d, 13.5) 1.79 (dd, 13.5, 10.0) 1.23a

1.30a

4.16 (dd, 8.0, 3.5) 1.66a 1.37a 1.76 (br d, 12.5) 1.26a 1.61 (dd, 13.5, 5.0) 1.94 (d, 8.5) 3.81 (dd, 8.5, 3.5) 5.52 (d, 3.5) 1.67a

Cui B-S et al. Hepatoprotective Saikosaponin Homologs …

5.52 br s

2.14 (t,11.5)

Planta Med 2014; 80: 1647–1656

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Table 1

1649

1650

Original Papers

Table 1 Continued Proton

1

2

3

4

5

6

7

8

H-6

4.38 (br d, 11.0) 4.53 (br d, 11.0) Glucose (at C-3 of Glu) 5.35 (d, 7.5) 4.03 (dd, 8.5, 7.5) 4.26 (dd, 8.5, 8.0) 4.25 (dd, 8.5, 8.5) 4.00a)

4.36a

1.54 (d, 6.5)

1.55 (d, 6.0)

1.47, (d, 6.0)

1.46 (d, 6.0)

4.36 m

1.52 (d, 5.5)

Glucose (at C-2 of Glu) 5.31 (d, 7.5) 4.10a

Glucose (at C-3 of Fuc) 5.02 (d, 8.5) 3.89 (t, 8.5)

Glucose (at C-3 of Fuc) 5.27 (d, 8.0) 4.02 (t, 8.0)

3.91a

4.21a

4.20a

4.27 (t, 9.5)

3.74a

4.06a

4.20a

4.12a

4.16 m

Glucose (at C-2 of Fuc) 5.25 (d, 7.5) 4.12 (dd, 9.0, 7.5) 4.15 (dd, 9.0, 9.0) 4.30 (dd, 9.0, 9.0) 3.75a

4.27 (br d, 10.5) 4.76 (br d, 10.5)

4.17a

4.23a

4.42a

4.83 (d, 10.0)

4.42a

H-1 H-2

Glucose (at C-6 of Glu) 5.06 (d, 8.0) 4.01 (t, 8.0)

4.76 (br d, 10.5) Glucose (at C-2 of Glu) 5.10 (d, 8.0) 4.05a 4.11a 4.16a 3.80a 4.34a 4.49 (br d, 13.5) Glucose (at C-6 of Glu) 5.01 (d, 8.0) 4.00 (t, 8.5)

H-3 H-4

4.22a 4.10 (t, 9.5)

4.19a 4.23a

4.22a 4.26a

H-5 H-6

3.93a 4.36 (br d, 10.5) 4.51 (br d, 10.5)

3.87a 4.32 (br d, 11.0) 4.43 (br d, 11.0)

3.97a 4.24a

H-1 H-2 H-3 H-4 H-5 H-6

4.38 (br, d 11.0) 4.53 (br, d 11.0)

H-1 H-2 H-3 H-4 H-5 H-6

a

4.45a

Glucose (at C-6 of Glu) 5.07 (d, 7.5) 4.06a

4.40a

Glucose (at C-3 of Fuc) 5.22 (d, 7.0) 3.96 (dd, 9.5, 7.0) 4.17a

Glucose (at C-2 of Glu) 5.31 (d, 7.5) 4.15a 3.91a

Glucose (at C-3 of Fuc) 5.02 (d, 7.0) 3.89 (dd, 8.5, 7.0) 4.21a

4.30 (t, 9.5)

3.76a

4.00 (dd, 9.0, 8.0) 4.09 (dd, 9.0, 6.5) 4.22a

4.20a

4.12a

4.32a

4.17a

4.81 (br d, 11.5)

4.66 (br d, 10.5)

4.77 (d, 10.5)

Glucose (at C-6 of Glu) 5.01 (d, 7.5) 4.00 (dd, 8.5, 7.5) 4.17a 4.47 (dd, 9.0, 9.0) 3.85 m 4.32a 4.46 (dd, 12.0, 1.5)

Glucose (at C-6 of Glu) 5.06 (d, 8.0) 4.05 (t, 8.0) 4.21a 4.10 (t, 10.5) 3.91a 4.36 (br d, 10.5) 4.50 (br d, 10.5)

Glucose (at C-2 of Glu) 5.10 (d, 7.0) 4.05a 4.11a 4.16a 3.80a 4.33a 4.48 (br d, 13.5) Glucose (at C-6 of Glu) 5.01 (d, 7.0) 4.00 (dd, 8.5, 7.0) 4.21a 4.23a 3.87a 4.33 (d, 10.5) 4.45a

Overlapped with other signals; assignments confirmed by 2D COSY, HSQC, and HMBC experiments; – not found

epoxide bridge between the quaternary carbons at C-13 (δC 83.6) and C-28 (δC 73.0). However, signals typical of an aldehyde group [δH 9.70 (1H, s) and δC 206.8 (C=O)] were observed at C-23, as de" Fig. 3). The triterpetermined by HMBC correlation analysis (l noid skeleton of 4 was identified as 3β,16β-dihydroxy-23-oxo13,28-epoxyolean-11-ene [15]. The three anomeric proton signals at δH 4.61, 5.07, and 5.27 and the three corresponding carbons at δC 104.7, 105.4, and 106.0, which were correlated in the HSQC spectrum, indicated a trisaccharide moiety. The two sets of " Tables 1 and 2) glucopyranosyl and fucopyranosyl signals (l were assigned by detailed analysis of the NMR data (1H, 13C, and HMBC) and comparison with the literature [16, 21]. The linkage between the fucopyranosyl and glucopyranosyls was established by analysis of the HMBC correlations between δH 4.61 (H-1′ of Fuc) and δC 81.3 (C-3 of the aglycone); δH 5.27 (H-1′′ of Glc) and δC 84.3 (C-3′ of Fuc); and δH 5.07 (H-1′′′ of Glc) and δC 70.0 (C-6′′ of Glc). The trisaccharide was elucidated as a [β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranosyl moi-

ety, and thus compound 4 was concluded to be 3β,16β-dihydroxy-23-oxo-13,28-epoxyolean-11-ene-3-O-[β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranoside. Comastomasaponin E (5) has the molecular formula C43H72O14, as determined by HR‑ESI‑MS analysis (m/z 835.4821 [M + Na]+). Thirty of the 43 carbons were assigned to the triterpenoid skeleton, 12 to a disaccharide moiety, and one to a methoxy group. The 1 H‑NMR spectrum showed six singlets corresponding to tertiary " Table methyl groups at δH 0.88, 0.99, 1.06, 1.10, 1.13, and 1.38 (l 1). These signals were correlated with six sp3 carbons at δC 13.8, " Table 2), which were correlated 18.3, 18.7, 24.3, 26.6, and 33.6 (l in the HSQC spectrum. The olefinic proton at δH 5.52 and the sp2 " Tables 1 and 2) are characteriscarbons at δC 122.8 and 148.6 (l tic of an olean-12-ene aglycone. Signals characteristic of hydroxymethyl groups at C-28 and C-23 were observed at δH 3.73, δH 4.38, and δC 68.9, and at δH 3.77, δH 4.40, and δC 65.5, respectively. The C-3 and C-16 signals were also assigned (δH 4.16 and 4.62, and δC 82.7 and 66.5). The substituted position of the me-

Cui B-S et al. Hepatoprotective Saikosaponin Homologs … Planta Med 2014; 80: 1647–1656

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4.45a

Original Papers

13

Agycone C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29 C-30 OCH3 C-1 C-2 C-3 C-4 C-5 C-6

C-1 C-2 C-3 C-4 C-5 C-6

C NMR spectroscopic data of compounds 1–8 (125 MHz in pyridine-d5). 1

2

3

4

5

6

7

8

38.5 26.0 81.8 43.7 47.2 17.5 31.6 42.2 53.0 36.2 132.1 131.1 84.0 45.6 36.1 64.0 47.0 52.1 37.7 31.6 34.7 25.7 64.0 13.0 18.7 20.0 20.8 73.0 33.6 23.8

38.0 25.6 82.3 43.3 47.5 17.2 31.1 41.7 52.5 35.7 131.8 130.5 83.4 45.1 35.6 63.5 46.5 51.6 37.3 31.1 34.2 25.3 64.4 12.4 18.2 19.5 20.3 72.5 33.2 23.3

38.6 26.1 82.0 43.7 47.4 17.5 31.6 42.2 53.1 36.2 132.2 131.1 84.0 45.6 36.1 64.0 47.0 52.1 37.7 31.6 34.7 25.7 64.1 12.9 18.7 20.0 20.8 73.0 33.6 23.8

37.8 25.1 81.3 55.4 47.4 19.9 31.0 42.3 52.5 35.4 131.3 131.2 83.6 45.4 35.9 63.7 46.8 51.9 37.5 31.4 34.5 25.5 206.8 9.5 17.9 19.7 20.7 72.8 33.4 23.6

Glucose 106.2 72.3 85.2 69.9 76.6 62.3 Glucose (1 → 3) 106.7 75.9 78.7 71.5 78.4 62.6

Glucose 103.5 83.5 76.4 71.2 77.6 62.3 Glucose (1 → 2) 105.6 76.1 78.1 70.9 77.8 69.7

Fucose 105.3 71.0 86.7 71.6 71.0 17.3 Glucose (1 → 3) 104.3 85.8 77.8 71.0 77.6 69.8 Glucose (1 → 2) 107.1 76.5 77.2 70.6 79.1 62.6 Glucose (1 → 6) 105.4 75.3 78.3 71.5 78.4 62.0

Fucose 105.4 71.0 84.3 71.9 70.9 17.1 Glucose (1 → 3) 106.0 75.1 78.2 71.6 77.1 70.0

40.5 26.7 82.7 44.0 48.7 18.7 33.5 41.4 52.5 38.4 76.3 122.8 148.6 44.2 37.2 66.5 44.2 44.3 47.3 31.5 34.6 26.2 65.5 13.8 18.3 18.7 26.6 68.9 33.6 24.3 54.4 Fucose 104.3 82.8 75.9 72.8 71.4 17.7 Glucose (1 → 2) 106.5 77.0 78.6 71.6 78.5 62.8

40.1 26.4 81.8 43.8 47.6 18.2 33.1 41.0 52.1 38.0 75.9 122.4 148.2 43.8 36.8 66.2 43.6 43.9 46.9 31.0 34.2 25.8 64.3 13.5 17.9 18.3 26.2 68.5 33.2 24.0 54.0 Fucose 106.0 71.5 84.9 72.1 71.0 17.2 Glucose (1 → 3) 106.1 75.2 78.3 71.7 77.3 70.1

40.0 26.5 82.7 43.9 48.3 18.3 33.2 41.0 52.0 38.0 76.0 122.6 148.1 44.0 36.8 66.2 43.6 43.9 47.0 31.1 34.2 25.9 64.5 13.4 17.9 18.3 26.3 68.6 33.3 24.0 54.2 Glucose 103.9 83.9 76.8 71.6 78.1 62.8 Glucose (1 → 2) 106.1 76.6 78.5 71.4 78.2 70.0

Glucose (1 → 6) 105.4 75.4 78.3 71.6 78.3 62.6

Glucose (1 → 6) 105.6 75.2 78.3 71.0 78.5 62.6

40.1 26.5 82.2 43.8 47.7 18.2 33.1 41.0 52.2 38.1 75.9 122.5 148.2 43.8 36.8 66.2 43.6 43.9 46.9 31.1 34.2 25.9 64.4 13.5 17.9 18.3 26.2 68.5 33.2 24.0 54.1 Fucose 105.3 71.0 86.7 71.5 71.1 17.3 Glucose (1 → 3) 104.3 85.9 77.8 71.0 77.7 69.8 Glucose (1 → 2) 107.1 76.5 77.2 70.6 79.1 62.6 Glucose (1 → 6) 105.4 75.3 78.3 71.5 78.4 62.0

C-1 C-2 C-3 C-4 C-5 C-6

C-1 C-2 C-3 C-4 C-5 C-6

Glucose (1 → 6) 105.0 74.7 77.9 70.6 78.0 62.1

Glucose (1 → 6) 104.7 75.3 78.2 71.4 78.2 62.4

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Table 2

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Original Papers

Fig. 2 The key HMBC correlations of compound 1. (Color figure available online only.)

Fig. 3 The key HMBC correlations of compound 4. (Color figure available online only.)

Fig. 4 The key HMBC correlations of compound 5. (Color figure available online only.)

thoxy [δH 3.22 (3H, s) and δC 54.4] group was established by analysis of the HMBC correlations between δH 3.22 and δC 76.3 (C-11 of the aglycone). The methoxy group was assigned to the α-position at C-11 because the coupling constants of H-11 (J = 8.5, 3.5 Hz) and H-9 (J = 8.5 Hz) were consistent with axial configurations for both protons [16, 21], and were further supported by the NOESY correlation between H-11β (3.81) and H-26β-CH3 (1.06). The triterpenoid skeleton was assigned as 3β,16β,23,28-tetrahydroxy-olean-12-ene; this assignment was further supported by " Fig. 4). The two anomeric protons HMBC correlation analysis (l at δH 4.94 and 5.25 and the two corresponding carbons at δC 104.3 and 106.5 indicated a disaccharide moiety. The two sets of " Tables 1 and glucopyranosyl and fucosyl signals were assigned (l 2) by analysis of the gCOSY, HSQC, and HMBC spectra. The linkage

between the glucopyranosyl and fucopyranosyl was established by analysis of the HMBC correlations between δH 4.94 (H-1′ of Fuc) and δC 82.7 (C-3 of the aglycone) and between δH 5.25 (H-1′ ′ of Glc) and δC 82.8 (C-2′ of Fuc). Therefore, compound 5 was determined to be 3β,16β,23,28-tetrahydroxy-13α-methoxy-olean12-ene-3-O-[β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyranoside. Comastomasaponin F (6) has the molecular formula C49H82O19, as determined by HR‑ESI‑MS analysis (m/z 997.5437 [M + Na]+). " Tables 1 and 2) revealed Detailed analysis of the NMR data (l that the aglycone signals of 6 and 5 were nearly identical. The saccharide signals, however, displayed significant differences. The three anomeric proton signals at δH 4.94, 5.01, and 5.22 and the three corresponding carbon signals at δC 105.4, 106.0, and 106.1 indicated a trisaccharide moiety. The two sets of glucopyr" Tables 1 and 2) by analysis of the anosyl signals were assigned (l gCOSY, HSQC, and HMBC spectra. The linkage between the glucopyranosyls and fucopyranosyl was established by analysis of the HMBC correlations between δH 4.94 (H-1′ of Fuc) and δC 81.8 (C-3 of the aglycone); δH 5.22 (H-1′′ of Glc) and δC 84.9 (C-3′ of Fuc); and δH 5.01 (H-1′′ of Glc) and δC 70.1 (C-6′′ of Glc). The trisaccharide was elucidated as a [β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranosyl moiety, and thus 6 was determined to be 3β,16β,23,28-tetrahydroxy-13α-methoxy-olean-12-ene-3-O-[β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranoside. Comastomasaponin G (7) has the molecular formula C49H82O20, as determined by HR‑ESI‑MS (m/z 1013.5302 [M + Na]+). Detailed " Tables 1 and 2) revealed that the analysis of the NMR data (l aglycone signals of 7 and 5 were nearly identical. The saccharide signals, however, displayed significant differences. The three anomeric proton signals at δH 5.02, 5.06, and 5.31 and the three corresponding carbons signal at δC 103.9, 105.6, and 106.6 indicated a trisaccharide moiety. The three sets of glucopyranosyl sig" Tables 1 and 2) by analysis of the gCOSY, nals were assigned (l HSQC, and HMBC spectra. The linkage between the glucopyranosyls was established by analysis of the HMBC correlations between δH 5.02 (H-1′ of Glc) and δC 82.7 (C-3 of the aglycone); δH 5.31 (H-1′′ of Glc) and δC 83.9 (C-2′ of Glc); and δH 5.06 (H-1′′′ of Glc) and δC 70.0 (C-6′′ of Glc). The trisaccharide was elucidated as a [β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl-(1 → 2)-β-Dglucopyranosyl moiety, and thus the structure of 7 was concluded to be 3β,16β,23,28-tetrahydroxy-13α-methoxy-olean12-ene-3-O-[β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyranosyl(1 → 2)-β-D-glucopyranoside. Comastomasaponin H (8) has the molecular formula C55H92O24, as determined by HR‑ESI‑MS analysis (m/z 1159.5878 [M + Na]+). " Tables 1 and 2) revealed Detailed analysis of the NMR data (l that the aglycone signals of 8 and 5 were nearly identical. The saccharide signals, however, displayed significant differences. The four anomeric proton signals at δH 4.92, 5.01, 5.02 and 5.10 and the four corresponding carbon signals at δC 104.3, 105.3, 105.4, and 107.1 indicated a tetrasaccharide moiety. The linkage between the fucopyranosyl and glucopyranosyls was established by analysis of the HMBC correlations between δH 4.92 (H-1′ of Fuc) and δC 82.2 (C-3 of the aglycone); δH 5.02 (H-1′′ of Glc) and δC 86.7 (C-3′ of Fuc); δH 5.10 (H-1′′′ of Glc) and δC 85.9 (C-2′′ of Glc); and δH 5.01 (H-1′′′′ of Glc) and δC 69.8 (C-6′′ of Glc). The tetrasaccharide was elucidated as a [β-D-glucopyranosyl-(1 → 6)-βD-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranosyl group, and thus 8 was determined to be 3β,16β,23,28-tetrahydroxy-13α-methoxy-olean-12-ene-3-O-[β-

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1652

Original Papers

Table 3 Hepatoprotective activity of compounds 1–17 against D-galactosamine-induced toxicity in WB-F344 cellsa.

a

Compound

Cell survival ratec

Normal Control Bicyclolb 1 4 9 10 11 12 13 Normal Control Bicyclolb 3 5 6 14 15 16 17 Normal Control Bicyclolb 2 7 8

100.78 ± 0.02 53.44 ± 0.03 68.54 ± 0.04** 74.59 ± 0.07*** 57.46 ± 0.06 74.59 ± 0.04*** 72.58 ± 0.04*** 86.68 ± 0.04*** 75.60 ± 0.05*** 41.35 ± 0.06 100.83 ± 0.02 44.40 ± 0.05 68.58 ± 0.09** 39.35 ± 0.03 73.62 ± 0.03*** 67.57 ± 0.07** 63.54 ± 0.05** 58.50 ± 0.04* 48.42 ± 0.04 69.59 ± 0.04** 100.85 ± 0.08 57.51 ± 0.08 71.62 ± 0.04* 66.58 ± 0.05 72.63 ± 0.04* 72.63 ± 0.06*

Inhibitiond 47.34 31.90 44.68 8.49 44.68 40.43 70.22 46.81 − 25.54 56.43 37.53 − 8.95 51.78 41.60 33.92 24.99 7.12 44.64 43.34 32.56 20.93 34.89 34.89

Results are expressed as means ± SD (n = 3; for normal and control, n = 6); * p < 0.05,

** p < 0.01, *** p < 0.001. Compounds were tested at 1 × 10−5 M. b Positive control substance bicyclol. c In contrast to normal group (%). d In contrast to control group (%)

The aerial portions of C. pedunculatum were collected from Gonghe County (3000–4000 m elevation), Qinghai province, China, in August 2006 and identified by Prof. Pengcheng Lin from Qinghai University for Nationalities. A voucher specimen (HMH200608A) was deposited in the College of Life and Environmental Science, Central University for Nationalities, Beijing.

123.87 ppm) as an internal standard. Mass spectra were obtained on a Mass Agilent 1100 Series LC‑MSD-Trap-SL spectrometer (ESI‑MS) and 6210 ESI‑TOF spectrometer (HR‑ESI‑MS). Silica gel 60 (QingDaohaiyangChem, 200–400 mesh), RP-18 (Alltech Bulk Higt Capacity C18, 45–75 µm), and Sephadex LH-20 (Amersham Pharmacia Biotech) were used for column chromatography (CC). Precoated silica gel plates (Qing Dao hai yang Chem, GF254) and precoated RP-18F254 plates (Merck) were used for analytical thin-layer chromatography. Medium-pressure liquid chromatography (MPLC) was performed using HEPF02 (H&E). High-performance liquid chromatography (HPLC) was performed using a Lab Alliance Prep-100 pump equipped with a Lab Alliance Model-201 UV‑vis detector at 210 nm and a semipreparative reversed-phase column (Grace, Allsphere ODS-25 µm, 250 × 10 mm). HPLC was performed on an SHIMADZU LC-20AT HPLC pump with an SPD-M20A detector at 250 nm. D-glucose, L-glucose, D-fucose, and L-fucose were from Alfa Aesar A Johnson Matthey Company, and L-cysteine methyl ester hydrochloride and o-toly isothiocyanate were from J&K Scientific Ltd.

General experimental procedures

Extraction and isolation

Optical rotation was measured in MeOH solution in a JASCO P2000 digital polarimeter with a sodium lamp (589 nm). The UV spectra were obtained using a JASCO V-650 spectrophotometer. IR spectra were acquired on a Nicolet 5700 spectrophotometer. NMR spectra were recorded on a Varian Unity Inova 500 and Bruker AVANCE III HD 600 FT‑NMR spectrometer. Chemical shifts are reported in parts per million (δ), and coupling constants (J) are expressed in Hertz, using residual C5D5 N (δH 7.22 and δC

The air-dried aerial portions of C. pedunculatum (5.0 kg) were extracted with 70% aqueous ethanol (3 × 40 L) under reflux for 1 h. The combined extracts were concentrated in vacuo, suspended in H2O (3 L), and partitioned sequentially with petroleum (3 × 3 L), EtOAc (3 × 3 L), and n-BuOH (3 × 3 L). The n-BuOH extract (230 g) was subjected to CC on AB-8 macroporous resin (10 L; column, 10 × 150 cm) and eluted with 20 % (80 L), 40% (80 L), and 60 % (60 L) aqueous ethanol. After removing the solvent, the 60 %

Materials and Methods !

Plant materials

Cui B-S et al. Hepatoprotective Saikosaponin Homologs …

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D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 3)-β-D-fucopyranoside. Compounds 1–17 were evaluated for their hepatoprotective activity against D-galactosamine-induced toxicity in WB-F344 cells, using the hepatoprotective drug bicyclol as the positive control [22]. Compounds 1, 5–12, 14, 15, and 17 exhibited potent " Table 3). hepatoprotective activity (l The cytotoxic activities of compounds 1–17 were evaluated against five different human tumor cell lines in in vitro assays, using the drug paclitaxel as the positive control. Only compound 11 showed substantial cytotoxic activity against HCT-8, Bel-7402, BGC-825, A549, and A2780 cells, with IC50 values of 4.08, 2.04, 3.12, 1.33, and 2.35 µM, and the IC50 values of 3.50, 12.32, 3.29, 7.86, and 2.34 nM for the positive control paclitaxel, respectively. The other compounds did not display significant cytotoxic activity, with IC50 values > 10 µM. Saikosaponins represent a group of triterpene saponins derived from Bupleurum falcatum L. (Umbelliferae) [11], which is a medicinal plant that has been used for treating various inflammatory and infectious diseases by the Chinese for over one thousand years. Saikosaponins were isolated from medicinal plants such as Bupleurum spp., Heteromorpha spp., and Scrophularia scorodonia. They were the major active components present in Bupleurum spp. and have been reported to possess various biological activities, specifically antihepatitis, antinephritis, antihepatoma, antiinflammation, immunomodulation, and antibacterial effects [12– 14]. Herein, we report the triterpenoid saponins and saikosaponin homologs from the aerial portions of C. pedunculatum. It is the first time saikosaponins have been found in the Gentianaceae family. The compounds were evaluated for hepatoprotective activity against D-galactosamine-induced toxicity and cytotoxic activity against five human tumor cell lines in vitro. Twelve compounds exhibited potent hepatoprotective activity and one compound displayed cytotoxic activity against HCT-8, Bel-7402, BGC825, A549, and A2780 human tumor cell lines. The results of the biological activities evaluation indicated the triterpenoid saponins, isolated from C. pedunculatum, were the major bioactive components present in C. pedunculatum. Our results provide basic information for a better understanding of the effect of the herb C. pedunculatum.

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aqueous ethanol fraction (25.7 g) was chromatographed over reversed-phase silica gel by MPLC [ODS (mesh, 45–75 µm, 200 g); column, 3.7 × 46 cm; 15 mL/min; twice] and eluted in a step gradient manner with MeOH‑H2O (20 : 80, 1 L; 30 : 70, 2 L; 35 : 65, 2 L; 40 : 60, 2 L; 45 : 55, 2 L; 50 : 50, 3 L; 55 : 45, 4 L; 60 : 40, 4 L; 65 : 35, 4 L; 70 : 30, 2 L; 80 : 20, 2 L; 100 : 0, 3 L) to afford 12 subfractions. Subfraction 3 (1.70 g) was subjected to Sephadex LH20 CC (100 g; column, 2.5 × 60 cm), eluted with CHCl3-MeOH (1 : 1; 2 L), and further separated by preparative HPLC [Allsphere C18 column, (250 × 10 mm, 5 µm)] in MeOH‑H2O (70 : 30, 2.0 mL/ min) to afford 15 (11 mg, 55.4 min, 210 nm) and 16 (12 mg, 23.3 min, 210 nm). Subfraction 4 (3.63 g) was fractionated over silica gel by MPLC (mesh, 38 µm, 200 g; column, 3.7 × 46 cm; 15 mL/min) with CHCl3-MeOH (6 : 1, 5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 0 : 1, each 0.5 L) to yield a mixture (864 mg) that was further separated by RP-18 preparative HPLC in MeOH‑H2O (60 : 40, 2.0 mL/ min) to isolate 6 (11 mg, 24.9 min, 210 nm), and MeOH‑H2O (70 : 30, 2.0 mL/min) to isolate 14 (164 mg, 35.5 min, 210 nm). Subfraction 5 (3.23 g) was subjected to Sephadex LH-20 [100 g; column, 2.5 × 60 cm] and eluted with MeOH (2 L) to provide two main subfractions. Subfractions 5–1 (438 mg) and 5–2 (211 mg) were purified by RP-18 preparative HPLC in MeOH‑H2O (65 : 35, 2.0 mL/min) to provide 2 (23 mg, 14.5 min, 210 nm) and 3 (18 mg, 43.5 min, 210 nm), and MeOH‑H2O (60 : 40, 2.0 mL/min) to provide 7 (8 mg, 65.6 min, 210 nm). Subfraction 6 (1.88 g) was further separated via RP-18 silica gel by MPLC [ODS (mesh, 45– 75 µm, 50 g); column, 1.5 × 46 cm; 10.0 mL/min] in MeOH‑H2O mixtures of increasing polarity to give ten subfractions. Subfraction 6–2 (612 mg), which was eluted with MeOH‑H2O (1 : 1, 2 L), was subjected to Sephadex LH-20 (50 g; column, 1.5 × 60 cm) to yield a mixture (376 mg) that was separated by RP-18 HPLC in MeOH‑H2O (65 : 35, 2.0 mL/min) to give 8 (15 mg, 28.6 min, 210 nm). Subfraction 7 (3.43 g) was fractionated over silica gel by MPLC (mesh, 45–75 µm, 100 g; column, 3.7 × 46 cm, 10.0 mL/ min) in CHCl3-MeOH (10 : 1, 8 : 1, 7 : 1, 5 : 1, 3 : 1, 1 : 1, 0 : 1 each 0.5 L) to yield seven subfractions. Subfractions 7–3 (149 mg) and 7–6 (243 mg) were further purified by RP-18 HPLC in MeOH‑H2O (70 : 30, 2.0 mL/min) to afford 1 (20 mg, 16.4 min, 210 nm), and MeOH‑H2O (64 : 36, 2.0 mL/min) to afford 5 (9 mg, 55.4 min, 210 nm,) and 17 (18 mg, 57.3 min, 210 nm). Subfraction 8 (4.92 g) was submitted to RP-18 by MPLC [ODS (mesh, 45– 75 µm, 200 g); column, 3.7 × 46 cm, 10 mL/min], eluted with MeOH‑H2O (40 : 60, 50 : 50, 60 : 40, 70 : 30, each 0.5 L), and further separated by RP-18 HPLC in MeOH‑H2O (65 : 35, 2.0 mL/min) to isolate 10 (7 mg, 90.2 min, 210 nm), 11 (246 mg, 91.8 min, 210 nm,), and 13 (47 mg, 103.0 min, 210 nm,), and MeOH : H2O (70 : 30, 2.0 mL/min) to isolate 9 (7 mg, 88.7 min, 210 nm,) and 12 (36 mg, 38.1 min, 210 nm). Subfraction 9 (1.10 g) was chromatographed on silica gel by MPLC (mesh, 38 µm, 10 g; column, 1.5 × 10 cm, 10.0 mL/min), eluted with CHCl3-MeOH (9 : 1, 8 : 1,7 : 1, 6 : 1, 5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 0 : 1 each 0.5 L), further separated by RP-18 HPLC, and then eluted with MeOH‑H2O (70: 30, 2.0 mL/min) to isolate 4 (7 mg, 21.3 min, 210 nm).

Absolute configurations of the sugars The absolute configurations of glucose and fucose were determined according to a reported procedure [19]. Compound 1 (2 mg) was hydrolyzed by 1 M HCl (1 mL) at 100 °C for 2 h. The reaction mixture was dried under vacuum. After the addition of water, the acidic solution was evaporated to again remove HCl. After drying under vacuum, the residue was dissolved in pyridine (0.4 mL) containing L-cysteine methyl ester hydrochloride (2 mg) and heated at 60 °C for 2 h. o-Toylisothiocyanate (2 µL) was then added and the mixture was heated at 60 °C for 2 h. Each reaction mixture was directly analyzed by reversedphase HPLC using a SHIMADZU LC-20AT HPLC pump with an SPD-M20A detector at 250 nm. A COSMOSIL packed column 5C18-AR‑II 4.6 ID × 150 mm HPLC column was used [temp, 35 °C; flow, 0.8 ml/min; eluate, CH3CN–H2O (25 : 75) containing 50 mM H3PO4]. The HPLC column was washed with MeOH after each injection. The reaction conditions for D- and L-glucose were the same as described above. Retention times for authentic sugar derivatives, D-glucose (11.62 min), and L-glucose (10.61 min) were used for comparison with retention times from reaction mixtures for the saponin. A peak at 11.52 min of the sugar derivatives from 1 coincided with the derivatives of D-glucose. The absolute configuration sugars of compounds 2–8 were identified by the same method as 1. Retention times of authentic samples were detected at 11.57 min (D-glucose) for 2 and 7, and 11.54 min (D-glucose) and 15.73 min (D-fucose, 15.78 min, L-fucose, 17.56 min) for 3–6 and 8, respectively.

Hepatoprotective activities against D-galactosamineinduced cytotoxicity in WB-F344 cells [9] The hepatoprotective activities of compounds 1–17 were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in WB-F344 cells. A cell suspension of 1 × 104 cells in 200 µL of Dulbeccoʼs Modified Eagleʼs medium containing fetal calf serum (3 %), penicillin (100 units/mL), and streptomycin (100 µg/mL) was placed in a 96-well microplate and precultured for 24 h at 37 °C under a 5 % CO2 atmosphere. Fresh medium (200 µL) containing the positive control bicyclol (purity > 99 %, Beijing Union Pharmaceutical Factory) and the test samples were added, and the cells were cultured for 1 h. The cultured cells were exposed to 40 mM D-galactosamine for 24 h. The cytotoxic effects of the test samples were simultaneously measured in the absence of D-galactosamine. After 24 h, the medium was then replaced with a medium containing 0.5 mg/ mL MTT. After incubation for 3.5 h, the medium was removed, and 150 µL of DMSO was added to dissolve the formazan crystals. The optical density (OD) of the formazan solution was measured at 492 nm using a microplate reader. Inhibition (%) was determined using the following formula: Inhibition (%) = [(OD(sample) – OD(control))/(OD(normal) – OD(control))] × 100

Statistical analysis Acid hydrolysis A solution of each compound (5 mg) in 0.21 N HCl (5 mL) was heated at 90 °C for 5 h. After extraction with EtOAc (3 × 5 mL), each mixture was diluted with H2O and extracted with EtOAc (3 × 5 mL). The aqueous layer was evaporated and cryodessicated, and the residue was analyzed by TLC over silica gel (CHCl3MeOH‑H2O, 6 : 4 : 1) and compared with authentic samples.

Studentʼs t-test for unpaired observations between the control and test samples was performed to identify significant differences, and p values less than 0.05 were considered significantly different.

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Human colon cancer (HCT-8), hepatoma (Bel-7402), stomach cancer (BGC-825), ovarian cancer (A2780), lung cancer (A549), and epithelial WISH cell lines (Susan Hayflick Wistar Institute) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. Cultures were incubated at 37 °C in a humidified 5% CO2 atmosphere. HCT-8, Bel-7402, BGC-823, A2780, A549, and human epithelial WISH cells were seeded in 96-well microtiter plates at a concentration of 1200 cells/well. After 24 h, compounds 1–17 were added to the cells. After 96 h of drug treatment, cell viability was determined by measuring the metabolic conversion of MTT into purple formazan crystals by the active cells. The MTT assay results were monitored at 570 nm using an MK 3 Wellscan (Labsystem DROGON) plate reader. All compounds were dissolved in 100 % DMSO to yield a final DMSO concentration of 0.1% in each well and tested at five different concentrations. The concentration of each compound was tested in three parallel wells. The positive control was paclitaxel (purity > 99 %, Beijing Union Pharmaceutical Factory). IC50 values were calculated using Microsoft Excel software. Comastomasaponin A (1): white amorphous powder (99.6 %, purity, HPLC); [α]20 D + 59.5 (c 0.051, MeOH); UV (MeOH) λmax (log ε) 202 (3.92); IR (Microscope) νmax 3404, 2943, 2870, 1640, 1466, 1451, 1385, 1365, 1305, 1258, 1155, 1075 cm−1; 1H NMR and 13C " Tables 1 and 2); ESI‑MS m/z 819.4 [M + Na]+; HR‑ENMR data (l SI‑MS m/z 819.4507 [M + Na]+ (calcd. for C42H68O14Na, 819.4501). Comastomasaponin B (2): white amorphous powder (96.5%, purity, HPLC); [α]20 D + 18.8 (c 0.062, MeOH); UV (MeOH) λmax (log ε) 203 (3.92); IR (Microscope) νmax 3359, 2925, 2874, 1619, 1453, 1419, 1385, 1366, 1264, 1165, 1075 cm−1; 1H NMR and 13C NMR " Tables 1 and 2); ESI‑MS m/z 981.6 [M + Na]+; HR‑ESI‑MS data (l m/z 981.5037 [M + Na]+ (calcd. for C48H78O19Na, 981.5030). Comastomasaponin C (3): white amorphous powder (97.8 %, purity, HPLC); [α]20 D + 33.9 (c 0.055, MeOH); UV (MeOH) λmax (log ε) 202 (3.78); IR (Microscope) νmax 3395, 2931, 2875, 1643, 1466, 1436, 1381, 1366, 1329, 1277, 1246, 1162, 1122, 1067, " Tables 1 and 2); ESI‑MS 1040 cm−1; 1H NMR and 13C NMR data (l m/z 1127.6 [M + Na]+; HR‑ESI‑MS m/z 1127.5611 [M + Na]+ (calcd. for C54H88O23Na, 1127.5609). Comastomasaponin D (4): white amorphous powder (96.3 %, purity, HPLC); [α]20 D + 16.9 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 211 (3.40), 279 (2.46); IR (Microscope) νmax 3369, 2934, 2874, 1716, 1641, 1450, 1385, 1365, 1306, 1166, 1066 cm−1; 1H NMR " Tables 1 and 2); ESI‑MS m/z 963.6 and 13C NMR data (l + [M + Na] ; HR‑ESI‑MS m/z 963.4923 [M + Na]+ (calcd. for C48H76O18Na, 963.4924). Comastomasaponin E (5): white amorphous powder (96.5 %, purity, HPLC); [α]20 D − 4.8 (c 0.044, MeOH); UV (MeOH) λmax (log ε) 202 (4.09); IR (Microscope) νmax 3374, 2945, 1645, 1451, 1386, " Tables 1 1364, 1165, 1075 cm−1; 1H NMR and 13C NMR data (l + and 2); ESI‑MS m/z 835.5 [M + Na] ; HR‑ESI‑MS m/z 835.4821 [M + Na]+ (calcd. for C43H72O14Na, 835.4814). Comastomasaponin F (6): white amorphous powder (98.6%, purity, HPLC); [α]20 D − 6.4 (c 0.065, MeOH); UV (MeOH) λmax (log ε) 203 (3.91); IR (Microscope) νmax 3389, 2944, 1644, 1452, 1385, 1365, 1313, 1263, 1166, 1072 cm−1; 1H NMR and 13C NMR data " Tables 1 and 2); ESI‑MS m/z 997.6 [M + Na]+; HR‑ESI‑MS m/z (l 997.5347 [M + Na]+ (calcd. for C49H82O19Na, 997.5343).

Comastomasaponin G (7): white amorphous powder (97.2 %, purity, HPLC); [α]20 D − 10.7 (c 0.016, MeOH); UV (MeOH) λmax (log ε) 202 (4.05); IR (Microscope) νmax 3335, 2925, 2854, 1655, 1597, 1459, 1416, 1386, 1317, 1264, 1166, 1076 cm−1; 1H NMR and 13C " Tables 1 and 2); ESI‑MS m/z 1013.6 [M + Na]+; HRNMR data (l ESI‑MS m/z 1013.5302 [M + Na]+ (calcd. for C49H82O20Na, 1013.5292). Comastomasaponin H (8): white, amorphous powder (99.5%, purity, HPLC); [α]20 D − 7.5 (c 0.056, MeOH); UV (MeOH) λmax (log ε) 203 (4.00); IR (Microscope) νmax 3383, 2942, 1641, 1450, 1381, 1364, 1313, 1262, 1168, 1071 cm−1; 1H NMR and 13C NMR data " Tables 1 and 2); ESI‑MS m/z 1159.6 [M + Na]+; HR‑ESI‑MS m/z (l 1159.5878 [M + Na]+ (calcd. for C55H92O24Na, 1159.5871).

Supporting information UV, IR, MS, 1H NMR, and 13C NMR spectra as well as 2D NMR correlation spectra of compounds 1–8 are available as Supporting Information.

Acknowledgements !

We gratefully acknowledge the financial support of the National Science and Technology Project of China (2012ZX0930100 2001003). We are grateful to Prof. Xiaoguang Chen for the human cancer cells screening. We thank Mr. Jianbei Li for the HR‑ESI‑MS analyses.

Conflict of Interest !

The authors declare that they have no conflict of interest.

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Assessment of the inhibitory activity of compounds 1–17 against human cancer cell lines [9]

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