Original Papers
Cytotoxic Triterpenoid Glycosides from the Roots of Camellia oleifera
Authors
Xia Li 1, Jianping Zhao 2, Cuiping Peng 1, Zhong Chen 1, Yanli Liu 1, Qiongming Xu 1, 2, Ikhlas A. Khan 1, 2, Shilin Yang 1
Affiliations
1 2
Key words " Camellia oleifera l " Theaceae l " triterpenoid saponins l " oleiferosides A–H l " cytotoxic activities l
College of Pharmaceutical Science, Soochow University, Suzhou, China National Center for Natural Products Research, and Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Mississippi, USA
Abstract !
Eight new triterpenoid saponins, oleiferosides A– H (1–8), were isolated from the EtOH extract of the roots of Camellia oleifera. Their structures were elucidated by a combination of 1D and 2D NMR techniques, mass spectrometry, and chemical methods. All were characterized to be oleanane-type saponins with sugar moieties linked to C-3 of the aglycone. Cytotoxic activities of these saponins were evaluated against four hu-
Introduction !
received revised accepted
Dec. 19, 2013 February 26, 2014 March 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368428 Planta Med 2014; 80: 590–598 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Qiongming Xu, Ph.D. College of Pharmaceutical Science Soochow University Suzhou 215123 China Phone: + 8 65 12 69 56 14 21 Fax: + 8 65 12 65 88 20 89 [email protected]
Belonging to the Theaceae family, the Camellia Linn genus comprises 120 species. The plants grow in the tropics and subtropics, with 65 species found in China. The plant Camellia oleifera C. Abel has been widely cultivated as an oil crop in many parts of China, including Hunan, Jiangxi, Anhui, Henan, Zhejiang, and Fujian provinces. The roots of C. oleifera have been used in traditional Chinese medicine for the treatment of the common cold, measles, ardent fever, urinary tract infection, nephritis, edema, and threatened abortion [1] due to their antimicrobial [2], antioxidant, and antitumor activities [3]. Previous studies on the constituents from the Camellia genus led to the isolation of different compounds such as bibenzyl glycosides [4], flavonoids [5], triterpenoids, and glycosides [6–8]. In our efforts to investigate biologically active compounds from plant sources, the EtOH extract of the roots of C. oleifera was found to exhibit significant cytotoxicities against A549 human lung adenocarcinoma, B16 mouse melanoma, BEL-7402 human hepatocarcinoma, and MCF-7 human breast carcinoma cell lines. Our previous phytochemical study on the roots of C. oleifera led to the discovery of three new triterpenes and six known triterpenes, which showed moderate cytotoxic activities [9].
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man tumor cell lines (A549, B16, BEL-7402, and MCF-7) by using the MTT in vitro assay. Compound 3 exhibited potent cytotoxic activitiy against all the tested cell lines with IC50 values < 10 µM. Compounds 1, 2, 4, and 5 showed moderate cytotoxic activities toward the tested cell lines. Supporting information available online at http://www.thieme-connect.de/products
A continuous search for cytotoxic agents led us to isolate eight new triterpenoid saponins (1–8) from the roots of C. oleifera. Herein we describe the isolation, structure elucidation, and biological activities of these saponins. The structures of " Fig. 1. compounds 1–8 are shown in l
Results and Discussion !
Compound 1 was separated as a white amorphous powder. The negative ion HR‑ESI‑MS of 1 illustrated a quasimolecular ion [M – H]− peak at m/z 1315.5968, indicating the molecular formula of C63H96O29. The IR spectrum showed the presence of a hydroxyl group (3454 cm−1), an aldehyde group (2816, 2718, 1736 cm−1), and an α,βunsaturated ester group (1604, 1601 cm−1). The 13 " Table 1) spectrum showed the resoC‑NMR (l nances of 63 carbons, ascribable to ten methyls, ten methylenes, thirty-one methines, and twelve quaternary carbons as revealed by the HSQC ex" Table 2) spectrum periment. The 1H‑NMR (l showed six methyl signals at δ 0.88 (Me-25), 1.04 (Me-26), 1.18 (Me-29), 1.41 (Me-30), 1.53 (Me24) and 1.90 (Me-27), one isolated oxymethylene giving signals at δ 3.57 (1H, d, J = 11.0 Hz) and 3.82 (1H, d, J = 11.0 Hz), five oxymethines at δ 4.09 (1H, dd, J = 11.0, 4.5 Hz, H-3), 4.26 (1H, brs, H-15), 4.54
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(1H, brs, H-16), 6.78 (1H, d, J = 10.5 Hz, H-21), and 6.41 (1H, d, J = 10.5 Hz, H-22), one olefinic proton signal at δ 5.58 (1H, brs, H12), and an aldehyde signal at δ 9.94 (1H, brs, H-23). Furthermore, the signals of two angeloyl (Ang) groups at δ [6.07 (1H, dq, J = 7.5, 1.5 Hz, 21-O‑Ang-3′), 2.18 (3H, d, J = 7.5 Hz, 21-O‑Ang-4′), and 2.09 (3H, s, 21-O‑Ang-5′)], and δ [5.88 (1H, dq, J = 7.5, 1.5 Hz, 22-O‑Ang-3″), 2.05 (3H, d, J = 7.5 Hz, 22-O‑Ang-4″), and 1.83 (3H, s, 22-O‑Ang-5″)] were observed. The NMR data were comparable to those published in the literature [9], suggesting that compound 1 had the 21β,22α-O-diangeloyl-3β,15α,16α,28-tetrahydroxyolean-12-en-23-al type of aglycone. The locations of the two Ang groups at C-21 and C-22 were confirmed by the HMBC " Fig. 2). The relative configuration of compound 1 experiment (l " Fig. 2). The crosswas established from its NOESY spectrum (l peaks between H-21 at δH 6.78 and H-29 at δH 1.18, as well as those between H-22 at δH 6.41 and H-30 at δH 1.41, and H-28 at δH 3.57, 3.82, suggested that H-21 and H-22 are α- and β-oriented, respectively, which means that the two Ang groups at C-21 and C-22 are β- and α-equatorial, respectively. The H-15 at δH 4.26 correlated with H-28 at δH 3.57, 3.82 and H-18 at δH 3.15, in-
Structures of compounds 1–8.
dicating that the 15-OH group is α-configured. The CHO group was established at position 23, which was confirmed by the correlations between H-23 at δH 9.94, C-4 at δC 55.4, and C-24 at δC 11.3 in the HMBC spectrum, as well as the correlation between H3 at δH 4.09 and H-23 at δH 9.94 in the NOESY spectrum. The anomeric proton signals at δH 4.80 (1H, d, J = 6.5 Hz, H-1 of glucuronic acid), 5.80 (1H, d, J = 7.5 Hz, H-1 of galactose 1), 5.09 (1H, d, J = 7.5 Hz, H-1 of xylose), and 5.95 (1H, d, J = 7.5 Hz, H-1 of galactose 1′), which showed the HSQC correlation to δC 104.3 (C-1 of glucuronic acid), 101.8 (C-1 of galactose 1), 107.9 (C-1 of xylose), and 103.1 (C-1 of galactose 1′), respectively, indicated the presence of four sugar residues. Acid hydrolysis of 1 and GC analysis revealed one unit of D-glucuronic acid, two units of D-galactose, and one unit of D-xylose. The HMBC correlations between δH 4.80 (H-1 of glucuronic acid) and δC 84.7 (C-3 of the aglycone) " Fig. 2) indicated that the glycosidic chain was located at C-3 (l of the aglycone. The spectroscopic data of sugar units were very similar to those sugar units of camellenodiol 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside [10], which indicated Li X et al. Cytotoxic Triterpenoid Glycosides …
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Fig. 1
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the glycosidic chain located at C-3 of the aglycone was β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl. The linkage of the glycosidic chain was also supported by the HMBC correlations between δH 5.80 (H-1 of galactose 1) and δC 84.9 (C-3 of glucuronic acid), between δH 5.09 (H-1 of xylose) and δC 84.1 (C-2 of galactose 1), and between δH 5.95 (H-1 of galactose 1′) and δC 78.2 (C-2 of glucuronic acid). The β-anomeric configurations of the four sugar units were deduced from the observation of their large 3 JH-1, H-2 coupling constants. Thus, the structure of 1 was established as 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12en-23-al 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside, and named oleiferoside A. Compound 2 was isolated as white amorphous powder. The quasimolecular ion [M – H]− peak at m/z 1317.6122 revealed its molecular formula to be C63H98 O29. The IR spectrum indicated that the presence of a hydroxyl group (3456 cm−1), an aldehyde group (2820, 2718, 1740 cm−1), a C=O group (1720 cm−1), and an α,β-unsaturated ester group (1601 cm−1). The NMR data of compound 2 were similar to those of compound 1, and the main differences arose from the significant upfield shifts of the C-2″ (− 87.6 ppm) signal at δ 41.7 and the C-3″ (− 109.7 ppm) signal at δ 27.1. The analyses of the 1H-1H COSY, HSQC, and HMBC spectra revealed that a 2-methylbutanoyl (MB) group was attached to C-22 in compound 2, where in compound 1 it was an Ang group. Thus, the structure of compound 2 was assigned to be 21β-O-angeloyloxy-15α,16α,28-trihydroxy-22α-O-(2-methylbutanoyloxy)olean-12-en-23-al 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside, and named oleiferoside B. Compound 3 was obtained as white amorphous powder. It exhibited a molecular formula of C58H92O26 deduced from its [M – H]− ion peak at m/z 1203.5740. The IR spectrum indicated the presence of hydroxyl groups (3452 cm−1) and an α,β-unsaturated ester group (1602 cm−1). The 13C‑NMR spectrum of compound 3 was similar to that of compound 1 except for the disappearance of a series of signals at δC 168.0, 129.1, 137.7, 21.3, and 16.2, corresponding to one angeloyl group. The HMBC correlation between H-22 at δH 6.27 and C-1′ at δC 168.2 indicated the remaining angeloyl group was located at C-22. Besides, the other difference was the significant chemical shift change of C-23, due to the aldehyde group in compound 1 being replaced by a methyl group in compound 3. The assignment was confirmed by the observation of HMBC correlations between C-23 at δC 28.1 and Me-24 at δH 1.17, as well as between C-24 at δC 17.0 and Me-23 at δH 1.29. Moreover, the structure of the aglycone of 3 was also confirmed by comparing its spectroscopic data with those of eryngiosides H [11]. Therefore, the structure of 3 was elucidated as 22α-O-angeloyloxy-15α,16α,28-trihydroxyolean-12-ene 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside, and named oleiferoside C. Compound 4 was obtained as white amorphous powder. The negative HR‑ESI‑MS illustrated a quasimolecular ion [M – H]− peak at m/z 1329.6129, which indicated a molecular formula of C64H98O29. The IR spectrum showed the presence of a hydroxyl group (3453 cm−1), an aldehyde group (2816, 2719, 1736 cm−1), and an α,β-unsaturated ester group (1604, 1601 cm−1). The NMR spectroscopic data of compound 4 were similar to those of compound 1, except for the presence of an additional signal at δC 52.3 corresponding to one OCH3 group. The OCH3 group was located Li X et al. Cytotoxic Triterpenoid Glycosides …
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at C-6 of the glucuronopyranosyl group, which was supported by the HMBC correlation between δC 170.0 (C-6 of glucuronic acid) and δH 3.78 (OCH3). Moreover, the order and linkage of the glycosidic chain was also supported by comparing its spectroscopic data with those of camellioside H [10], 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-6-methoxy-β-D-glucuronopyranoside. Therefore, the structure of compound 4 was elucidated as 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-en-23-al 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-6′-methoxy-β-D-glucuronopyranoside, and named oleiferoside D. Compound 5 was isolated as white amorphous powder. Its molecular formula of C51H80O19 was proposed on the basis of the [M – H]− ion peak at m/z 995.5257 in the HR‑ESI‑MS spectrum. The IR spectrum indicated the presence of a hydroxyl group (3456 cm−1), a C=O group (1720 cm−1), and an α,β-unsaturated ester group (1601 cm−1). Comparison of its NMR data with those of compound 2 revealed that the aglycone structure of compound 5 was similar to that of compound 2, and the main differences arose from the significant upfield shifts of the C-23 (− 145.8 ppm) signal at δ 64.4 and the C-4 (− 11.7 ppm) signal at δ 43.6, which were attributed to the replacement of the 23-CHO group in compound 2 by the 23-CH2OH group in compound 5. Thus, the aglycone of compound 5 was elucidated as 21β-O-angeloyl3β,15α,16α,23α,28-pentahydroxy-22α-O-(2-methylbutanoyl) olean-12-ene, which was identified to be a new aglycone. The 1H‑NMR spectrum of compound 5 indicated two sugar residues, which were identified as D-glucuronic acid and L-arabinose by acid hydrolysis and GC analysis. The spectroscopic data of the sugar units were very similar to the sugar units of gordonoside [12], α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranosyl, which indicated that the glycosidic chain was α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranosyl. The location and linkage of the glycosidic chain were also identified by the HMBC correlations between δH 5.34 (H-1 of glucuronic acid) and δC 81.3 (C-3 of aglycone), and between δH 5.38 (H-1 of arabinose) and δC 85.8 (C3 of glucuronic acid). The β-anomeric configuration of the glucuronopyranosyl unit was determined from the observation of the large 3JH-1, H-2 coupling constant, and the α-anomeric configuration of the arabinopyranosyl unit was assigned according to the NOESY correlations between δ 5.38 (H-1 of arabinose) and δ " Fig. 2). On 4.58 (H-3 of arabinose) and 4.38 (H-4 of arabinose) (l the basis of the above analysis, the structure of compound 5 was elucidated as 21β-O-angeloyloxy-15α,16α,23,28-tetrahydroxy22α-O-(2-methylbutanoyloxy)olean-12-ene 3β-O-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside, and named oleiferoside E. Compound 6 was obtained as white amorphous powder. Its molecular formula of C54H82O19 was determined according to the [M – H]− ion peak at m/z 1033.5325. The IR spectrum indicated the presence of a hydroxyl group (3456 cm−1), a C=O group (1720 cm−1) and an α,β-unsaturated ester group (1601, 1602 cm−1). The NMR spectroscopic data of compound 6 were similar to those of TR-saponins [13], a triterpenoid glycoside isolated from Gordonia chrysandra roots, except for the presence of an additional signal at δC 52.0 corresponding to one OCH3 group. The OCH3 group appeared to be located at C-6 of the glucuronopyranosyl group, which was supported by the HMBC correlation between δC 170.2 (C-6 of glucuronic acid) and δH 3.78 (OCH3). The glycosidic chain was located at C-3 of the aglycone, and was inferred to be α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyra-
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nosyl methyl ester, which was established by the HMBC correlations between δH 5.26 (H-1 of glucuronic acid) and δC 82.0 (C-3 of aglycone), and between δH 5.37 (H-1 of arabinose) and δC 85.5 (C3 of glucuronic acid). The β-anomeric configuration of the glucuronopyranosyl unit was determined on the basis of the observation of the large 3JH-1, H-2 coupling constant, and the α-anomeric configuration of the arabinopyranosyl unit was indicated by NOESY correlations between δ 5.37 (H-1 of arabinose) and δ " Fig. 2). 4.26 (H-3 of arabinose) and 4.34 (H-4 of arabinose) (l Thus, compound 6 was elucidated as 16α-acetoxy-21β,22α-O-diangeloyloxy-23,28-dihydroxyolean-12-ene 3β-O-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside methyl ester, and named oleiferoside F. Compound 7 was obtained as white amorphous powder. Its molecular formula of C60H92O23 was determined by the observation of the [M + Na]+ ion peak at m/z 1203.5953 in the HR‑ESI‑MS spectrum. The IR spectrum indicated the presence of a hydroxyl group (3456 cm−1), a C=O group (1720 cm−1), and an α,β-unsaturated ester group (1601, 1602 cm−1). The NMR spectroscopic data of compound 7 were similar to those of compound 6, except for the presence of additional signals at δC 103.7, 72.2, 72.4, 74.0, 70.0, and 18.5, corresponding to one rhamnopyranosyl group. The acid hydrolysis and GC analysis confirmed its presence. The HMBC correlations between δH 5.25 (H-1 of glucuronic acid) and δC 82.0 (C-3 of aglycone), between δH 5.26 (H-1 of arabinose) and δC 86.2 (C-3 of glucuronic acid), and between δH 6.11 (H-1 of rhamnose) and δC 80.4 (C-3 of arabinose), indicated that the glycosidic chain was located at C-3 of the aglycone and the sugar
units were connected by α-L-rhamnopyranosyl-(1 → 3)-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranosyl methyl ester, which was identified to be a new sugar chain. The β-anomeric configuration of the glucuronopyranosyl unit was determined from the observation of the large 3JH-1, H-2 coupling constant, and the α-anomeric configurations of the arabinopyranosyl unit and the rhamnopyranosyl unit were assigned according to the NOESY correlations between δ 5.26 (H-1 of arabinose) and δ 4.33 (H-3 of arabinose) and 4.53 (H-4 of arabinose), and between " Fig. 2). δ 6.11 (H-1 of rhamnose) and 4.38 (H-4 of rhamnose) (l Thus, the structure of compound 7 was elucidated as 16α-acetoxy-21β,22α-O-diangeloyloxy-23,28-dihydroxyolean-12-ene 3β-O-α-L-rhamnopyranosyl-(1 → 3)-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside methyl ester, and named oleiferoside G. Compound 8 was obtained as white amorphous powder. Its negative ion HR‑ESI‑MS showed a quasimolecular ion peak [M – H]− at m/z 1181.6082, which inferred the molecular formula of C60H94O23. The IR spectrum indicated the presence of hydroxyl groups (3456 cm−1), a C=O group (1720, 1724 cm−1), an and α,βunsaturated ester group (1601 cm−1). The NMR spectroscopic data of compound 8 were similar to those of compound 7. The significant upfield shift of the C-2″ (− 86.7 ppm) signal at δ 41.6 and the C-3″ (− 111.3 ppm) signal at δ 26.7 suggested the presence of the 22α-O‑MB group in compound 8 in place of the 22α-O‑Ang group in compound 7. Further analyses of the NMR data led to the structure assignment of compound 8 as 16α-acetoxy-21β-Oangeloyloxy-23,28-dihydroxy-22α-O-(2-methylbutanoyloxy)
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
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Fig. 2 Key HMBC and NOESY correlations of compounds 1–8.
Original Papers
olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1 → 3)-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside methyl ester, named oleiferoside H. The aglycone of oleiferoside H was reported for the first time. In conclusion, eight new compounds were isolated and identified from 70 % EtOH of C. oleifera, which showed cytotoxic activity. All of the isolated triterpenoid glycosides possessed an olean-12-ene skeleton and the oligosaccharidic chains were linked to C-3. Of these compounds, the aglycones of compounds 5 and 8 were first discovered and so were the sugar chains of compounds 7 and 8. Cytotoxic activities of the isolated saponins were evaluated against four human tumor cell lines (A549, B16, BEL-7402, and MCF-7) by using the MTT in vitro assay. Compound 3 exhibited potent cytotoxic activity against all the tested cell lines with IC50 values < 10 µM, and compounds 1, 2, 4, and 5 showed moderate cytotoxic activities toward the tested cell lines. Among these compounds, compound 3 was the most potent against all the tested cell lines, which suggests the importance of the free hydroxy group at C-21 in mediating cytotoxicity when comparing its structure with others. Besides, the activities of compound 1 were stronger than those of compound 2, which suggests that the C-21-O‑Ang substitute made a more efficient contribution than the C-21-O‑MB substitute to the cytotoxic activity. Furthermore, these results also suggest that the free hydroxy group at C16 might play a role in mediating cytotoxicity because of the acetylation of the hydroxy group at C-16 (i.e., 6, 7, and 8 resulted in activity decreases).
Materials and Methods !
General experimental procedures The specific rotation values were determined with a Perkin-Elmer model 241 polarimeter. IR spectra were recorded on a Perkin-Elmer 983 G spectrometer. 1H, 13C NMR, and 2D NMR spectra were recorded on a Varian Inova 500 spectrometer in C5D5 N using tetramethylsilane (TMS) as the internal standard. HR‑ESI‑MS spectra were determined on a Micromass Q-TOF2 spectrometer. HPLC analysis and separation were performed on a Shimadzu HPLC system composed of a LC-20AT pump with a SPD-20A detector (Shimadzu Corp.), and the wavelength for detection was 203 nm. MPLC separation was performed on a Büchi flash chromatography system composed of a C-650 pump with a flash column (460 × 26 mm i. d., Büchi Corp.). The HPLC column (250 × 9.4 mm i. d., 5 µm, Agilent Zorbax SB‑C18 semipreparative) was purchased from Agilent Corp. Silica gel (200–300 mesh) for column chromatography and precoated silica gel TLC plates were purchased from Qingdao Marine Chemical Factory. ODS for MPLC was purchased from Merck KGaA. Sephadex LH-20 for column chromatography was purchased from GE Corp. GC was conducted on a GC-14C (Shimadzu Corp.) with a flame ionization detector (FID). Compounds on the TLC were colored by 10 % sulfuric acid alcohol solution.
Plant material The roots of C. oleifera were collected in Qichun, Hubei Province of China in November 2011, and identified by Professor Xiao-Ran Li at Soochow University. A voucher sample (No. 11-11-06-01) was deposited in the herbarium of the College of Pharmacy, Soochow University.
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Extraction and isolation Air-dried roots (9.3 kg) of C. oleifera were crushed with a grinder into fine debris, and extracted two times with 70 % EtOH (2 × 50 L) under reflux for 2 h. The solvent was subsequently removed under reduced pressure to give the residue (0.45 kg), which was then dissolved in distilled water and passed through a D101 macroporous resin column (i. d. 30 cm × 200 cm, Xiʼan Sunresin New Materials Co. Ltd.) eluted with a gradient of EtOH aqueous (EtOH‑H2O 0 %, 30 %, 60%, 80 %, each 40 L). The 80 % EtOH eluate (25 g) was further vacuum chromatographed on a silica gel column (18 × 30 cm) eluted with a gradient of CHCl3-MeOH (100 : 0, 90 : 10, 80 : 20, 70 : 30, 60 : 40, 50 : 50, 40 : 60, 30 : 70, 0 : 100, each 5.0 L). The CHCl3-MeOH (80 : 20) eluate (1.0 g) was then chromatographed by MPLC over an ODS column eluted with MeOH‑H2O (60 : 40, 70 : 30, 80 : 20, 90 : 10, 100 : 0, each 500 mL) at 20.0 mL/ min to afford 5 fractions. Fraction 3 (500–1000 mL, 125 mg) was separated by semipreparative HPLC over an ODS column eluted with MeOH‑H2O (85 : 15) to yield compounds 8 (15 mg, tR 35.25 min), 7 (9 mg, tR 36.28 min), and 6 (9 mg, tR 40.32 min). The CHCl3-MeOH (70 : 30) eluate (6.8 g) was chromatographed by MPLC eluted with MeOH‑H2O (40 : 60, 50 : 50, 60 : 40, 70 : 30, 80 : 20, 100 : 0, each 800 mL) at 20.0 mL/min to afford 10 fractions. Fraction 4 (1500–2000 mL, 420 mg) was separated by semipreparative HPLC over an ODS column eluted with MeOH‑H2O (72 : 28) to yield compounds 3 (56 mg, tR 27.5 min), 1 (90 mg, tR 39.0 min), and 2 (32 mg, tR 37.5 min). The separation of fraction 8 (142 mg) was also performed on semipreparative HPLC using MeOH‑H2O (78: 22, v/v) to yield compounds 4 (15 mg, tR 52.5 min) and 5 (16 mg, tR 44.5 min). The purities of the above saponins were > 90%, as determined by HPLC. Oleiferoside A (1): white amorphous powder; [α]20 D + 6.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 211 (4.30), 254 (4.15) nm; IR νmax 3454, 2962, 2924, 2851, 2816, 2718, 1736, 1604, 1601, 1466, 1368, 1184, 1041 cm−1; 1H NMR (pyridine-d5, 500 MHz) " Tables 1 and 13C NMR (pyridine-d5, 125 MHz) data are given in l and 2; HRESIMS m/z 1315.5968 [M – H]− (calcd. for C63H95O29, 1315.5959). Oleiferoside B (2): white amorphous powder; [α]20 D + 8.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 210 (4.29), 254 (4.05) nm; IR νmax 3456, 2962, 2920, 2855, 2820, 2718, 1740, 1720, 1601, 1460, 1378, 1168, 1046 cm−1; 1H NMR (pyridine-d5, 500 MHz) " Tables 1 and 13C NMR (pyridine-d5, 125 MHz) data are given in l − and 2; HRESIMS m/z 1317.6122 [M – H] (calcd. for C63H97O29, 1317.6116). Oleiferoside C (3): white amorphous powder; [α]20 D + 2.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 208 (4.29), 254 (3.82) nm; IR νmax 3452, 2962, 2910, 2845, 1720, 1602, 1460, 1375, 1170, 1049 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyri" Tables 1 and 2; HRESIMS dine-d5, 125 MHz) data are given in l − m/z 1203.5740 [M – H] (calcd. for C58H91O26, 1203.5799). Oleiferoside D (4): white amorphous powder; [α]20 D + 6.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 211 (4.30), 254 (4.15) nm; IR νmax 3453, 2962, 2924, 2851, 2816, 2719, 1736, 1604, 1601, 1461, 1362, 1186, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) " Tables 1 and 13C NMR (pyridine-d5, 125 MHz) data are given in l − and 2; HRESIMS m/z 1329.6129 [M – H] (calcd. for C64H97O29, 1329.6116). Oleiferoside E (5): white amorphous powder; [α]20 D + 6.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 206 (4.26), 254 (4.22) nm; IR νmax 3456, 2963, 2925, 2850, 1720, 1601, 1460, 1372, 1161, 1042 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyri-
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C NMR (pyridine-d5, 125 MHz) spectroscopic data for compounds 1–8.
Position
1
2
3
4
5
6
7
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 21-O‑Ang 1′ 2′ 3′ 4′ 5′ 22-O‑Ang 1″ 2″ 3″ 4″ 5″ 22-O‑MB 1″ 2″ 3″ 4″ 5″ OAc-1″′ 2″′ Sugar units 1 2 3 4 5 6 OMe
38.5 25.5 84.7 55.4 47.2 20.9 36.2 41.9 48.4 36.4 24.1 125.3 143.9 47.9 67.7 73.7 48.6 41.2 47.0 36.6 78.8 73.5 210.3 11.3 16.2 17.7 21.5 63.3 29.7 20.5
38.5 25.5 84.6 55.3 47.1 20.9 36.1 41.8 47.9 36.4 24.1 125.3 143.9 48.6 67.6 73.2 48.4 41.0 46.9 36.5 78.8 73.2 210.2 11.3 16.1 17.7 21.4 63.0 29.6 20.4
39.1 26.8 89.7 39.8 55.7 19.0 37.2 41.7 47.3 37.2 24.2 125.1 144.7 48.0 67.7 75.2 45.4 42.0 47.2 32.2 36.9 72.9 28.1 17.0 16.0 17.8 21.5 63.9 33.7 25.4
38.5 25.3 84.5 55.3 47.1 20.9 36.1 41.8 48.4 36.4 24.1 125.2 143.9 47.9 67.6 73.7 48.5 41.1 47.0 36.5 78.7 73.5 210.0 11.2 16.1 17.6 20.8 63.3 29.6 20.4
39.1 26.2 81.3 43.6 47.4 18.7 36.5 41.7 47.4 37.1 24.2 125.6 143.9 47.9 67.7 73.2 48.6 41.0 46.9 36.5 78.8 73.3 64.4 11.9 16.6 17.8 21.4 63.1 29.6 20.4
38.6 26.0 82.0 43.4 47.1 17.8 32.6 39.9 46.8 36.5 23.7 125.0 140.8 41.0 30.6 72.0 47.0 39.4 46.8 36.0 77.9 72.4 63.8 13.5 16.0 16.7 26.9 63.3 29.3 19.6
38.6 26.0 82.0 43.4 47.1 17.8 32.6 39.9 46.8 36.5 23.7 125.0 140.8 41.0 30.6 72.0 46.8 39.4 47.0 36.0 77.9 72.5 63.8 13.5 16.0 16.7 26.9 63.3 29.3 19.6
38.6 26.0 82.0 43.4 47.2 17.8 32.6 39.9 46.9 36.5 23.7 125.0 140.9 41.0 30.9 71.4 46.9 39.4 47.0 36.0 78.0 72.5 63.8 13.5 16.0 16.7 26.9 63.5 29.3 19.6
168.0 129.1 137.7 16.2 21.3
167.8 128.8 138.8 16.3 21.3
167.5 129.0 137.6 16.1 21.4
167.8 128.9 138.7 16.2 21.2
167.6 128.4 138.0 15.6 20.8
167.1 128.4 138.3 15.8 20.8
167.4 128.1 139.3 16.0 20.9
167.1 128.3 138.3 15.8 20.7
167.6 128.3 138.0 15.6 20.7
1 2 3 4 5 6
168.3 129.3 136.8 15.9 20.9
168.2 129.7 136.6 16.0 21.1
168.4 129.2 136.7 15.8 21.2
176.8 41.7 27.1 12.0 16.9
176.8 41.6 27.0 11.9 16.8
GlcA 104.3 78.2 84.9 69.9 77.2 172.6
GlcA 104.3 78.2 85.0 69.9 77.3 172.5
GlcA 105.7 78.6 84.8 70.0 77.5 172.6
Gal 1 101.8 84.1 75.2 71.1 77.3 62.1
Gal 1 101.8 84.1 75.2 71.0 77.2 62.0
Gal 1 102.0 84.0 75.2 71.4 77.2 62.0
GlcAOMe 104.1 78.1 85.0 69.8 77.2 170.0 52.3 Gal 1 101.8 84.0 75.2 70.7 76.6 61.9
GlcA 105.7 74.5 85.8 71.6 78.1 170.5 Ara 105.4 72.8 74.5 69.3 67.2
169.8 21.9 GlcAOMe 106.0 74.5 85.5 71.1 76.7 170.2 52.0 Ara 105.8 72.8 74.3 69.1 67.0
Li X et al. Cytotoxic Triterpenoid Glycosides …
169.8 21.9 GlcAOMe 106.4 74.4 86.2 71.0 76.6 170.2 52.0 Ara 105.9 72.1 80.4 69.2 67.9
175.8 41.6 26.7 11.8 16.6 169.7 21.9 GlcAOMe 106.4 74.4 86.2 71.0 76.6 170.1 52.0 Ara 105.9 72.1 80.4 69.2 67.9 continued
Planta Med 2014; 80: 590–598
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Table 1
595
596
Original Papers
Position 1 2 3 4 5 6 1 2 3 4 5 6 a
1
2
3
4
Xyl 107.9 76.5 78.4 71.0 67.7
Xyl 107.9 76.5 78.4 71.0 67.8
Xyl 107.8 76.1 78.5 70.8 67.7
Xyl 107.9 76.5 78.5 71.0 67.8
Gal 1′ 103.1 74.0 75.5 70.7 76.6 62.3
Gal 1′ 103.1 74.0 75.5 70.7 76.6 62.3
Gal 1′ 102.5 73.0 76.6 69.8 78.5 63.2
Gal 1′ 103.2 73.9 75.5 70.6 76.5 62.3
5
6
7
8
Rha 103.7 72.2 72.4 74.0 70.0 18.5
Rha 103.7 72.2 72.4 74.0 70.0 18.5
Chemical shifts are in ppm, and the assignments were based on HSQC, HMBC, and NOESY spectra. b nd = not detected
Table 2
1
H NMR (pyridine-d5, 500 MHz) spectroscopic data for compounds 1–8.
Position 1 2 3 5 6 7 9 11 12 15 16 18 19 21 22 23 24 25 26 27 28 29 30 21-O‑Ang 3′ 4′ 5′ 22-O‑Ang 3″ 4″ 5″ 22-O‑MB 2″ 3″ 4″ 5″ OAc-2″′
1
2
3
4
5
6
7
8
1.08 m, 1.48 m 1.94 m, 2.21 m 4.09 dd (11.0, 4.5) 1.83 m 1.41 m, 1.48 m 2.17 m, 2.18 m 1.53 m 1.90 m, 1.94 m 5.58 brs 4.26 brs 4.54 brs 3.15 m 1.48 m, 3.15 m 6.78 d (10.5) 6.41 d (10.5) 9.94 brs
0.97 m, 1.49 m 1.92 m, 2.21 m 4.11 dd (11.0, 4.5) 1.83 m 1.10 m, 1.49 m 2.16 m, 2.18 m 1.49 m 1.86 m, 1.94 m 5.56 brs 4.23 brs 4.46 brs 3.15 m 1.49 m, 3.13 m 6.73 d (10.5) 6.34 d (10.5) 9.96 brs
0.87 m, 1.48 m 1.88 m, 2.21 m 3.33 dd (11.0, 4.5) 0.90 m 1.47 m, 1.66 m 2.11 m, 2.88 m 1.80 m 1.85 m, 2.03 m 5.57 brs 4.34 brs 4.62 brs 3.11 m 1.41 m, 2.96 m 2.11 m, 2.88 m 6.27 m 1.29 s
0.99 m, 1.52 m 1.91 m, 2.19 m 4.10 dd (11.0, 4.5) 1.85 m 1.12 m, 1.50 m 2.06 m, 2.10 m 1.47 m 1.85 m, 1.98 m 5.58 brs 4.27 brs 4.51 brs 3.16 m 1.52 m, 3.16 m 6.79 d (10.5) 6.40 d (10.5) 9.95 brs
1.53 s 0.88 s 1.04 s 1.90 s 3.57 d (11.0) 3.82 d (11.0) 1.18 s 1.41 s
1.54 s 0.88 s 1.04 s 1.89 s 3.55 d (11.0) 3.82 d (11.0) 1.17 s 1.38 s
1.17 s 0.92 s 1.11 s 1.95 s 3.72 d (10.0) 3.87 d (10.0) 1.14 s 1.36 s
1.52 s 0.90 s 1.05 s 1.90 s 3.60 d (11.0) 3.84 d (11.0) 1.19 s 1.42 s
1.01 m, 1.55 m 2.06 m, 2.35 m 4.38 dd (11.0, 4.5) 1.86 m 1.52 m, 1.92 m 2.17 m, 2.33 m 1.90 m 1.90 m, 2.01 m 5.60 brs 4.26 brs 4.46 brs 3.15 m 1.48 m, 3.15 m 6.71 d (10.5) 6.32 d (10.5) 3.77 d (11.0) 4.38 d (11.0) 1.04 s 1.09 s 1.15 s 1.90 s 3.56 d (11.0) 3.87 d (11.0) 1.15 s 1.39 s
1.02 m, 1.51 m 2.01 m, 2.23 m 4.35 dd (11.0, 4.5) 1.70 m 1.34 m, 1.76 m 1.24 m, 1.54 m 1.82 m 1.87 m, 1.95 m 5.49 brs 1.69 m, 1.92 m 5.59 brs 3.13 m 1.54 m, 2.72 m 6.06 d (10.5) 6.36 d (10.0) 3.88 d (11.0) 4.41 d (11.0) 1.00 s 0.96 s 0.86 s 1.46 s 3.53 d (10.0) 3.70 d (10.0) 1.14 s 1.37 s
1.04 m, 1.52 m 2.06 m, 2.25 m 4.36 dd (11.0, 4.5) 1.70 m 1.29 m, 1.75 m 1.20 m, 1.54 m 1.83 m 1.88 m, 1.99 m 5.50 brs 1.70 m, 1.93 m 5.59 brs 3.15 m 1.52 m, 2.72 m 6.06 d (10.5) 6.36 d (10.0) 3.84 d (11.0) 4.42 d (11.0) 1.00 s 0.96 s 0.87 s 1.47 s 3.53 d (10.0) 3.80 d (10.0) 1.14 s 1.37 s
1.15 m,1.54 m 2.05 m, 2.23 m 4.34 dd (11.0, 4.5) 1.70 m 1.39 m, 1.74 m 1.22 m, 1.58 m 1.84 m 1.87 m, 1.96 m 5.49 brs 1.62 m, 1.95 m 5.67 brs 3.13 m 1.54 m, 2.71 m 5.99 d (10.5) 6.28 d (10.0) 3.85 d (11.0) 4.41 d (11.0) 1.00 s 0.97 s 0.89 s 1.49 s 3.58 d (10.0) 3.74 d (10.0) 1.13 s 1.35 s
6.07 dq (7.5, 1.5) 2.18 d (7.5) 2.09 s
6.16 dq (7.5, 1.5) 2.25 d (7.5) 2.12 s
6.05 dq (7.5, 1.5) 2.17 d (7.5) 2.10 s
6.15 dq (5.5, 1.5) 2.24 d (7.5) 2.12 s
6.04 dq (7.5, 1.5) 2.11 d (7.5) 2.10 s
6.07 dq (7.5, 1.5) 2.11 d (7.5) 2.10 s
6.16 dq (7.5, 1.5) 2.20 d (7.5) 2.07 s
5.99 dq (7.5, 1.5) 2.13 d (7.5) 2.04 s
5.99 dq (7.5, 1.5) 2.10 d (7.5) 2.04 s
5.88 dq (7.5, 1.5) 2.05 d (7.5) 1.83 s
5.94 dq (7.5, 1.5) 2.12 d (7.5) 1.93 s
5.88 dq (7.5, 1.5) 2.05 d (7.5) 1.83 s
2.15 m 1.30 m, 1.67 m 0.75 t (7.5) 1.10 d (6.5)
Li X et al. Cytotoxic Triterpenoid Glycosides …
2.16 m 1.31 m, 1.67 m 0.77 t (7.5) 1.04 d (6.5) 2.57 s
Planta Med 2014; 80: 590–598
2.57 s
2.55 m 1.55 m, 1.86 m 0.98 t (7.5) 1.26 d (6.5) 2.60 s continued
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Table 1 Continued
Original Papers
597
Position
1
2
3
4
5
6
7
8
Sugar units 1 2 3 4 5 OMe
GlcA 4.80 d (6.5) 4.57 m 4.41 m 4.57 m 4.50 m
GlcA 4.83 d (6.5) 4.57 m 4.39 m 4.58 m 4.49 m
GlcA 4.90 d (7.5) 4.72 m 4.52 m 4.53 m 4.39 m
GlcA 5.34 d (7.0) 4.26 m 4.30 m 4.58 m 4.60 m
Gal 1 5.80 d (7.5) 4.57 m 4.39 m 4.50 m 4.18 m 4.45 m, 4.46 m Xyl 5.09 d (7.5) 4.27 m 4.11 m 4.41 m 3.50 m, 4.45 m
Gal 1 5.80 d (7.5) 4.58 m 4.37 m 4.53 m 4.20 m 4.40 m, 4.39 m Xyl 5.10 d (7.0) 4.28 m 4.11 m 4.39 m 3.51 m, 4.18 m
Gal 1 5.80 d (7.0) 4.58 m 4.31 m 4.52 m 4.52 m 4.46 m, 4.47 m Xyl 5.13 d (7.5) 4.37 m 4.10 m 4.47 m 3.53 m, 4.42 m
GlcAOMe 4.84 d (10.0) 4.55 m 4.35 m 4.57 m 4.18 m 3.78 s Gal 1 5.74 d (10.0) 4.57 m 4.35 m 4.59 m 4.39 m 4.43 m, 4.45 m Xyl 5.12 d (7.5) 4.29 m 4.13 m 4.42 m 3.59 m, 4.53 m
GlcAOMe5.26 d (7.0) 4.16 m 4.24 m 4.43 m 4.50 m 3.78 s Ara 5.37 d (6.5) 4.53 m 4.26 m 4.34 m 3.81 m, 4.45 m
GlcAOMe 5.25 d (7.0) 4.12 m 4.23 m 4.40 m 4.50 m 3.77 s Ara 5.26 d (7.0) 4.71 m 4.33 m 4.53 m 3.87 m, 4.35 m
GlcAOMe 5.24 d (7.0) 4.12 m 4.32 m 4.40 m 4.50 m 3.78 s Ara 5.24 d (7.0) 4.67 m 4.43 m 4.52 m 3.87 m, 4.36 m
Rha 6.11 brs 4.79 m 4.63 m 4.38 m 4.69 m 1.74 d (5.0)
Rha 6.10 brs 4.78 m 4.61 m 4.37 m 4.63 m 1.75 d (5.0)
Gal 1′ 5.95 d (7.5) 4.50 m 4.45 m 4.57 m 4.55 m 4.57 m, 4.55 m
Gal 1′ 5.96 d (7.5) 4.52 m 4.46 m 4.58 m 4.55 m 4.58 m, 4.57 m
Gal 1′ 6.08 d (7.5) 4.17 m 4.19 m 4.21 m 4.53 m 4.37 m, 4.57 m
Gal 1′ 5.86 d (7.5) 4.51 m 4.45 m 4.46 m 4.53 m 4.55 m, 4.57 m
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Ara 5.38 d (6.5) 4.26 m 4.58 m 4.38 m 3.89 m, 4.46 m
Chemical shifts are in ppm and coupling constants (J) in Hz are given in parentheses. The assignments were based on HSQC, HMBC, and NOESY spectra
" Tables 1 and 2; HRESIMS dine-d5, 125 MHz) data are given in l m/z 995.5257 [M – H]− (calcd. for C51H79O19, 995.5216). Oleiferoside F (6): white amorphous powder; [α]20 D + 5.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 208 (4.29), 254 (3.82) nm; IR νmax 3456, 2962, 2910, 2845, 1720, 1602, 1601, 1441, 1382, 1168, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR " Tables 1 and 2; HRE(pyridine-d5, 125 MHz) data are given in l SIMS m/z 1033.5325 [M – H]− (calcd. for C54H81O19, 1033.5372). Oleiferoside G (7): white amorphous powder; [α]20 D + 6.4 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 211 (4.18), 254 (4.34) nm; IR νmax 3456, 2965, 2918, 2849, 1720, 1602, 1601, 1441, 1382, 1168, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR " Tables 1 and 2; HRE(pyridine-d5, 125 MHz) data are given in l SIMS m/z 1203.5953 [M + Na]+ (calcd. for C60H92O23 Na, 1203.5927). Oleiferoside H (8): white amorphous powder; [α]20 D + 8.4 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 210 (4.18), 254 (4.32) nm; IR νmax 3456, 2963, 2912, 2843, 1720, 1724, 1601, 1441, 1382, 1158, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR " Tables 1 and 2; HRE(pyridine-d5, 125 MHz) data are given in l SIMS m/z 1181.6082 [M – H]− (calcd. for C60H93O23, 1181.6108).
Acid hydrolysis and sugar analysis of compounds 1–8 Each saponin (2 mg) was dissolved in 2 N HCl (2 mL) and stirred at 80 °C for 4 h. The reaction mixture was extracted with chloroform, and the aqueous layer was evaporated to give a mixture of monosaccharides. The residue was dissolved in anhydrous pyridine (1 mL) followed by the addition of 2 mg of L-cysteine methyl ester hydrochloride (Tokyo Chemical Industry, 99 %). After heating at 60 °C for 2 h, the solvent was evaporated under N2, and
0.2 mL trimethylsilylimidazole (Tokyo Chemical Industry, 99 %) was added. Then the mixture was heated at 60 °C for another 2 h and partitioned between n-hexane and water. The organic layer was investigated by GC under the following conditions: capillary column, HP-5 (30 m × 0.25 mm × 0.25 µm; Dikma); FID detector with a temperature of 280 °C; injection temperature 250 °C; initial temperature 160 °C, then raised to 280 °C at 5 °C/min, final temperature maintained for 10 min; carrier gas, N2. The standard sugars experienced the same reaction and GC conditions. The retention times of persilylated D-glucuronic acid, D-glucuronic acid methyl ester,D-galactose, D-xylose, and L-arabinose were 19.770 min, 24.460 min, 25.643 min, 32.257 min, and 33.284 min, respectively.
Cytotoxicity assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay was performed to evaluate the cytotoxic activities of the isolated saponins against A549, B16, BEL7402, and MCF-7 human tumor cell lines (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The amount of formazan was determined by photometry at 570 nm. Cells were plated into 96-well flat-bottomed cultured plates at a concentration of 2 × 105 cells per well in complete RPMI 1640 culture medium. After twenty-four hours, the medium containing fetal calf serum was removed and the test solutions were applied to the cells in different final concentrations at 0.625, 1.25, 2.5, 5, 10, 20, 50, and 100 µM. After 24 h, the MTT solution was added to the wells and the plates were incubated at 37 °C for 4 h. The positive control group was treated with norcantharidin (purity higher than 99.0 % as determined by HPLC; Nanjing Zelang Medical
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
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Table 2 Continued
Original Papers
Table 3 In vitro cytotoxic activities of compounds 1–8 (IC50 values in µM). Com-
Cell line IC50 (µM)
pound
A549
B16
7402
MCF-7
1 2 3 4 5 6 7 8 Norcantharidin
14.95 ± 0.40 22.23 ± 0.25 5.73 ± 0.09 20.23 ± 1.12 33.27 ± 0.28 89.91 ± 15.78 89.99 ± 3.29 99.41 ± 5.28 4.16 ± 0.28
17.56 ± 0.77 28.32 ± 2.04 6.04 ± 0.04 25.46 ± 1.58 31.80 ± 2.15 57.21 ± 3.84 63.74 ± 0.99 80.24 ± 6.06 3.93 ± 0.22
15.76 ± 0.33 28.32 ± 0.16 6.45 ± 0.07 33.49 ± 5.33 35.11 ± 2.31 71.91 ± 11.57 92.67 ± 11.60 91.87 ± 14.74 3.45 ± 0.26
14.25 ± 1.90 24.15 ± 1.37 6.45 ± 0.48 17.33 ± 2.07 26.98 ± 2.79 77.33 ± 15.71 90.31 ± 13.37 85.79 ± 12.52 5.17 ± 0.38
Technology Co. Ltd.). The amount of formazan was determined by photometry at 570 nm. The results are expressed as the percentage of absorbance in the control cells in comparison to that in the drug-treated cells. The IC50 values (50 % inhibitory concentration) " Table 3. The experiment was of compounds 1–8 are shown in l performed in triplicate for each sample.
Supporting information Various spectra for compounds 1–8 are available as Supporting Information.
Acknowledgements !
This work was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors thank Dr. Jon Parcher at the University of Mississippi for helpful discussions.
Conflict of Interest !
All authors of this paper confirm that they have no conflicts of interest.
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
References 1 Takemoto T, Miyasi T, Kusano G. Flavones and other compounds of Boehmeria tricuspis and B. holosericea. Phytochemistry 1975; 14: 2534 2 Hu JL, Nie SP, Huang DF, Li C, Xie MY, Wan Y. Antimicrobial activity of saponin-rich fraction from Camellia oleifera cake and its effect on cell viability of mouse macrophage RAW 264.7. J Sci Food Agric 2012; 92: 2443–2449 3 Jin XC, Ning Y. Antioxidant and antitumor activities of the polysaccharide from seed cake of Camellia oleifera Abel. Int J Biol Macromol 2012; 51: 364–368 4 Chen YL, Tang L, Feng BM, Shi LY, Wang HG, Wang YQ. New bibenzyl glycosides from leaves of Camellia oleifera Abel. with cytotoxic activities. Fitoterapia 2011; 82: 481–484 5 Ye Y, Guo Y, Luo YT. Anti-Inflammatory and Analgesic Activities of a Novel Biflavonoid from Shells of Camellia oleifera. Int J Mol Sci 2012; 13: 12 401–12 411 6 Yoshikawa M, Morikawa T, Yamamoto K, Matsuda H. Floratheasaponins A–C, acylated oleanane-type triterpene oligoglycosides with anti-hyperlipidemic activities from flowers of the tea plant (Camellia sinensis). J Nat Prod 2005; 68: 1360–1365 7 Sugimoto S, Yoshikawa M, Nakamura S, Matsuda H. Medicinal flowers. XXV. Structures of floratheasaponin J and chakanoside II from Japanese tea flower, flower buds of Camellia sinensis. Heterocycles 2009; 78: 1023–1029 8 Yoshikawa M, Sugimoto S, Katou Y, Nakamura S, Wang T, Yamashita C, Matsuda H. Acylated oleanane-type triterpene saponins with acceleration of gastrointestinal transit and inhibitory effect on pancreatic lipase from flower buds of chinese tea plant (Camellia sinensis). Chem Biodivers 2009; 6: 903–915 9 Wang SL, Chen Z, Tong XJ, Liu YL, Li X, Xu QM, Li XR, Yang SL. Triterpenoids from the Roots of Camellia oleifera C. Abel and Their Cytotoxic Activities. Helv Chim Acta 2013; 96: 1126–1133 10 Thao NTP, Hung TM, Cuong TD, Kim JC, Kim EH, Jin SE, Na MK, Lee YM, Kim YH, Choi JS, Min BS. 28-Nor-oleanane-type triterpene saponins from Camellia japonica and their inhibitory activity on LPS-induced NO production in macrophage RAW264.7 cells. Bioorg Med Chem Lett 2010; 20: 7435–7439 11 Zhang ZZ, Li SY, Qwnby S, Wang P, Yuan W, Zhang WL, Beasley RS. Phenolic compounds and rare polyhydroxylated triterpenoid saponins from Eryngium yuccifolium. Phytochemistry 2008; 69: 2070–2080 12 Yu L, Yang JZ, Chen XG, Shi JG, Zhang DM. Cytotoxic triterpenoid glycosides from the foots of Gordonia chrysandra. J Nat Prod 2009; 72: 866– 870 13 Lu Y, Umeda T, Yagi A, Sakata K, Chaudhuri T, Ganguly DK, Sarma S. Triterpenoid saponins from the roots of tea plant (Camellia sinensis var. assamica). Phytochemistry 2000; 53: 941–946
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598
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Cytotoxic Triterpenoid Glycosides from the Roots of Camellia oleifera
Authors
Xia Li 1, Jianping Zhao 2, Cuiping Peng 1, Zhong Chen 1, Yanli Liu 1, Qiongming Xu 1, 2, Ikhlas A. Khan 1, 2, Shilin Yang 1
Affiliations
1 2
Key words " Camellia oleifera l " Theaceae l " triterpenoid saponins l " oleiferosides A–H l " cytotoxic activities l
College of Pharmaceutical Science, Soochow University, Suzhou, China National Center for Natural Products Research, and Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Mississippi, USA
Abstract !
Eight new triterpenoid saponins, oleiferosides A– H (1–8), were isolated from the EtOH extract of the roots of Camellia oleifera. Their structures were elucidated by a combination of 1D and 2D NMR techniques, mass spectrometry, and chemical methods. All were characterized to be oleanane-type saponins with sugar moieties linked to C-3 of the aglycone. Cytotoxic activities of these saponins were evaluated against four hu-
Introduction !
received revised accepted
Dec. 19, 2013 February 26, 2014 March 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368428 Planta Med 2014; 80: 590–598 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Qiongming Xu, Ph.D. College of Pharmaceutical Science Soochow University Suzhou 215123 China Phone: + 8 65 12 69 56 14 21 Fax: + 8 65 12 65 88 20 89 [email protected]
Belonging to the Theaceae family, the Camellia Linn genus comprises 120 species. The plants grow in the tropics and subtropics, with 65 species found in China. The plant Camellia oleifera C. Abel has been widely cultivated as an oil crop in many parts of China, including Hunan, Jiangxi, Anhui, Henan, Zhejiang, and Fujian provinces. The roots of C. oleifera have been used in traditional Chinese medicine for the treatment of the common cold, measles, ardent fever, urinary tract infection, nephritis, edema, and threatened abortion [1] due to their antimicrobial [2], antioxidant, and antitumor activities [3]. Previous studies on the constituents from the Camellia genus led to the isolation of different compounds such as bibenzyl glycosides [4], flavonoids [5], triterpenoids, and glycosides [6–8]. In our efforts to investigate biologically active compounds from plant sources, the EtOH extract of the roots of C. oleifera was found to exhibit significant cytotoxicities against A549 human lung adenocarcinoma, B16 mouse melanoma, BEL-7402 human hepatocarcinoma, and MCF-7 human breast carcinoma cell lines. Our previous phytochemical study on the roots of C. oleifera led to the discovery of three new triterpenes and six known triterpenes, which showed moderate cytotoxic activities [9].
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
man tumor cell lines (A549, B16, BEL-7402, and MCF-7) by using the MTT in vitro assay. Compound 3 exhibited potent cytotoxic activitiy against all the tested cell lines with IC50 values < 10 µM. Compounds 1, 2, 4, and 5 showed moderate cytotoxic activities toward the tested cell lines. Supporting information available online at http://www.thieme-connect.de/products
A continuous search for cytotoxic agents led us to isolate eight new triterpenoid saponins (1–8) from the roots of C. oleifera. Herein we describe the isolation, structure elucidation, and biological activities of these saponins. The structures of " Fig. 1. compounds 1–8 are shown in l
Results and Discussion !
Compound 1 was separated as a white amorphous powder. The negative ion HR‑ESI‑MS of 1 illustrated a quasimolecular ion [M – H]− peak at m/z 1315.5968, indicating the molecular formula of C63H96O29. The IR spectrum showed the presence of a hydroxyl group (3454 cm−1), an aldehyde group (2816, 2718, 1736 cm−1), and an α,βunsaturated ester group (1604, 1601 cm−1). The 13 " Table 1) spectrum showed the resoC‑NMR (l nances of 63 carbons, ascribable to ten methyls, ten methylenes, thirty-one methines, and twelve quaternary carbons as revealed by the HSQC ex" Table 2) spectrum periment. The 1H‑NMR (l showed six methyl signals at δ 0.88 (Me-25), 1.04 (Me-26), 1.18 (Me-29), 1.41 (Me-30), 1.53 (Me24) and 1.90 (Me-27), one isolated oxymethylene giving signals at δ 3.57 (1H, d, J = 11.0 Hz) and 3.82 (1H, d, J = 11.0 Hz), five oxymethines at δ 4.09 (1H, dd, J = 11.0, 4.5 Hz, H-3), 4.26 (1H, brs, H-15), 4.54
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(1H, brs, H-16), 6.78 (1H, d, J = 10.5 Hz, H-21), and 6.41 (1H, d, J = 10.5 Hz, H-22), one olefinic proton signal at δ 5.58 (1H, brs, H12), and an aldehyde signal at δ 9.94 (1H, brs, H-23). Furthermore, the signals of two angeloyl (Ang) groups at δ [6.07 (1H, dq, J = 7.5, 1.5 Hz, 21-O‑Ang-3′), 2.18 (3H, d, J = 7.5 Hz, 21-O‑Ang-4′), and 2.09 (3H, s, 21-O‑Ang-5′)], and δ [5.88 (1H, dq, J = 7.5, 1.5 Hz, 22-O‑Ang-3″), 2.05 (3H, d, J = 7.5 Hz, 22-O‑Ang-4″), and 1.83 (3H, s, 22-O‑Ang-5″)] were observed. The NMR data were comparable to those published in the literature [9], suggesting that compound 1 had the 21β,22α-O-diangeloyl-3β,15α,16α,28-tetrahydroxyolean-12-en-23-al type of aglycone. The locations of the two Ang groups at C-21 and C-22 were confirmed by the HMBC " Fig. 2). The relative configuration of compound 1 experiment (l " Fig. 2). The crosswas established from its NOESY spectrum (l peaks between H-21 at δH 6.78 and H-29 at δH 1.18, as well as those between H-22 at δH 6.41 and H-30 at δH 1.41, and H-28 at δH 3.57, 3.82, suggested that H-21 and H-22 are α- and β-oriented, respectively, which means that the two Ang groups at C-21 and C-22 are β- and α-equatorial, respectively. The H-15 at δH 4.26 correlated with H-28 at δH 3.57, 3.82 and H-18 at δH 3.15, in-
Structures of compounds 1–8.
dicating that the 15-OH group is α-configured. The CHO group was established at position 23, which was confirmed by the correlations between H-23 at δH 9.94, C-4 at δC 55.4, and C-24 at δC 11.3 in the HMBC spectrum, as well as the correlation between H3 at δH 4.09 and H-23 at δH 9.94 in the NOESY spectrum. The anomeric proton signals at δH 4.80 (1H, d, J = 6.5 Hz, H-1 of glucuronic acid), 5.80 (1H, d, J = 7.5 Hz, H-1 of galactose 1), 5.09 (1H, d, J = 7.5 Hz, H-1 of xylose), and 5.95 (1H, d, J = 7.5 Hz, H-1 of galactose 1′), which showed the HSQC correlation to δC 104.3 (C-1 of glucuronic acid), 101.8 (C-1 of galactose 1), 107.9 (C-1 of xylose), and 103.1 (C-1 of galactose 1′), respectively, indicated the presence of four sugar residues. Acid hydrolysis of 1 and GC analysis revealed one unit of D-glucuronic acid, two units of D-galactose, and one unit of D-xylose. The HMBC correlations between δH 4.80 (H-1 of glucuronic acid) and δC 84.7 (C-3 of the aglycone) " Fig. 2) indicated that the glycosidic chain was located at C-3 (l of the aglycone. The spectroscopic data of sugar units were very similar to those sugar units of camellenodiol 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside [10], which indicated Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
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Fig. 1
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the glycosidic chain located at C-3 of the aglycone was β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl. The linkage of the glycosidic chain was also supported by the HMBC correlations between δH 5.80 (H-1 of galactose 1) and δC 84.9 (C-3 of glucuronic acid), between δH 5.09 (H-1 of xylose) and δC 84.1 (C-2 of galactose 1), and between δH 5.95 (H-1 of galactose 1′) and δC 78.2 (C-2 of glucuronic acid). The β-anomeric configurations of the four sugar units were deduced from the observation of their large 3 JH-1, H-2 coupling constants. Thus, the structure of 1 was established as 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12en-23-al 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside, and named oleiferoside A. Compound 2 was isolated as white amorphous powder. The quasimolecular ion [M – H]− peak at m/z 1317.6122 revealed its molecular formula to be C63H98 O29. The IR spectrum indicated that the presence of a hydroxyl group (3456 cm−1), an aldehyde group (2820, 2718, 1740 cm−1), a C=O group (1720 cm−1), and an α,β-unsaturated ester group (1601 cm−1). The NMR data of compound 2 were similar to those of compound 1, and the main differences arose from the significant upfield shifts of the C-2″ (− 87.6 ppm) signal at δ 41.7 and the C-3″ (− 109.7 ppm) signal at δ 27.1. The analyses of the 1H-1H COSY, HSQC, and HMBC spectra revealed that a 2-methylbutanoyl (MB) group was attached to C-22 in compound 2, where in compound 1 it was an Ang group. Thus, the structure of compound 2 was assigned to be 21β-O-angeloyloxy-15α,16α,28-trihydroxy-22α-O-(2-methylbutanoyloxy)olean-12-en-23-al 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside, and named oleiferoside B. Compound 3 was obtained as white amorphous powder. It exhibited a molecular formula of C58H92O26 deduced from its [M – H]− ion peak at m/z 1203.5740. The IR spectrum indicated the presence of hydroxyl groups (3452 cm−1) and an α,β-unsaturated ester group (1602 cm−1). The 13C‑NMR spectrum of compound 3 was similar to that of compound 1 except for the disappearance of a series of signals at δC 168.0, 129.1, 137.7, 21.3, and 16.2, corresponding to one angeloyl group. The HMBC correlation between H-22 at δH 6.27 and C-1′ at δC 168.2 indicated the remaining angeloyl group was located at C-22. Besides, the other difference was the significant chemical shift change of C-23, due to the aldehyde group in compound 1 being replaced by a methyl group in compound 3. The assignment was confirmed by the observation of HMBC correlations between C-23 at δC 28.1 and Me-24 at δH 1.17, as well as between C-24 at δC 17.0 and Me-23 at δH 1.29. Moreover, the structure of the aglycone of 3 was also confirmed by comparing its spectroscopic data with those of eryngiosides H [11]. Therefore, the structure of 3 was elucidated as 22α-O-angeloyloxy-15α,16α,28-trihydroxyolean-12-ene 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucuronopyranoside, and named oleiferoside C. Compound 4 was obtained as white amorphous powder. The negative HR‑ESI‑MS illustrated a quasimolecular ion [M – H]− peak at m/z 1329.6129, which indicated a molecular formula of C64H98O29. The IR spectrum showed the presence of a hydroxyl group (3453 cm−1), an aldehyde group (2816, 2719, 1736 cm−1), and an α,β-unsaturated ester group (1604, 1601 cm−1). The NMR spectroscopic data of compound 4 were similar to those of compound 1, except for the presence of an additional signal at δC 52.3 corresponding to one OCH3 group. The OCH3 group was located Li X et al. Cytotoxic Triterpenoid Glycosides …
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at C-6 of the glucuronopyranosyl group, which was supported by the HMBC correlation between δC 170.0 (C-6 of glucuronic acid) and δH 3.78 (OCH3). Moreover, the order and linkage of the glycosidic chain was also supported by comparing its spectroscopic data with those of camellioside H [10], 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-6-methoxy-β-D-glucuronopyranoside. Therefore, the structure of compound 4 was elucidated as 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-en-23-al 3β-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-6′-methoxy-β-D-glucuronopyranoside, and named oleiferoside D. Compound 5 was isolated as white amorphous powder. Its molecular formula of C51H80O19 was proposed on the basis of the [M – H]− ion peak at m/z 995.5257 in the HR‑ESI‑MS spectrum. The IR spectrum indicated the presence of a hydroxyl group (3456 cm−1), a C=O group (1720 cm−1), and an α,β-unsaturated ester group (1601 cm−1). Comparison of its NMR data with those of compound 2 revealed that the aglycone structure of compound 5 was similar to that of compound 2, and the main differences arose from the significant upfield shifts of the C-23 (− 145.8 ppm) signal at δ 64.4 and the C-4 (− 11.7 ppm) signal at δ 43.6, which were attributed to the replacement of the 23-CHO group in compound 2 by the 23-CH2OH group in compound 5. Thus, the aglycone of compound 5 was elucidated as 21β-O-angeloyl3β,15α,16α,23α,28-pentahydroxy-22α-O-(2-methylbutanoyl) olean-12-ene, which was identified to be a new aglycone. The 1H‑NMR spectrum of compound 5 indicated two sugar residues, which were identified as D-glucuronic acid and L-arabinose by acid hydrolysis and GC analysis. The spectroscopic data of the sugar units were very similar to the sugar units of gordonoside [12], α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranosyl, which indicated that the glycosidic chain was α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranosyl. The location and linkage of the glycosidic chain were also identified by the HMBC correlations between δH 5.34 (H-1 of glucuronic acid) and δC 81.3 (C-3 of aglycone), and between δH 5.38 (H-1 of arabinose) and δC 85.8 (C3 of glucuronic acid). The β-anomeric configuration of the glucuronopyranosyl unit was determined from the observation of the large 3JH-1, H-2 coupling constant, and the α-anomeric configuration of the arabinopyranosyl unit was assigned according to the NOESY correlations between δ 5.38 (H-1 of arabinose) and δ " Fig. 2). On 4.58 (H-3 of arabinose) and 4.38 (H-4 of arabinose) (l the basis of the above analysis, the structure of compound 5 was elucidated as 21β-O-angeloyloxy-15α,16α,23,28-tetrahydroxy22α-O-(2-methylbutanoyloxy)olean-12-ene 3β-O-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside, and named oleiferoside E. Compound 6 was obtained as white amorphous powder. Its molecular formula of C54H82O19 was determined according to the [M – H]− ion peak at m/z 1033.5325. The IR spectrum indicated the presence of a hydroxyl group (3456 cm−1), a C=O group (1720 cm−1) and an α,β-unsaturated ester group (1601, 1602 cm−1). The NMR spectroscopic data of compound 6 were similar to those of TR-saponins [13], a triterpenoid glycoside isolated from Gordonia chrysandra roots, except for the presence of an additional signal at δC 52.0 corresponding to one OCH3 group. The OCH3 group appeared to be located at C-6 of the glucuronopyranosyl group, which was supported by the HMBC correlation between δC 170.2 (C-6 of glucuronic acid) and δH 3.78 (OCH3). The glycosidic chain was located at C-3 of the aglycone, and was inferred to be α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyra-
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nosyl methyl ester, which was established by the HMBC correlations between δH 5.26 (H-1 of glucuronic acid) and δC 82.0 (C-3 of aglycone), and between δH 5.37 (H-1 of arabinose) and δC 85.5 (C3 of glucuronic acid). The β-anomeric configuration of the glucuronopyranosyl unit was determined on the basis of the observation of the large 3JH-1, H-2 coupling constant, and the α-anomeric configuration of the arabinopyranosyl unit was indicated by NOESY correlations between δ 5.37 (H-1 of arabinose) and δ " Fig. 2). 4.26 (H-3 of arabinose) and 4.34 (H-4 of arabinose) (l Thus, compound 6 was elucidated as 16α-acetoxy-21β,22α-O-diangeloyloxy-23,28-dihydroxyolean-12-ene 3β-O-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside methyl ester, and named oleiferoside F. Compound 7 was obtained as white amorphous powder. Its molecular formula of C60H92O23 was determined by the observation of the [M + Na]+ ion peak at m/z 1203.5953 in the HR‑ESI‑MS spectrum. The IR spectrum indicated the presence of a hydroxyl group (3456 cm−1), a C=O group (1720 cm−1), and an α,β-unsaturated ester group (1601, 1602 cm−1). The NMR spectroscopic data of compound 7 were similar to those of compound 6, except for the presence of additional signals at δC 103.7, 72.2, 72.4, 74.0, 70.0, and 18.5, corresponding to one rhamnopyranosyl group. The acid hydrolysis and GC analysis confirmed its presence. The HMBC correlations between δH 5.25 (H-1 of glucuronic acid) and δC 82.0 (C-3 of aglycone), between δH 5.26 (H-1 of arabinose) and δC 86.2 (C-3 of glucuronic acid), and between δH 6.11 (H-1 of rhamnose) and δC 80.4 (C-3 of arabinose), indicated that the glycosidic chain was located at C-3 of the aglycone and the sugar
units were connected by α-L-rhamnopyranosyl-(1 → 3)-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranosyl methyl ester, which was identified to be a new sugar chain. The β-anomeric configuration of the glucuronopyranosyl unit was determined from the observation of the large 3JH-1, H-2 coupling constant, and the α-anomeric configurations of the arabinopyranosyl unit and the rhamnopyranosyl unit were assigned according to the NOESY correlations between δ 5.26 (H-1 of arabinose) and δ 4.33 (H-3 of arabinose) and 4.53 (H-4 of arabinose), and between " Fig. 2). δ 6.11 (H-1 of rhamnose) and 4.38 (H-4 of rhamnose) (l Thus, the structure of compound 7 was elucidated as 16α-acetoxy-21β,22α-O-diangeloyloxy-23,28-dihydroxyolean-12-ene 3β-O-α-L-rhamnopyranosyl-(1 → 3)-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside methyl ester, and named oleiferoside G. Compound 8 was obtained as white amorphous powder. Its negative ion HR‑ESI‑MS showed a quasimolecular ion peak [M – H]− at m/z 1181.6082, which inferred the molecular formula of C60H94O23. The IR spectrum indicated the presence of hydroxyl groups (3456 cm−1), a C=O group (1720, 1724 cm−1), an and α,βunsaturated ester group (1601 cm−1). The NMR spectroscopic data of compound 8 were similar to those of compound 7. The significant upfield shift of the C-2″ (− 86.7 ppm) signal at δ 41.6 and the C-3″ (− 111.3 ppm) signal at δ 26.7 suggested the presence of the 22α-O‑MB group in compound 8 in place of the 22α-O‑Ang group in compound 7. Further analyses of the NMR data led to the structure assignment of compound 8 as 16α-acetoxy-21β-Oangeloyloxy-23,28-dihydroxy-22α-O-(2-methylbutanoyloxy)
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
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Fig. 2 Key HMBC and NOESY correlations of compounds 1–8.
Original Papers
olean-12-ene 3β-O-α-L-rhamnopyranosyl-(1 → 3)-α-L-arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside methyl ester, named oleiferoside H. The aglycone of oleiferoside H was reported for the first time. In conclusion, eight new compounds were isolated and identified from 70 % EtOH of C. oleifera, which showed cytotoxic activity. All of the isolated triterpenoid glycosides possessed an olean-12-ene skeleton and the oligosaccharidic chains were linked to C-3. Of these compounds, the aglycones of compounds 5 and 8 were first discovered and so were the sugar chains of compounds 7 and 8. Cytotoxic activities of the isolated saponins were evaluated against four human tumor cell lines (A549, B16, BEL-7402, and MCF-7) by using the MTT in vitro assay. Compound 3 exhibited potent cytotoxic activity against all the tested cell lines with IC50 values < 10 µM, and compounds 1, 2, 4, and 5 showed moderate cytotoxic activities toward the tested cell lines. Among these compounds, compound 3 was the most potent against all the tested cell lines, which suggests the importance of the free hydroxy group at C-21 in mediating cytotoxicity when comparing its structure with others. Besides, the activities of compound 1 were stronger than those of compound 2, which suggests that the C-21-O‑Ang substitute made a more efficient contribution than the C-21-O‑MB substitute to the cytotoxic activity. Furthermore, these results also suggest that the free hydroxy group at C16 might play a role in mediating cytotoxicity because of the acetylation of the hydroxy group at C-16 (i.e., 6, 7, and 8 resulted in activity decreases).
Materials and Methods !
General experimental procedures The specific rotation values were determined with a Perkin-Elmer model 241 polarimeter. IR spectra were recorded on a Perkin-Elmer 983 G spectrometer. 1H, 13C NMR, and 2D NMR spectra were recorded on a Varian Inova 500 spectrometer in C5D5 N using tetramethylsilane (TMS) as the internal standard. HR‑ESI‑MS spectra were determined on a Micromass Q-TOF2 spectrometer. HPLC analysis and separation were performed on a Shimadzu HPLC system composed of a LC-20AT pump with a SPD-20A detector (Shimadzu Corp.), and the wavelength for detection was 203 nm. MPLC separation was performed on a Büchi flash chromatography system composed of a C-650 pump with a flash column (460 × 26 mm i. d., Büchi Corp.). The HPLC column (250 × 9.4 mm i. d., 5 µm, Agilent Zorbax SB‑C18 semipreparative) was purchased from Agilent Corp. Silica gel (200–300 mesh) for column chromatography and precoated silica gel TLC plates were purchased from Qingdao Marine Chemical Factory. ODS for MPLC was purchased from Merck KGaA. Sephadex LH-20 for column chromatography was purchased from GE Corp. GC was conducted on a GC-14C (Shimadzu Corp.) with a flame ionization detector (FID). Compounds on the TLC were colored by 10 % sulfuric acid alcohol solution.
Plant material The roots of C. oleifera were collected in Qichun, Hubei Province of China in November 2011, and identified by Professor Xiao-Ran Li at Soochow University. A voucher sample (No. 11-11-06-01) was deposited in the herbarium of the College of Pharmacy, Soochow University.
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
Extraction and isolation Air-dried roots (9.3 kg) of C. oleifera were crushed with a grinder into fine debris, and extracted two times with 70 % EtOH (2 × 50 L) under reflux for 2 h. The solvent was subsequently removed under reduced pressure to give the residue (0.45 kg), which was then dissolved in distilled water and passed through a D101 macroporous resin column (i. d. 30 cm × 200 cm, Xiʼan Sunresin New Materials Co. Ltd.) eluted with a gradient of EtOH aqueous (EtOH‑H2O 0 %, 30 %, 60%, 80 %, each 40 L). The 80 % EtOH eluate (25 g) was further vacuum chromatographed on a silica gel column (18 × 30 cm) eluted with a gradient of CHCl3-MeOH (100 : 0, 90 : 10, 80 : 20, 70 : 30, 60 : 40, 50 : 50, 40 : 60, 30 : 70, 0 : 100, each 5.0 L). The CHCl3-MeOH (80 : 20) eluate (1.0 g) was then chromatographed by MPLC over an ODS column eluted with MeOH‑H2O (60 : 40, 70 : 30, 80 : 20, 90 : 10, 100 : 0, each 500 mL) at 20.0 mL/ min to afford 5 fractions. Fraction 3 (500–1000 mL, 125 mg) was separated by semipreparative HPLC over an ODS column eluted with MeOH‑H2O (85 : 15) to yield compounds 8 (15 mg, tR 35.25 min), 7 (9 mg, tR 36.28 min), and 6 (9 mg, tR 40.32 min). The CHCl3-MeOH (70 : 30) eluate (6.8 g) was chromatographed by MPLC eluted with MeOH‑H2O (40 : 60, 50 : 50, 60 : 40, 70 : 30, 80 : 20, 100 : 0, each 800 mL) at 20.0 mL/min to afford 10 fractions. Fraction 4 (1500–2000 mL, 420 mg) was separated by semipreparative HPLC over an ODS column eluted with MeOH‑H2O (72 : 28) to yield compounds 3 (56 mg, tR 27.5 min), 1 (90 mg, tR 39.0 min), and 2 (32 mg, tR 37.5 min). The separation of fraction 8 (142 mg) was also performed on semipreparative HPLC using MeOH‑H2O (78: 22, v/v) to yield compounds 4 (15 mg, tR 52.5 min) and 5 (16 mg, tR 44.5 min). The purities of the above saponins were > 90%, as determined by HPLC. Oleiferoside A (1): white amorphous powder; [α]20 D + 6.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 211 (4.30), 254 (4.15) nm; IR νmax 3454, 2962, 2924, 2851, 2816, 2718, 1736, 1604, 1601, 1466, 1368, 1184, 1041 cm−1; 1H NMR (pyridine-d5, 500 MHz) " Tables 1 and 13C NMR (pyridine-d5, 125 MHz) data are given in l and 2; HRESIMS m/z 1315.5968 [M – H]− (calcd. for C63H95O29, 1315.5959). Oleiferoside B (2): white amorphous powder; [α]20 D + 8.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 210 (4.29), 254 (4.05) nm; IR νmax 3456, 2962, 2920, 2855, 2820, 2718, 1740, 1720, 1601, 1460, 1378, 1168, 1046 cm−1; 1H NMR (pyridine-d5, 500 MHz) " Tables 1 and 13C NMR (pyridine-d5, 125 MHz) data are given in l − and 2; HRESIMS m/z 1317.6122 [M – H] (calcd. for C63H97O29, 1317.6116). Oleiferoside C (3): white amorphous powder; [α]20 D + 2.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 208 (4.29), 254 (3.82) nm; IR νmax 3452, 2962, 2910, 2845, 1720, 1602, 1460, 1375, 1170, 1049 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyri" Tables 1 and 2; HRESIMS dine-d5, 125 MHz) data are given in l − m/z 1203.5740 [M – H] (calcd. for C58H91O26, 1203.5799). Oleiferoside D (4): white amorphous powder; [α]20 D + 6.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 211 (4.30), 254 (4.15) nm; IR νmax 3453, 2962, 2924, 2851, 2816, 2719, 1736, 1604, 1601, 1461, 1362, 1186, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) " Tables 1 and 13C NMR (pyridine-d5, 125 MHz) data are given in l − and 2; HRESIMS m/z 1329.6129 [M – H] (calcd. for C64H97O29, 1329.6116). Oleiferoside E (5): white amorphous powder; [α]20 D + 6.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 206 (4.26), 254 (4.22) nm; IR νmax 3456, 2963, 2925, 2850, 1720, 1601, 1460, 1372, 1161, 1042 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyri-
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C NMR (pyridine-d5, 125 MHz) spectroscopic data for compounds 1–8.
Position
1
2
3
4
5
6
7
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 21-O‑Ang 1′ 2′ 3′ 4′ 5′ 22-O‑Ang 1″ 2″ 3″ 4″ 5″ 22-O‑MB 1″ 2″ 3″ 4″ 5″ OAc-1″′ 2″′ Sugar units 1 2 3 4 5 6 OMe
38.5 25.5 84.7 55.4 47.2 20.9 36.2 41.9 48.4 36.4 24.1 125.3 143.9 47.9 67.7 73.7 48.6 41.2 47.0 36.6 78.8 73.5 210.3 11.3 16.2 17.7 21.5 63.3 29.7 20.5
38.5 25.5 84.6 55.3 47.1 20.9 36.1 41.8 47.9 36.4 24.1 125.3 143.9 48.6 67.6 73.2 48.4 41.0 46.9 36.5 78.8 73.2 210.2 11.3 16.1 17.7 21.4 63.0 29.6 20.4
39.1 26.8 89.7 39.8 55.7 19.0 37.2 41.7 47.3 37.2 24.2 125.1 144.7 48.0 67.7 75.2 45.4 42.0 47.2 32.2 36.9 72.9 28.1 17.0 16.0 17.8 21.5 63.9 33.7 25.4
38.5 25.3 84.5 55.3 47.1 20.9 36.1 41.8 48.4 36.4 24.1 125.2 143.9 47.9 67.6 73.7 48.5 41.1 47.0 36.5 78.7 73.5 210.0 11.2 16.1 17.6 20.8 63.3 29.6 20.4
39.1 26.2 81.3 43.6 47.4 18.7 36.5 41.7 47.4 37.1 24.2 125.6 143.9 47.9 67.7 73.2 48.6 41.0 46.9 36.5 78.8 73.3 64.4 11.9 16.6 17.8 21.4 63.1 29.6 20.4
38.6 26.0 82.0 43.4 47.1 17.8 32.6 39.9 46.8 36.5 23.7 125.0 140.8 41.0 30.6 72.0 47.0 39.4 46.8 36.0 77.9 72.4 63.8 13.5 16.0 16.7 26.9 63.3 29.3 19.6
38.6 26.0 82.0 43.4 47.1 17.8 32.6 39.9 46.8 36.5 23.7 125.0 140.8 41.0 30.6 72.0 46.8 39.4 47.0 36.0 77.9 72.5 63.8 13.5 16.0 16.7 26.9 63.3 29.3 19.6
38.6 26.0 82.0 43.4 47.2 17.8 32.6 39.9 46.9 36.5 23.7 125.0 140.9 41.0 30.9 71.4 46.9 39.4 47.0 36.0 78.0 72.5 63.8 13.5 16.0 16.7 26.9 63.5 29.3 19.6
168.0 129.1 137.7 16.2 21.3
167.8 128.8 138.8 16.3 21.3
167.5 129.0 137.6 16.1 21.4
167.8 128.9 138.7 16.2 21.2
167.6 128.4 138.0 15.6 20.8
167.1 128.4 138.3 15.8 20.8
167.4 128.1 139.3 16.0 20.9
167.1 128.3 138.3 15.8 20.7
167.6 128.3 138.0 15.6 20.7
1 2 3 4 5 6
168.3 129.3 136.8 15.9 20.9
168.2 129.7 136.6 16.0 21.1
168.4 129.2 136.7 15.8 21.2
176.8 41.7 27.1 12.0 16.9
176.8 41.6 27.0 11.9 16.8
GlcA 104.3 78.2 84.9 69.9 77.2 172.6
GlcA 104.3 78.2 85.0 69.9 77.3 172.5
GlcA 105.7 78.6 84.8 70.0 77.5 172.6
Gal 1 101.8 84.1 75.2 71.1 77.3 62.1
Gal 1 101.8 84.1 75.2 71.0 77.2 62.0
Gal 1 102.0 84.0 75.2 71.4 77.2 62.0
GlcAOMe 104.1 78.1 85.0 69.8 77.2 170.0 52.3 Gal 1 101.8 84.0 75.2 70.7 76.6 61.9
GlcA 105.7 74.5 85.8 71.6 78.1 170.5 Ara 105.4 72.8 74.5 69.3 67.2
169.8 21.9 GlcAOMe 106.0 74.5 85.5 71.1 76.7 170.2 52.0 Ara 105.8 72.8 74.3 69.1 67.0
Li X et al. Cytotoxic Triterpenoid Glycosides …
169.8 21.9 GlcAOMe 106.4 74.4 86.2 71.0 76.6 170.2 52.0 Ara 105.9 72.1 80.4 69.2 67.9
175.8 41.6 26.7 11.8 16.6 169.7 21.9 GlcAOMe 106.4 74.4 86.2 71.0 76.6 170.1 52.0 Ara 105.9 72.1 80.4 69.2 67.9 continued
Planta Med 2014; 80: 590–598
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Table 1
595
596
Original Papers
Position 1 2 3 4 5 6 1 2 3 4 5 6 a
1
2
3
4
Xyl 107.9 76.5 78.4 71.0 67.7
Xyl 107.9 76.5 78.4 71.0 67.8
Xyl 107.8 76.1 78.5 70.8 67.7
Xyl 107.9 76.5 78.5 71.0 67.8
Gal 1′ 103.1 74.0 75.5 70.7 76.6 62.3
Gal 1′ 103.1 74.0 75.5 70.7 76.6 62.3
Gal 1′ 102.5 73.0 76.6 69.8 78.5 63.2
Gal 1′ 103.2 73.9 75.5 70.6 76.5 62.3
5
6
7
8
Rha 103.7 72.2 72.4 74.0 70.0 18.5
Rha 103.7 72.2 72.4 74.0 70.0 18.5
Chemical shifts are in ppm, and the assignments were based on HSQC, HMBC, and NOESY spectra. b nd = not detected
Table 2
1
H NMR (pyridine-d5, 500 MHz) spectroscopic data for compounds 1–8.
Position 1 2 3 5 6 7 9 11 12 15 16 18 19 21 22 23 24 25 26 27 28 29 30 21-O‑Ang 3′ 4′ 5′ 22-O‑Ang 3″ 4″ 5″ 22-O‑MB 2″ 3″ 4″ 5″ OAc-2″′
1
2
3
4
5
6
7
8
1.08 m, 1.48 m 1.94 m, 2.21 m 4.09 dd (11.0, 4.5) 1.83 m 1.41 m, 1.48 m 2.17 m, 2.18 m 1.53 m 1.90 m, 1.94 m 5.58 brs 4.26 brs 4.54 brs 3.15 m 1.48 m, 3.15 m 6.78 d (10.5) 6.41 d (10.5) 9.94 brs
0.97 m, 1.49 m 1.92 m, 2.21 m 4.11 dd (11.0, 4.5) 1.83 m 1.10 m, 1.49 m 2.16 m, 2.18 m 1.49 m 1.86 m, 1.94 m 5.56 brs 4.23 brs 4.46 brs 3.15 m 1.49 m, 3.13 m 6.73 d (10.5) 6.34 d (10.5) 9.96 brs
0.87 m, 1.48 m 1.88 m, 2.21 m 3.33 dd (11.0, 4.5) 0.90 m 1.47 m, 1.66 m 2.11 m, 2.88 m 1.80 m 1.85 m, 2.03 m 5.57 brs 4.34 brs 4.62 brs 3.11 m 1.41 m, 2.96 m 2.11 m, 2.88 m 6.27 m 1.29 s
0.99 m, 1.52 m 1.91 m, 2.19 m 4.10 dd (11.0, 4.5) 1.85 m 1.12 m, 1.50 m 2.06 m, 2.10 m 1.47 m 1.85 m, 1.98 m 5.58 brs 4.27 brs 4.51 brs 3.16 m 1.52 m, 3.16 m 6.79 d (10.5) 6.40 d (10.5) 9.95 brs
1.53 s 0.88 s 1.04 s 1.90 s 3.57 d (11.0) 3.82 d (11.0) 1.18 s 1.41 s
1.54 s 0.88 s 1.04 s 1.89 s 3.55 d (11.0) 3.82 d (11.0) 1.17 s 1.38 s
1.17 s 0.92 s 1.11 s 1.95 s 3.72 d (10.0) 3.87 d (10.0) 1.14 s 1.36 s
1.52 s 0.90 s 1.05 s 1.90 s 3.60 d (11.0) 3.84 d (11.0) 1.19 s 1.42 s
1.01 m, 1.55 m 2.06 m, 2.35 m 4.38 dd (11.0, 4.5) 1.86 m 1.52 m, 1.92 m 2.17 m, 2.33 m 1.90 m 1.90 m, 2.01 m 5.60 brs 4.26 brs 4.46 brs 3.15 m 1.48 m, 3.15 m 6.71 d (10.5) 6.32 d (10.5) 3.77 d (11.0) 4.38 d (11.0) 1.04 s 1.09 s 1.15 s 1.90 s 3.56 d (11.0) 3.87 d (11.0) 1.15 s 1.39 s
1.02 m, 1.51 m 2.01 m, 2.23 m 4.35 dd (11.0, 4.5) 1.70 m 1.34 m, 1.76 m 1.24 m, 1.54 m 1.82 m 1.87 m, 1.95 m 5.49 brs 1.69 m, 1.92 m 5.59 brs 3.13 m 1.54 m, 2.72 m 6.06 d (10.5) 6.36 d (10.0) 3.88 d (11.0) 4.41 d (11.0) 1.00 s 0.96 s 0.86 s 1.46 s 3.53 d (10.0) 3.70 d (10.0) 1.14 s 1.37 s
1.04 m, 1.52 m 2.06 m, 2.25 m 4.36 dd (11.0, 4.5) 1.70 m 1.29 m, 1.75 m 1.20 m, 1.54 m 1.83 m 1.88 m, 1.99 m 5.50 brs 1.70 m, 1.93 m 5.59 brs 3.15 m 1.52 m, 2.72 m 6.06 d (10.5) 6.36 d (10.0) 3.84 d (11.0) 4.42 d (11.0) 1.00 s 0.96 s 0.87 s 1.47 s 3.53 d (10.0) 3.80 d (10.0) 1.14 s 1.37 s
1.15 m,1.54 m 2.05 m, 2.23 m 4.34 dd (11.0, 4.5) 1.70 m 1.39 m, 1.74 m 1.22 m, 1.58 m 1.84 m 1.87 m, 1.96 m 5.49 brs 1.62 m, 1.95 m 5.67 brs 3.13 m 1.54 m, 2.71 m 5.99 d (10.5) 6.28 d (10.0) 3.85 d (11.0) 4.41 d (11.0) 1.00 s 0.97 s 0.89 s 1.49 s 3.58 d (10.0) 3.74 d (10.0) 1.13 s 1.35 s
6.07 dq (7.5, 1.5) 2.18 d (7.5) 2.09 s
6.16 dq (7.5, 1.5) 2.25 d (7.5) 2.12 s
6.05 dq (7.5, 1.5) 2.17 d (7.5) 2.10 s
6.15 dq (5.5, 1.5) 2.24 d (7.5) 2.12 s
6.04 dq (7.5, 1.5) 2.11 d (7.5) 2.10 s
6.07 dq (7.5, 1.5) 2.11 d (7.5) 2.10 s
6.16 dq (7.5, 1.5) 2.20 d (7.5) 2.07 s
5.99 dq (7.5, 1.5) 2.13 d (7.5) 2.04 s
5.99 dq (7.5, 1.5) 2.10 d (7.5) 2.04 s
5.88 dq (7.5, 1.5) 2.05 d (7.5) 1.83 s
5.94 dq (7.5, 1.5) 2.12 d (7.5) 1.93 s
5.88 dq (7.5, 1.5) 2.05 d (7.5) 1.83 s
2.15 m 1.30 m, 1.67 m 0.75 t (7.5) 1.10 d (6.5)
Li X et al. Cytotoxic Triterpenoid Glycosides …
2.16 m 1.31 m, 1.67 m 0.77 t (7.5) 1.04 d (6.5) 2.57 s
Planta Med 2014; 80: 590–598
2.57 s
2.55 m 1.55 m, 1.86 m 0.98 t (7.5) 1.26 d (6.5) 2.60 s continued
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Table 1 Continued
Original Papers
597
Position
1
2
3
4
5
6
7
8
Sugar units 1 2 3 4 5 OMe
GlcA 4.80 d (6.5) 4.57 m 4.41 m 4.57 m 4.50 m
GlcA 4.83 d (6.5) 4.57 m 4.39 m 4.58 m 4.49 m
GlcA 4.90 d (7.5) 4.72 m 4.52 m 4.53 m 4.39 m
GlcA 5.34 d (7.0) 4.26 m 4.30 m 4.58 m 4.60 m
Gal 1 5.80 d (7.5) 4.57 m 4.39 m 4.50 m 4.18 m 4.45 m, 4.46 m Xyl 5.09 d (7.5) 4.27 m 4.11 m 4.41 m 3.50 m, 4.45 m
Gal 1 5.80 d (7.5) 4.58 m 4.37 m 4.53 m 4.20 m 4.40 m, 4.39 m Xyl 5.10 d (7.0) 4.28 m 4.11 m 4.39 m 3.51 m, 4.18 m
Gal 1 5.80 d (7.0) 4.58 m 4.31 m 4.52 m 4.52 m 4.46 m, 4.47 m Xyl 5.13 d (7.5) 4.37 m 4.10 m 4.47 m 3.53 m, 4.42 m
GlcAOMe 4.84 d (10.0) 4.55 m 4.35 m 4.57 m 4.18 m 3.78 s Gal 1 5.74 d (10.0) 4.57 m 4.35 m 4.59 m 4.39 m 4.43 m, 4.45 m Xyl 5.12 d (7.5) 4.29 m 4.13 m 4.42 m 3.59 m, 4.53 m
GlcAOMe5.26 d (7.0) 4.16 m 4.24 m 4.43 m 4.50 m 3.78 s Ara 5.37 d (6.5) 4.53 m 4.26 m 4.34 m 3.81 m, 4.45 m
GlcAOMe 5.25 d (7.0) 4.12 m 4.23 m 4.40 m 4.50 m 3.77 s Ara 5.26 d (7.0) 4.71 m 4.33 m 4.53 m 3.87 m, 4.35 m
GlcAOMe 5.24 d (7.0) 4.12 m 4.32 m 4.40 m 4.50 m 3.78 s Ara 5.24 d (7.0) 4.67 m 4.43 m 4.52 m 3.87 m, 4.36 m
Rha 6.11 brs 4.79 m 4.63 m 4.38 m 4.69 m 1.74 d (5.0)
Rha 6.10 brs 4.78 m 4.61 m 4.37 m 4.63 m 1.75 d (5.0)
Gal 1′ 5.95 d (7.5) 4.50 m 4.45 m 4.57 m 4.55 m 4.57 m, 4.55 m
Gal 1′ 5.96 d (7.5) 4.52 m 4.46 m 4.58 m 4.55 m 4.58 m, 4.57 m
Gal 1′ 6.08 d (7.5) 4.17 m 4.19 m 4.21 m 4.53 m 4.37 m, 4.57 m
Gal 1′ 5.86 d (7.5) 4.51 m 4.45 m 4.46 m 4.53 m 4.55 m, 4.57 m
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Ara 5.38 d (6.5) 4.26 m 4.58 m 4.38 m 3.89 m, 4.46 m
Chemical shifts are in ppm and coupling constants (J) in Hz are given in parentheses. The assignments were based on HSQC, HMBC, and NOESY spectra
" Tables 1 and 2; HRESIMS dine-d5, 125 MHz) data are given in l m/z 995.5257 [M – H]− (calcd. for C51H79O19, 995.5216). Oleiferoside F (6): white amorphous powder; [α]20 D + 5.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 208 (4.29), 254 (3.82) nm; IR νmax 3456, 2962, 2910, 2845, 1720, 1602, 1601, 1441, 1382, 1168, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR " Tables 1 and 2; HRE(pyridine-d5, 125 MHz) data are given in l SIMS m/z 1033.5325 [M – H]− (calcd. for C54H81O19, 1033.5372). Oleiferoside G (7): white amorphous powder; [α]20 D + 6.4 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 211 (4.18), 254 (4.34) nm; IR νmax 3456, 2965, 2918, 2849, 1720, 1602, 1601, 1441, 1382, 1168, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR " Tables 1 and 2; HRE(pyridine-d5, 125 MHz) data are given in l SIMS m/z 1203.5953 [M + Na]+ (calcd. for C60H92O23 Na, 1203.5927). Oleiferoside H (8): white amorphous powder; [α]20 D + 8.4 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 210 (4.18), 254 (4.32) nm; IR νmax 3456, 2963, 2912, 2843, 1720, 1724, 1601, 1441, 1382, 1158, 1040 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR " Tables 1 and 2; HRE(pyridine-d5, 125 MHz) data are given in l SIMS m/z 1181.6082 [M – H]− (calcd. for C60H93O23, 1181.6108).
Acid hydrolysis and sugar analysis of compounds 1–8 Each saponin (2 mg) was dissolved in 2 N HCl (2 mL) and stirred at 80 °C for 4 h. The reaction mixture was extracted with chloroform, and the aqueous layer was evaporated to give a mixture of monosaccharides. The residue was dissolved in anhydrous pyridine (1 mL) followed by the addition of 2 mg of L-cysteine methyl ester hydrochloride (Tokyo Chemical Industry, 99 %). After heating at 60 °C for 2 h, the solvent was evaporated under N2, and
0.2 mL trimethylsilylimidazole (Tokyo Chemical Industry, 99 %) was added. Then the mixture was heated at 60 °C for another 2 h and partitioned between n-hexane and water. The organic layer was investigated by GC under the following conditions: capillary column, HP-5 (30 m × 0.25 mm × 0.25 µm; Dikma); FID detector with a temperature of 280 °C; injection temperature 250 °C; initial temperature 160 °C, then raised to 280 °C at 5 °C/min, final temperature maintained for 10 min; carrier gas, N2. The standard sugars experienced the same reaction and GC conditions. The retention times of persilylated D-glucuronic acid, D-glucuronic acid methyl ester,D-galactose, D-xylose, and L-arabinose were 19.770 min, 24.460 min, 25.643 min, 32.257 min, and 33.284 min, respectively.
Cytotoxicity assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay was performed to evaluate the cytotoxic activities of the isolated saponins against A549, B16, BEL7402, and MCF-7 human tumor cell lines (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The amount of formazan was determined by photometry at 570 nm. Cells were plated into 96-well flat-bottomed cultured plates at a concentration of 2 × 105 cells per well in complete RPMI 1640 culture medium. After twenty-four hours, the medium containing fetal calf serum was removed and the test solutions were applied to the cells in different final concentrations at 0.625, 1.25, 2.5, 5, 10, 20, 50, and 100 µM. After 24 h, the MTT solution was added to the wells and the plates were incubated at 37 °C for 4 h. The positive control group was treated with norcantharidin (purity higher than 99.0 % as determined by HPLC; Nanjing Zelang Medical
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
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Table 2 Continued
Original Papers
Table 3 In vitro cytotoxic activities of compounds 1–8 (IC50 values in µM). Com-
Cell line IC50 (µM)
pound
A549
B16
7402
MCF-7
1 2 3 4 5 6 7 8 Norcantharidin
14.95 ± 0.40 22.23 ± 0.25 5.73 ± 0.09 20.23 ± 1.12 33.27 ± 0.28 89.91 ± 15.78 89.99 ± 3.29 99.41 ± 5.28 4.16 ± 0.28
17.56 ± 0.77 28.32 ± 2.04 6.04 ± 0.04 25.46 ± 1.58 31.80 ± 2.15 57.21 ± 3.84 63.74 ± 0.99 80.24 ± 6.06 3.93 ± 0.22
15.76 ± 0.33 28.32 ± 0.16 6.45 ± 0.07 33.49 ± 5.33 35.11 ± 2.31 71.91 ± 11.57 92.67 ± 11.60 91.87 ± 14.74 3.45 ± 0.26
14.25 ± 1.90 24.15 ± 1.37 6.45 ± 0.48 17.33 ± 2.07 26.98 ± 2.79 77.33 ± 15.71 90.31 ± 13.37 85.79 ± 12.52 5.17 ± 0.38
Technology Co. Ltd.). The amount of formazan was determined by photometry at 570 nm. The results are expressed as the percentage of absorbance in the control cells in comparison to that in the drug-treated cells. The IC50 values (50 % inhibitory concentration) " Table 3. The experiment was of compounds 1–8 are shown in l performed in triplicate for each sample.
Supporting information Various spectra for compounds 1–8 are available as Supporting Information.
Acknowledgements !
This work was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors thank Dr. Jon Parcher at the University of Mississippi for helpful discussions.
Conflict of Interest !
All authors of this paper confirm that they have no conflicts of interest.
Li X et al. Cytotoxic Triterpenoid Glycosides …
Planta Med 2014; 80: 590–598
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