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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Three new olean-type triterpenoid saponins from aerial parts of Eclipta prostrata (L.) a
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Feng-Min Xi , Chun-Tong Li , Jun-Ling Mi , Zhi-Jun Wu & WanSheng Chen
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Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, P.R. China b
Department of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, P.R. China Published online: 09 Sep 2013.
To cite this article: Feng-Min Xi, Chun-Tong Li, Jun-Ling Mi, Zhi-Jun Wu & Wan-Sheng Chen (2014) Three new olean-type triterpenoid saponins from aerial parts of Eclipta prostrata (L.), Natural Product Research: Formerly Natural Product Letters, 28:1, 35-40, DOI: 10.1080/14786419.2013.832674 To link to this article: http://dx.doi.org/10.1080/14786419.2013.832674
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Natural Product Research, 2014 Vol. 28, No. 1, 35–40, http://dx.doi.org/10.1080/14786419.2013.832674
Three new olean-type triterpenoid saponins from aerial parts of Eclipta prostrata (L.) Feng-Min Xia, Chun-Tong Lia, Jun-Ling Mib, Zhi-Jun Wua* and Wan-Sheng Chena* a
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Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China; bDepartment of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, P.R. China (Received 3 February 2013; final version received 6 July 2013) Three new olean-type triterpenoid saponins, namely 3-O-(2-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (1), 3-O-(6-O-acetyl-b-D glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (2) and 3-O-(b-D glucopyranosyl) oleanolic acid-28-O-(6-O-acetyl-b-D -glucopyranosyl) ester (3), were isolated from the aerial parts of Eclipta prostrata (L.). Their structures were elucidated based on 1D and 2D NMR and MS spectroscopic data. Keywords: Eclipta prostrata; chemical constituents; triterpenoid saponins
1. Introduction Eclipta prostrata (L.) is an annual herbaceous plant, which is one of four species of genus Eclipta from the family Asteraceae. It is growing widely in the tropical and subtropical areas of the world, and distributed throughout China, which is known as ‘Mohanlian’. The aerial parts of E. prostrata are one important traditional Chinese medicine and mainly used as a tonic for enriching the blood (China Pharmacopoeia Committee 2010). It has attracted a great deal of attention for its wide range of biological activities, such as antiinflammatory (Chandra et al. 1987; Arunachalam et al. 2009), antioxidant (Karthikumar et al. 2007), immunomodulatory (Jayathirthaa & Mishraa 2004), hepatoprotective (Saxena & Singh 1993) and hair growth promoting properties (Roy et al. 2008). Previous phytochemical investigations on E. prostrata revealed that it is rich in bioactive natural products, including triterpenes (Yahara et al. 1994, 1997), coumestans (Wagner et al. 1986), steroids, flavonoids (Sarg et al. 1981), thiopenes (Singh 1988), polyacetylenes (Singh & Bhargava 1922) and alkaloids (Abdel-Kader et al. 1998). As part of an ongoing search for bioactive natural products from traditional folk medicine, we have conducted a phytochemical investigation on the EtOH extract of the aerial parts of E. prostrata. This has led to the isolation of three new olean-type triterpenoid saponins (Figure 1), successively named 3-O-(2-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (1), 3-O-(6-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (2) and 3-O-(b-D -glucopyranosyl) oleanolic acid-28-O-(6-O-acetyl-b-D glucopyranosyl) ester (3). This paper reports the isolation and structure elucidation of the three compounds.
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
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Figure 1. Structures of compounds 1 – 3.
2. Results and discussion The 80% EtOH extract of air-dried aerial parts of E. prostrata was suspended in H2O and partitioned successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH-soluble extract was subjected to column chromatography (CC) and purified by semi-preparative high-performance liquid chromatography (HPLC) to yield three new terpenoid glycosides (1–3). Compound 1 was obtained as white amorphous powder. The negative HR-ESI-MS exhibited pseudo-molecular ion peak at m/z 883.4707 [M þ COOH]2 (calcd for 883.4698, C45H71O17), consistent with the formula C44H70O15, indicating 108 of unsaturation. The 1H NMR spectrum of 1 showed signals for eight tertiary methyl groups (dH 2.12, 1.81, 1.04, 1.09, 1.02, 0.98, 0.87 and 0.81), an olefinic proton [dH 5.29 (1H, m)] and two anomeric protons [dH 6.28 (1H, d, J ¼ 7.9 Hz) and 4.89 (1H, d, J ¼ 8.0 Hz)]. The 13C NMR spectrum of 1 exhibited 44 carbon resonances, classified into 9 quaternary carbons, 16 methines including 12 oxygen-substituted carbons, 11 methylenes including 2 oxygen-substituted carbons, and 8 methyls. The signals at dC 175.8, 169.9, 144.3, 122.5, 103.7, 95.7 and 21.1 were assigned to a carbonyl carbon (dC 175.8), a pair of olefinic carbons (dC 144.3, 122.5), two anomeric carbons (dC 103.7, 95.7) and an acetyl group (dC 169.9, 21.1). These spectroscopic data suggested that 1 was a typical olean-12-enetype triterpenoid glycoside. The assignment of 1H and 13C NMR spectroscopic data of 1 (see Table S1) was based on 1H – 1H COSY, HSQC and HMBC experiments. The 1H and 13C NMR data of 1 were in good agreement with those of eclalbasaponin I (Figure 1, 1a) (Yahara et al. 1994), except for the additional acetyl group (dC 169.9, 21.2) in 1. The position ester linkage of the acetyl group was confirmed by HMBC spectrum of 1. In the HMBC experiment (Figure S1), the correlation between H-20 (dH 5.53) and acetyl group (dC 169.9) was observed, indicating that the acetyl group was attached at the oxygen atom of C-20 of the b-D -glucopyranosyl. The absolute configurations of glucopyranosyls were further confirmed by acid hydrolysis, followed by chiral GC –MS analysis of trimethylsilylated derivatives of the resultant monosaccharides. The relative configurations of 1 were further confirmed by NOESY experiment, which showed NOE correlations between the following proton pairs: H-3 and H-5, H-23; H-5 and H-9, H-23; H-9 and H-27; H-16 and H-27; H-18 and H-30; H-24 and H-25; H-25 and H-26. Consequently, the structure of 1 was elucidated as 3-O-(2-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O(b-D -glucopyranosyl) ester. Compound 2 was isolated as white amorphous powder. Its negative HR-ESI-MS showed pseudo-molecular ion peaks at m/z 883.4714 [M þ COOH]2 (calcd for 883.4698, C45H71O17), quite close to peak of 1. The same formula C44H70O15 revealed that 1 and 2 were isomers. Comparing the 1H and 13C NMR spectroscopic data of 2 with 1 (Table S1), both were almost superimposable, except for some differences of the signals of Glc-I. The different signals of Glc-I in 2 may result from different substitutional positions of acetyl group. This hypothesis was confirmed unambiguously by the HMBC spectrum of 2. The HMBC experiment showed cross-peaks from
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H-60 (dH 4.89, 4.79) to the acetyl group (dC 170.7). This deduced that the acetyl group was located at the oxygen atom of C-60 of Glc-I. The absolute configurations of glucopyranosyls were further verified by acid hydrolysis and chiral GC–MS analysis. Therefore, the structure of 2 is assigned as 3-O-(6-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester. Compound 3 was obtained as white amorphous powder. Its molecular formula was determined as C44H70O15 on the basis of the negative HR-ESI-MS (883.4684 [M þ COOH]2, calcd for 883.4698), indicating that 3 was the isomer of 1 and 2. The 1H and 13C NMR spectroscopic data of 3 (Table S1) were quite similar to those of 1a, except that the signals of Glc-II showed some differences. Further analysis indicated that the major differences of signals of Glc-II between 3 and 1a could be explained by substitution of acetyl group. HMBC correlation between H-600 (dH 4.82, 4.73) and the acetyl group (dC 170.5) implied that the acetyl group was located at the oxygen atom of C-600 of Glc-II in 3. Hence, the structure of 3 is determined to be 3O-(b-D -glucopyranosyl) oleanolic acid-28-O-(6-O-acetyl-b-D -glucopyranosyl) ester. The structures of triterpenoid saponins from E. prostrata contain two types: oleanane-type and taraxstane-type, such as eclalbasaponin I–VI (Yahara et al. 1994) and eclalbasaponin VII – X (Yahara et al. 1997). The three new isolated triterpenoid saponins could be classified into olean-12-ene-type, which is common in the genus Eclipta. 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer 341 Polarimeter (Waltham, MA, USA). IR analyses were performed with a NEXUS 470 FT-IR spectrometer (Thermo Nicolet, USA). UV spectra were recorded on Shimadzu UV/VIS-240 recording spectrophotometer (Shimadzu Co., Japan). 1D and 2D NMR spectra were obtained on a Bruker Avance 600 NMR spectrometer (Bruker Co., Germany). HR-ESI-MS were acquired on an Agilent 6220 TOF LC-MS instrument (Agilent Technologies, MA, USA). GC – MS was conducted on a Thermo Finnigan Trace GC apparatus (Thermo Finnigan, CA, USA) using an L-Chirasil-Val column (25 m £ 0.32 mm). CC was performed by using silica gel (100 –200 and 200– 300 mesh), ODS (50 mm; YMC (YMC CO., Ltd, Japan)) and Sephadex LH-20 (40 – 70 mm) (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Semi-preparative HPLC isolation was achieved (Agilent Technologies, MA, USA) with an Agilent 1200 instrument with a refractive index detector, using a C18 column (250 mm £ 10 mm £ 5 mm, YMC) and eluting with MeOH – H2O at 2.0 mL/min. Precoated silica gel GF254 and HF254 plates were used for TLC, and zones were visualised under UV light (254 and 365 nm) or by spraying with 10% H2SO4 – EtOH followed by heating. 3.2. Plant material The plant of E. prostrata was collected from Xianning City, Hubei Province (GPS coordinates: N 29813.4840 , E 113850.7610 ), during July 2011, and identified by Prof. Lian-Na Sun (Department of Pharmacy, Second Military Medical University, Shanghai, China). A voucher specimen (No. 20110828) was deposited at the Department of Pharmacognosy, Second Military Medical University. 3.3. Extraction and isolation The air-dried aerial parts of E. prostrata (15.0 kg) were extracted three times with 80% EtOH under reflux. After removal of the solvent by evaporation in vacuum, the residue was suspended in water (20 L) and then successively partitioned with petroleum ether, EtOAc and n-BuOH, (3 £ 15 L). The n-BuOH-soluble part (550 g) was fractionated by silica gel CC eluted with CH2Cl2 –MeOH (100:1 ! 50:1 ! 10:1 ! 5:1) to give five fractions
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(Fr. 1 –Fr. 5). Fr. 5 (30 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH – H2O (50:1:0.1 ! 30:1:0.1 ! 20:1:0.1 ! 10:1:0.1) to afford eight subfractions (Fr. 5.1– Fr. 5.8). Fr. 5.3 (5.1 g) was applied to ODS CC eluted with MeOH –H2O (25% ! 50% ! 75%) to afford four subfractions (Fr. 5.3.1 –Fr. 5.3.4). Fr. 5.3.2 (1.0 g) was purified by semi-preparative HPLC (45% MeOH – H2O, 2 mL/min) to yield compounds 1 (32.1 mg, tR ¼ 35.4 min) and 2 (48.9 mg, tR ¼ 38.3 min). Fr. 5.4 (2.5 g) was subjected to Sephadex LH-20 eluted with MeOH –H2O (1:1) to yield four subfractions (Fr. 5.4.1 –Fr. 5.4.4). Fr. 5.4.2 (0.3 g) was purified by semi-preparative HPLC (40% MeOH –H2O, 2 mL/min) to give compound 3 (5.8 mg, tR ¼ 49.5 min).
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3.4. Acid hydrolysis of compounds Compounds 1, 2 and 3 (each 2.0 mg) were separately heated in 2 M HCl (1 mL) at 1208C for 1.0 h. The mixture was concentrated and the residue was dissolved in 1-trimethylsilyl imidazole and pyridine (0.2 mL), and the solution was stirred at 608C for 5 min. After drying the solution, the residue was partitioned between CH2Cl2 and H2O (1 mL, 1:1 v/v). The organic phase was submitted to GC – MS analysis using an L-Chirasil-Val column (0.32 mm £ 25 m). Temperatures of the injector and detector were 2008C for both. A temperature gradient system was used for the oven, starting at 1008C for 1 min and increasing up to 1808C at a rate of 58C/ min. The authentic samples were treated in the same manner with 1-trimethylsilyl imidazole in pyridine. Only D -glucose was identified by comparing the retention time with those of authentic D -glucose (tR ¼ 14.70 min) after treatment using the same method. 3.5. Characterisation of compounds Compound 1: amorphous powder; ½a25 D þ 71:6 (c ¼ 0.5, MeOH); IR (KBr) nmax 3434, 2946, 2881, 1732, 1640, 1464, 1389, 1376, 1308, 1248, 1167, 1077, 1028 cm211; 1H NMR (pyridined5, 600 MHz): d: 0.75 (1H, dd, J ¼ 12.0, 1.0 Hz, H-5), 0.81 (3H, s, H-25), 0.87 (3H, s, H-24), 0.89 (1H, m, H-1b), 0.98 (3H, s, H-29), 1.02 (3H, s, H-30), 1.04 (3H, s, H-23), 1.09 (3H, s, H-26), 1.23 (1H, m, H-21b), 1.27 (1H, m, H-6b), 1.35 (1H, m, H-19b), 1.35 (1H, m, H-7b), 1.40 (1H, m, H-1a), 1.43 (1H, m, H-6a), 1.57 (1H, m, H-7a), 1.72 (1H, m, H-2b), 1.73 (1H, m, H-9), 1.77 (1H, m, H-15b), 1.81 (3H, s, H-27), 1.94 (2H, m, H-11), 2.07 (1H, m, H-22b), 2.11 (1H, dd, J ¼ 13.2, 4.0 Hz, H-2a), 2.12 (3H, s, H-Ac), 2.37 (1H, m, H-22a), 2.41 (1H, m, H-21a), 2.53 (1H, dd, J ¼ 12.0, 2.6 Hz, H-15a), 2.77 (1H, t, J ¼ 13.7 Hz, H-19a), 3.22 (1H, dd, J ¼ 11.7, 4.4 Hz, H-3), 3.48 (1H, dd, J ¼ 13.7, 3.9 Hz, H-18), 3.97 (1H, m, H-50 ), 4.01 (1H, m, H-500 ), 4.12 (1H, t, J ¼ 8.4 Hz, H-200 ), 4.18 (1H, m, H-40 ), 4.24 (1H, m, H-300 ), 4.26 (1H, m, H-400 ), 4.27 (1H, m, H-30 ), 4.32 (1H, m, H-60 b), 4.35 (1H, m, H-600 b), 4.42 (1H, m, H-600 a), 4.53 (1H, m, H-60 a), 4.89 (1H, d, J ¼ 8.0 Hz, H-10 ), 5.29 (1H, br s, H-16a), 5.53 (1H, t, J ¼ 8.0 Hz, H-20 ), 5.58 (1H, t, J ¼ 3.0 Hz, H-12), 6.28 (1H, d, J ¼ 8.4 Hz, H-100 ). 13C NMR (pyridine-d5, 150 MHz): d: 38.4 (C-1), 26.1 (C-2), 88.9 (C-3), 38.9 (C-4), 55.5 (C-5), 18.3 (C6), 33.2 (C-7), 39.9 (C-8), 47.0 (C-9), 36.8 (C-10), 23.6 (C-11), 122.5 (C-12), 144.3 (C-13), 41.9 (C-14), 35.9 (C-15), 74.2 (C-16), 48.9 (C-17), 41.1 (C-18), 47.0 (C-19), 30.6 (C-20), 35.7 (C-21), 32.0 (C-22), 27.7 (C-23), 16.6 (C-24), 15.4 (C-25), 17.3 (C-26), 27.0 (C-27), 175.8 (C-28), 33.0 (C-29), 24.4 (C-30), 103.7 (C-10 ), 75.5 (C-20 ), 76.0 (C-30 ), 71.6 (C-40 ), 78.2 (C-50 ), 62.4 (C-60 ), 95.7 (C-100 ), 73.9 (C-200 ), 78.6 (C-300 ), 70.9 (C-400 ), 79.1 (C-500 ), 62.0 (C-600 ), 169.9 (C-Ac), 21.1 (C-Ac). HRESI-MS m/z 883.4707 [M þ COOH]2 (calcd for 883.4698, C45H71O17). Compound 2: amorphous powder; ½a25 D þ 74:8 (c ¼ 0.5, MeOH); IR (KBr) nmax 3436, 2945, 2926, 1735, 1637, 1460, 1389, 1370, 1306, 1247, 1168, 1077, 1034 cm21; 1H NMR (pyridine-d5, 600 MHz): d: 0.80 (1H, dd, J ¼ 12.1, 0.8 Hz, H-5), 0.87 (3H, s, H-25), 0.93 (3H, s, H-24), 0.97 (3H, s, H-29), 1.01 (3H, s, H-30), 1.04 (1H,dd, J ¼ 14.1, 3.2 Hz, H-1b), 1.09 (3H, s, H-26), 1.21 (1H, m, H-21b), 1.24 (3H, s, H-23), 1.25 (1H, m, H-6b), 1.35 (1H, m, H-19b), 1.35
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(1H, m, H-7b), 1.42 (1H, m, H-6a), 1.57 (1H, m, H-7a), 1.58 (1H, m, H-1a), 1.76 (1H, m, H-15b), 1.78 (1H, m, H-9), 1.81 (3H, s, H-27), 1.86 (1H, m, H-2b), 1.97 (2H, m, H-11), 2.06 (1H, m, H-22b), 2.12 (3H, s, H-Ac), 2.22 (1H, dd, J ¼ 13.3, 3.6 Hz, H-2a), 2.34 (1H, m, H-22a), 2.38 (1H, m, H-21a), 2.52 (1H, dd, J ¼ 11.7, 2.8 Hz, H-15a), 2.77 (1H, t, J ¼ 13.6 Hz, H-19a), 3.22 (1H, dd, J ¼ 11.7, 4.3 Hz, H-3), 3.48 (1H, dd, J ¼ 13.6, 4.0 Hz, H-18), 3.97 (1H, m, H-50 ), 3.98 (1H, m, H-40 ), 3.98 (1H, m, H-500 ), 4.02 (1H, m, H-20 ), 4.10 (1H, t, J ¼ 8.4 Hz, H-200 ), 4.18 (1H, m, H-30 ), 4.23 (1H, m, H-300 ), 4.26 (1H, m, H-400 ), 4.33 (1H, dd, J ¼ 11.8, 4.5 Hz, H-600 b), 4.41 (1H, dd, J ¼ 11.8, 2.2 Hz, H-600 a), 4.79 (1H, dd, J ¼ 11.4, 6.4 Hz, H-60 b), 4.83 (1H, d, J ¼ 7.8 Hz, H-10 ), 4.89 (1H, dd, J ¼ 11.4, 1.8 Hz, H-60 a), 5.28 (1H, br s, H-16a), 5.59 (1H, t, J ¼ 3.5 Hz, H-12), 6.26 (1H, d, J ¼ 8.4 Hz, H-100 ). 13C NMR (pyridine-d5, 150 MHz): d: 38.7 (C-1), 26.4 (C-2), 89.1 (C-3), 39.2 (C-4), 55.8 (C-5), 18.3 (C-6), 33.2 (C-7), 39.9 (C-8), 47.0 (C-9), 36.8 (C-10), 23.6 (C-11), 122.5 (C-12), 144.2 (C-13), 41.9 (C-14), 35.8 (C-15), 74.1 (C-16), 48.9 (C-17), 41.1 (C-18), 47.0 (C-19), 30.6 (C-20), 35.7 (C-21), 32.0 (C-22), 27.9 (C-23), 16.7 (C-24), 15.4 (C-25), 17.3 (C-26), 27.0 (C-27), 175.8 (C-28), 33.0 (C-29), 24.4 (C-30), 106.7 (C-10 ), 75.3 (C-20 ), 78.2 (C-30 ), 71.5 (C-40 ), 74.6 (C-50 ), 64.6 (C-60 ), 95.6 (C-100 ), 73.9 (C-200 ), 78.5 (C-300 ), 70.8 (C-400 ), 79.1 (C-500 ), 61.9 (C-600 ), 170.7 (C-Ac), 20.6 (C-Ac). HRESI-MS m/z 883.4714 [M þ COOH]2 (calcd for 883.4698, C45H71O17). Compound 3: amorphous powder; ½a25 D þ 75:0 (c ¼ 0.2, MeOH); IR (KBr) nmax 3411, 2944, 2873, 1732, 1456, 1388, 1363, 1233, 1165, 1076, 1028 cm21; 1H NMR (pyridine-d5, 600 MHz): d: 0.81 (1H, dd, J ¼ 12.8, 1.0 Hz, H-5), 0.91 (3H, s, H-25), 0.93 (1H,dd, J ¼ 14.1, 3.2 Hz, H-1b), 0.98 (3H, s, H-29), 1.00 (3H, s, H-24), 1.03 (3H, s, H-30), 1.11 (3H, s, H-26), 1.24 (1H, m, H-21b), 1.29 (3H, s, H-23), 1.31 (1H, m, H-6b), 1.34 (1H, m, H-19b), 1.38 (1H, m, H-7b), 1.45 (1H, m, H-6a), 1.47 (1H, m, H-1a), 1.59 (1H, m, H-7a), 1.77 (1H, m, H-15b), 1.77 (1H, m, H-9), 1.82 (1H, m, H-2b), 1.83 (3H, s, H-27), 1.88 (3H, s, H-Ac), 1.97 (2H, m, H-11), 2.16 (1H, m, H-22b), 2.24 (1H, dd, J ¼ 13.6, 3.6 Hz, H-2a), 2.35 (1H, m, H-22a), 2.40 (1H, m, H-21a), 2.51 (1H, dd, J ¼ 15.3, 3.1 Hz, H-15a), 2.78 (1H, t, J ¼ 13.6 Hz, H-19a), 3.40 (1H, dd, J ¼ 11.7, 4.3 Hz, H-3), 3.51 (1H, dd, J ¼ 13.6, 4.5 Hz, H-18), 4.00 (1H, m, H-50 ), 4.03 (1H, m, H-20 ), 4.07 (1H, m, H-400 ), 4.09 (1H, m, H-500 ), 4.15 (1H, t, J ¼ 8.4 Hz, H-200 ), 4.20 (1H, m, H-40 ), 4.23 (1H, m, H-300 ), 4.24 (1H, m, H-30 ), 4.40 (1H, dd, J ¼ 11.5, 5.5 Hz, H-60 b), 4.57 (1H, dd, J ¼ 11.5, 2.3 Hz, H-60 a), 4.73 (1H, dd, J ¼ 11.8, 5.3 Hz, H-600 b), 4.82 (1H, dd, J ¼ 11.8, 1.7 Hz, H-600 a), 4.93 (1H, d, J ¼ 7.7 Hz, H-10 ), 5.25 (1H, br s, H-16a), 5.60 (1H, t, J ¼ 3.2 Hz, H-12), 6.25 (1H, d, J ¼ 8.4 Hz, H-100 ). 13C NMR (pyridine-d5, 150 MHz): d: 38.6 (C-1), 26.3 (C-2), 88.6 (C-3), 39.2 (C-4), 55.6 (C-5), 18.2 (C-6), 33.2 (C-7), 39.8 (C-8), 46.9 (C-9), 36.7 (C-10), 23.5 (C-11), 122.5 (C-12), 144.1 (C-13), 41.8 (C-14), 35.8 (C-15), 74.1 (C-16), 48.9 (C-17), 41.0 (C-18), 46.9 (C-19), 30.5 (C-20), 35.6 (C-21), 31.9 (C-22), 27.9 (C-23), 16.8 (C-24), 15.4 (C-25), 17.3 (C-26), 26.9 (C-27), 175.8 (C-28), 32.9 (C-29), 24.3 (C-30), 106.6 (C-10 ), 75.5 (C-20 ), 78.3 (C-30 ), 71.5 (C-40 ), 78.4 (C-50 ), 62.7 (C-60 ), 95.3 (C-100 ), 73.7 (C-200 ), 78.0 (C-300 ), 70.7 (C-400 ), 75.8 (C-500 ), 64.1 (C-600 ), 170.5 (C-Ac), 20.4 (C-Ac). HR-ESI-MS m/z 883.4684 [M þ COOH]2 (calcd for 883.4698, C45H71O17). Supplementary material Supplementary material relating to this article is available online, alongside Table S1 and Figure S1 and 1D, 2D NMR spectra and HR-ESI-MS, IR data of compounds 1– 3. Acknowledgements The work was financially supported by the National Natural Science Foundation of the People’s Republic of China (Grant No. 81274032) and the Scientific Foundation of Shanghai (Grant Nos 11DZ1971301 and 08DZ1971600).
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References Abdel-Kader MS, Bahler BD, Malone S, Werkhoven MCM, Troon FV, Wisse JH, Bursuker I, Neddermann KM, Mamber SW, Kingston DGI, David GI. 1998. DNA damaging steroidal alkaloids from Eclipta alba from the suriname rainforest. J Nat Prod. 61:1202–1208. Arunachalam G, Subramanian N, Pazhani GP, Ravichandran V. 2009. Antiinflammatory activity of methanolic extract of Eclipta prostrata L. (Astearaceae). Afr J Pharm Pharmacol. 3:97–100. Chandra T, Sadique J, Somasundaram S. 1987. Effects of Eclipta alba on inflammation and liver injury. Fitoterapia. 58:23–32. China Pharmacopoeia Committee. 2010. Chinese Pharmacopoeia. Vol. 1. Beijing: Chemical Industry Press. p. 352. Jayathirthaa MG, Mishraa SH. 2004. Preliminary immunomodulatory activities of methanol extracts of Eclipta alba and Centella asiatica. Phytomedicine. 11:361–365. Karthikumar S, Vigneswari K, Jegatheesan K. 2007. Screening of antibacterial and antioxidant activities of leaves of Eclipta prostrata (L.). Sci Res Essays. 2:101–104. Roy RK, Thakur M, Dixit VK. 2008. Hair growth promoting activity of Eclipta alba in male albino rats. Arch Dermatol Res. 300:357–364. Sarg TM, Salam NAA, El-Domiaty M, Khafagy SM. 1981. The steroid, triterpenoid and flavonoid constituents of Eclipta alba (L.) Hassk (Compositae) grown in Egypt. Sci Pharm. 49:262–264. Saxena AK, Singh BA. 1993. Hepatoprotective effects of Eclipta alba on subceellular levels in rats. J Ethnopharmacol. 40:155–161. Singh P. 1988. Naturally occurring thiophene derivatives from Eclipta alba species. Bioact Mol. 7:179–186. Singh P, Bhargava S. 1922. A dithienyl acetylene ester from Eclipta erecta. Phytochemistry. 31:2883–2884. Wagner H, Geyer B, Kiso Y, Hikino H, Rao GS. 1986. Coumestans as the main active principles of the liver drugs Eclipta alba and Wedelia calendulacea. Planta Med. 52:370– 374. Yahara S, Ding N, Nohara T. 1994. Oleanane glycosides from Eclipta alba. Chem Pharm Bull. 42:1336– 1338. Yahara S, Ding N, Nohara T, Masuda K, Ageta H. 1997. Taraxastane glycosides from Eclipta alba. Phytochemistry. 40:131–135.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Three new olean-type triterpenoid saponins from aerial parts of Eclipta prostrata (L.) a
a
b
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Feng-Min Xi , Chun-Tong Li , Jun-Ling Mi , Zhi-Jun Wu & WanSheng Chen
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Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, P.R. China b
Department of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, P.R. China Published online: 09 Sep 2013.
To cite this article: Feng-Min Xi, Chun-Tong Li, Jun-Ling Mi, Zhi-Jun Wu & Wan-Sheng Chen (2014) Three new olean-type triterpenoid saponins from aerial parts of Eclipta prostrata (L.), Natural Product Research: Formerly Natural Product Letters, 28:1, 35-40, DOI: 10.1080/14786419.2013.832674 To link to this article: http://dx.doi.org/10.1080/14786419.2013.832674
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Natural Product Research, 2014 Vol. 28, No. 1, 35–40, http://dx.doi.org/10.1080/14786419.2013.832674
Three new olean-type triterpenoid saponins from aerial parts of Eclipta prostrata (L.) Feng-Min Xia, Chun-Tong Lia, Jun-Ling Mib, Zhi-Jun Wua* and Wan-Sheng Chena* a
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Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China; bDepartment of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, P.R. China (Received 3 February 2013; final version received 6 July 2013) Three new olean-type triterpenoid saponins, namely 3-O-(2-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (1), 3-O-(6-O-acetyl-b-D glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (2) and 3-O-(b-D glucopyranosyl) oleanolic acid-28-O-(6-O-acetyl-b-D -glucopyranosyl) ester (3), were isolated from the aerial parts of Eclipta prostrata (L.). Their structures were elucidated based on 1D and 2D NMR and MS spectroscopic data. Keywords: Eclipta prostrata; chemical constituents; triterpenoid saponins
1. Introduction Eclipta prostrata (L.) is an annual herbaceous plant, which is one of four species of genus Eclipta from the family Asteraceae. It is growing widely in the tropical and subtropical areas of the world, and distributed throughout China, which is known as ‘Mohanlian’. The aerial parts of E. prostrata are one important traditional Chinese medicine and mainly used as a tonic for enriching the blood (China Pharmacopoeia Committee 2010). It has attracted a great deal of attention for its wide range of biological activities, such as antiinflammatory (Chandra et al. 1987; Arunachalam et al. 2009), antioxidant (Karthikumar et al. 2007), immunomodulatory (Jayathirthaa & Mishraa 2004), hepatoprotective (Saxena & Singh 1993) and hair growth promoting properties (Roy et al. 2008). Previous phytochemical investigations on E. prostrata revealed that it is rich in bioactive natural products, including triterpenes (Yahara et al. 1994, 1997), coumestans (Wagner et al. 1986), steroids, flavonoids (Sarg et al. 1981), thiopenes (Singh 1988), polyacetylenes (Singh & Bhargava 1922) and alkaloids (Abdel-Kader et al. 1998). As part of an ongoing search for bioactive natural products from traditional folk medicine, we have conducted a phytochemical investigation on the EtOH extract of the aerial parts of E. prostrata. This has led to the isolation of three new olean-type triterpenoid saponins (Figure 1), successively named 3-O-(2-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (1), 3-O-(6-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester (2) and 3-O-(b-D -glucopyranosyl) oleanolic acid-28-O-(6-O-acetyl-b-D glucopyranosyl) ester (3). This paper reports the isolation and structure elucidation of the three compounds.
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
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Figure 1. Structures of compounds 1 – 3.
2. Results and discussion The 80% EtOH extract of air-dried aerial parts of E. prostrata was suspended in H2O and partitioned successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH-soluble extract was subjected to column chromatography (CC) and purified by semi-preparative high-performance liquid chromatography (HPLC) to yield three new terpenoid glycosides (1–3). Compound 1 was obtained as white amorphous powder. The negative HR-ESI-MS exhibited pseudo-molecular ion peak at m/z 883.4707 [M þ COOH]2 (calcd for 883.4698, C45H71O17), consistent with the formula C44H70O15, indicating 108 of unsaturation. The 1H NMR spectrum of 1 showed signals for eight tertiary methyl groups (dH 2.12, 1.81, 1.04, 1.09, 1.02, 0.98, 0.87 and 0.81), an olefinic proton [dH 5.29 (1H, m)] and two anomeric protons [dH 6.28 (1H, d, J ¼ 7.9 Hz) and 4.89 (1H, d, J ¼ 8.0 Hz)]. The 13C NMR spectrum of 1 exhibited 44 carbon resonances, classified into 9 quaternary carbons, 16 methines including 12 oxygen-substituted carbons, 11 methylenes including 2 oxygen-substituted carbons, and 8 methyls. The signals at dC 175.8, 169.9, 144.3, 122.5, 103.7, 95.7 and 21.1 were assigned to a carbonyl carbon (dC 175.8), a pair of olefinic carbons (dC 144.3, 122.5), two anomeric carbons (dC 103.7, 95.7) and an acetyl group (dC 169.9, 21.1). These spectroscopic data suggested that 1 was a typical olean-12-enetype triterpenoid glycoside. The assignment of 1H and 13C NMR spectroscopic data of 1 (see Table S1) was based on 1H – 1H COSY, HSQC and HMBC experiments. The 1H and 13C NMR data of 1 were in good agreement with those of eclalbasaponin I (Figure 1, 1a) (Yahara et al. 1994), except for the additional acetyl group (dC 169.9, 21.2) in 1. The position ester linkage of the acetyl group was confirmed by HMBC spectrum of 1. In the HMBC experiment (Figure S1), the correlation between H-20 (dH 5.53) and acetyl group (dC 169.9) was observed, indicating that the acetyl group was attached at the oxygen atom of C-20 of the b-D -glucopyranosyl. The absolute configurations of glucopyranosyls were further confirmed by acid hydrolysis, followed by chiral GC –MS analysis of trimethylsilylated derivatives of the resultant monosaccharides. The relative configurations of 1 were further confirmed by NOESY experiment, which showed NOE correlations between the following proton pairs: H-3 and H-5, H-23; H-5 and H-9, H-23; H-9 and H-27; H-16 and H-27; H-18 and H-30; H-24 and H-25; H-25 and H-26. Consequently, the structure of 1 was elucidated as 3-O-(2-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O(b-D -glucopyranosyl) ester. Compound 2 was isolated as white amorphous powder. Its negative HR-ESI-MS showed pseudo-molecular ion peaks at m/z 883.4714 [M þ COOH]2 (calcd for 883.4698, C45H71O17), quite close to peak of 1. The same formula C44H70O15 revealed that 1 and 2 were isomers. Comparing the 1H and 13C NMR spectroscopic data of 2 with 1 (Table S1), both were almost superimposable, except for some differences of the signals of Glc-I. The different signals of Glc-I in 2 may result from different substitutional positions of acetyl group. This hypothesis was confirmed unambiguously by the HMBC spectrum of 2. The HMBC experiment showed cross-peaks from
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H-60 (dH 4.89, 4.79) to the acetyl group (dC 170.7). This deduced that the acetyl group was located at the oxygen atom of C-60 of Glc-I. The absolute configurations of glucopyranosyls were further verified by acid hydrolysis and chiral GC–MS analysis. Therefore, the structure of 2 is assigned as 3-O-(6-O-acetyl-b-D -glucopyranosyl) oleanolic acid-28-O-(b-D -glucopyranosyl) ester. Compound 3 was obtained as white amorphous powder. Its molecular formula was determined as C44H70O15 on the basis of the negative HR-ESI-MS (883.4684 [M þ COOH]2, calcd for 883.4698), indicating that 3 was the isomer of 1 and 2. The 1H and 13C NMR spectroscopic data of 3 (Table S1) were quite similar to those of 1a, except that the signals of Glc-II showed some differences. Further analysis indicated that the major differences of signals of Glc-II between 3 and 1a could be explained by substitution of acetyl group. HMBC correlation between H-600 (dH 4.82, 4.73) and the acetyl group (dC 170.5) implied that the acetyl group was located at the oxygen atom of C-600 of Glc-II in 3. Hence, the structure of 3 is determined to be 3O-(b-D -glucopyranosyl) oleanolic acid-28-O-(6-O-acetyl-b-D -glucopyranosyl) ester. The structures of triterpenoid saponins from E. prostrata contain two types: oleanane-type and taraxstane-type, such as eclalbasaponin I–VI (Yahara et al. 1994) and eclalbasaponin VII – X (Yahara et al. 1997). The three new isolated triterpenoid saponins could be classified into olean-12-ene-type, which is common in the genus Eclipta. 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer 341 Polarimeter (Waltham, MA, USA). IR analyses were performed with a NEXUS 470 FT-IR spectrometer (Thermo Nicolet, USA). UV spectra were recorded on Shimadzu UV/VIS-240 recording spectrophotometer (Shimadzu Co., Japan). 1D and 2D NMR spectra were obtained on a Bruker Avance 600 NMR spectrometer (Bruker Co., Germany). HR-ESI-MS were acquired on an Agilent 6220 TOF LC-MS instrument (Agilent Technologies, MA, USA). GC – MS was conducted on a Thermo Finnigan Trace GC apparatus (Thermo Finnigan, CA, USA) using an L-Chirasil-Val column (25 m £ 0.32 mm). CC was performed by using silica gel (100 –200 and 200– 300 mesh), ODS (50 mm; YMC (YMC CO., Ltd, Japan)) and Sephadex LH-20 (40 – 70 mm) (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Semi-preparative HPLC isolation was achieved (Agilent Technologies, MA, USA) with an Agilent 1200 instrument with a refractive index detector, using a C18 column (250 mm £ 10 mm £ 5 mm, YMC) and eluting with MeOH – H2O at 2.0 mL/min. Precoated silica gel GF254 and HF254 plates were used for TLC, and zones were visualised under UV light (254 and 365 nm) or by spraying with 10% H2SO4 – EtOH followed by heating. 3.2. Plant material The plant of E. prostrata was collected from Xianning City, Hubei Province (GPS coordinates: N 29813.4840 , E 113850.7610 ), during July 2011, and identified by Prof. Lian-Na Sun (Department of Pharmacy, Second Military Medical University, Shanghai, China). A voucher specimen (No. 20110828) was deposited at the Department of Pharmacognosy, Second Military Medical University. 3.3. Extraction and isolation The air-dried aerial parts of E. prostrata (15.0 kg) were extracted three times with 80% EtOH under reflux. After removal of the solvent by evaporation in vacuum, the residue was suspended in water (20 L) and then successively partitioned with petroleum ether, EtOAc and n-BuOH, (3 £ 15 L). The n-BuOH-soluble part (550 g) was fractionated by silica gel CC eluted with CH2Cl2 –MeOH (100:1 ! 50:1 ! 10:1 ! 5:1) to give five fractions
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(Fr. 1 –Fr. 5). Fr. 5 (30 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH – H2O (50:1:0.1 ! 30:1:0.1 ! 20:1:0.1 ! 10:1:0.1) to afford eight subfractions (Fr. 5.1– Fr. 5.8). Fr. 5.3 (5.1 g) was applied to ODS CC eluted with MeOH –H2O (25% ! 50% ! 75%) to afford four subfractions (Fr. 5.3.1 –Fr. 5.3.4). Fr. 5.3.2 (1.0 g) was purified by semi-preparative HPLC (45% MeOH – H2O, 2 mL/min) to yield compounds 1 (32.1 mg, tR ¼ 35.4 min) and 2 (48.9 mg, tR ¼ 38.3 min). Fr. 5.4 (2.5 g) was subjected to Sephadex LH-20 eluted with MeOH –H2O (1:1) to yield four subfractions (Fr. 5.4.1 –Fr. 5.4.4). Fr. 5.4.2 (0.3 g) was purified by semi-preparative HPLC (40% MeOH –H2O, 2 mL/min) to give compound 3 (5.8 mg, tR ¼ 49.5 min).
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3.4. Acid hydrolysis of compounds Compounds 1, 2 and 3 (each 2.0 mg) were separately heated in 2 M HCl (1 mL) at 1208C for 1.0 h. The mixture was concentrated and the residue was dissolved in 1-trimethylsilyl imidazole and pyridine (0.2 mL), and the solution was stirred at 608C for 5 min. After drying the solution, the residue was partitioned between CH2Cl2 and H2O (1 mL, 1:1 v/v). The organic phase was submitted to GC – MS analysis using an L-Chirasil-Val column (0.32 mm £ 25 m). Temperatures of the injector and detector were 2008C for both. A temperature gradient system was used for the oven, starting at 1008C for 1 min and increasing up to 1808C at a rate of 58C/ min. The authentic samples were treated in the same manner with 1-trimethylsilyl imidazole in pyridine. Only D -glucose was identified by comparing the retention time with those of authentic D -glucose (tR ¼ 14.70 min) after treatment using the same method. 3.5. Characterisation of compounds Compound 1: amorphous powder; ½a25 D þ 71:6 (c ¼ 0.5, MeOH); IR (KBr) nmax 3434, 2946, 2881, 1732, 1640, 1464, 1389, 1376, 1308, 1248, 1167, 1077, 1028 cm211; 1H NMR (pyridined5, 600 MHz): d: 0.75 (1H, dd, J ¼ 12.0, 1.0 Hz, H-5), 0.81 (3H, s, H-25), 0.87 (3H, s, H-24), 0.89 (1H, m, H-1b), 0.98 (3H, s, H-29), 1.02 (3H, s, H-30), 1.04 (3H, s, H-23), 1.09 (3H, s, H-26), 1.23 (1H, m, H-21b), 1.27 (1H, m, H-6b), 1.35 (1H, m, H-19b), 1.35 (1H, m, H-7b), 1.40 (1H, m, H-1a), 1.43 (1H, m, H-6a), 1.57 (1H, m, H-7a), 1.72 (1H, m, H-2b), 1.73 (1H, m, H-9), 1.77 (1H, m, H-15b), 1.81 (3H, s, H-27), 1.94 (2H, m, H-11), 2.07 (1H, m, H-22b), 2.11 (1H, dd, J ¼ 13.2, 4.0 Hz, H-2a), 2.12 (3H, s, H-Ac), 2.37 (1H, m, H-22a), 2.41 (1H, m, H-21a), 2.53 (1H, dd, J ¼ 12.0, 2.6 Hz, H-15a), 2.77 (1H, t, J ¼ 13.7 Hz, H-19a), 3.22 (1H, dd, J ¼ 11.7, 4.4 Hz, H-3), 3.48 (1H, dd, J ¼ 13.7, 3.9 Hz, H-18), 3.97 (1H, m, H-50 ), 4.01 (1H, m, H-500 ), 4.12 (1H, t, J ¼ 8.4 Hz, H-200 ), 4.18 (1H, m, H-40 ), 4.24 (1H, m, H-300 ), 4.26 (1H, m, H-400 ), 4.27 (1H, m, H-30 ), 4.32 (1H, m, H-60 b), 4.35 (1H, m, H-600 b), 4.42 (1H, m, H-600 a), 4.53 (1H, m, H-60 a), 4.89 (1H, d, J ¼ 8.0 Hz, H-10 ), 5.29 (1H, br s, H-16a), 5.53 (1H, t, J ¼ 8.0 Hz, H-20 ), 5.58 (1H, t, J ¼ 3.0 Hz, H-12), 6.28 (1H, d, J ¼ 8.4 Hz, H-100 ). 13C NMR (pyridine-d5, 150 MHz): d: 38.4 (C-1), 26.1 (C-2), 88.9 (C-3), 38.9 (C-4), 55.5 (C-5), 18.3 (C6), 33.2 (C-7), 39.9 (C-8), 47.0 (C-9), 36.8 (C-10), 23.6 (C-11), 122.5 (C-12), 144.3 (C-13), 41.9 (C-14), 35.9 (C-15), 74.2 (C-16), 48.9 (C-17), 41.1 (C-18), 47.0 (C-19), 30.6 (C-20), 35.7 (C-21), 32.0 (C-22), 27.7 (C-23), 16.6 (C-24), 15.4 (C-25), 17.3 (C-26), 27.0 (C-27), 175.8 (C-28), 33.0 (C-29), 24.4 (C-30), 103.7 (C-10 ), 75.5 (C-20 ), 76.0 (C-30 ), 71.6 (C-40 ), 78.2 (C-50 ), 62.4 (C-60 ), 95.7 (C-100 ), 73.9 (C-200 ), 78.6 (C-300 ), 70.9 (C-400 ), 79.1 (C-500 ), 62.0 (C-600 ), 169.9 (C-Ac), 21.1 (C-Ac). HRESI-MS m/z 883.4707 [M þ COOH]2 (calcd for 883.4698, C45H71O17). Compound 2: amorphous powder; ½a25 D þ 74:8 (c ¼ 0.5, MeOH); IR (KBr) nmax 3436, 2945, 2926, 1735, 1637, 1460, 1389, 1370, 1306, 1247, 1168, 1077, 1034 cm21; 1H NMR (pyridine-d5, 600 MHz): d: 0.80 (1H, dd, J ¼ 12.1, 0.8 Hz, H-5), 0.87 (3H, s, H-25), 0.93 (3H, s, H-24), 0.97 (3H, s, H-29), 1.01 (3H, s, H-30), 1.04 (1H,dd, J ¼ 14.1, 3.2 Hz, H-1b), 1.09 (3H, s, H-26), 1.21 (1H, m, H-21b), 1.24 (3H, s, H-23), 1.25 (1H, m, H-6b), 1.35 (1H, m, H-19b), 1.35
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(1H, m, H-7b), 1.42 (1H, m, H-6a), 1.57 (1H, m, H-7a), 1.58 (1H, m, H-1a), 1.76 (1H, m, H-15b), 1.78 (1H, m, H-9), 1.81 (3H, s, H-27), 1.86 (1H, m, H-2b), 1.97 (2H, m, H-11), 2.06 (1H, m, H-22b), 2.12 (3H, s, H-Ac), 2.22 (1H, dd, J ¼ 13.3, 3.6 Hz, H-2a), 2.34 (1H, m, H-22a), 2.38 (1H, m, H-21a), 2.52 (1H, dd, J ¼ 11.7, 2.8 Hz, H-15a), 2.77 (1H, t, J ¼ 13.6 Hz, H-19a), 3.22 (1H, dd, J ¼ 11.7, 4.3 Hz, H-3), 3.48 (1H, dd, J ¼ 13.6, 4.0 Hz, H-18), 3.97 (1H, m, H-50 ), 3.98 (1H, m, H-40 ), 3.98 (1H, m, H-500 ), 4.02 (1H, m, H-20 ), 4.10 (1H, t, J ¼ 8.4 Hz, H-200 ), 4.18 (1H, m, H-30 ), 4.23 (1H, m, H-300 ), 4.26 (1H, m, H-400 ), 4.33 (1H, dd, J ¼ 11.8, 4.5 Hz, H-600 b), 4.41 (1H, dd, J ¼ 11.8, 2.2 Hz, H-600 a), 4.79 (1H, dd, J ¼ 11.4, 6.4 Hz, H-60 b), 4.83 (1H, d, J ¼ 7.8 Hz, H-10 ), 4.89 (1H, dd, J ¼ 11.4, 1.8 Hz, H-60 a), 5.28 (1H, br s, H-16a), 5.59 (1H, t, J ¼ 3.5 Hz, H-12), 6.26 (1H, d, J ¼ 8.4 Hz, H-100 ). 13C NMR (pyridine-d5, 150 MHz): d: 38.7 (C-1), 26.4 (C-2), 89.1 (C-3), 39.2 (C-4), 55.8 (C-5), 18.3 (C-6), 33.2 (C-7), 39.9 (C-8), 47.0 (C-9), 36.8 (C-10), 23.6 (C-11), 122.5 (C-12), 144.2 (C-13), 41.9 (C-14), 35.8 (C-15), 74.1 (C-16), 48.9 (C-17), 41.1 (C-18), 47.0 (C-19), 30.6 (C-20), 35.7 (C-21), 32.0 (C-22), 27.9 (C-23), 16.7 (C-24), 15.4 (C-25), 17.3 (C-26), 27.0 (C-27), 175.8 (C-28), 33.0 (C-29), 24.4 (C-30), 106.7 (C-10 ), 75.3 (C-20 ), 78.2 (C-30 ), 71.5 (C-40 ), 74.6 (C-50 ), 64.6 (C-60 ), 95.6 (C-100 ), 73.9 (C-200 ), 78.5 (C-300 ), 70.8 (C-400 ), 79.1 (C-500 ), 61.9 (C-600 ), 170.7 (C-Ac), 20.6 (C-Ac). HRESI-MS m/z 883.4714 [M þ COOH]2 (calcd for 883.4698, C45H71O17). Compound 3: amorphous powder; ½a25 D þ 75:0 (c ¼ 0.2, MeOH); IR (KBr) nmax 3411, 2944, 2873, 1732, 1456, 1388, 1363, 1233, 1165, 1076, 1028 cm21; 1H NMR (pyridine-d5, 600 MHz): d: 0.81 (1H, dd, J ¼ 12.8, 1.0 Hz, H-5), 0.91 (3H, s, H-25), 0.93 (1H,dd, J ¼ 14.1, 3.2 Hz, H-1b), 0.98 (3H, s, H-29), 1.00 (3H, s, H-24), 1.03 (3H, s, H-30), 1.11 (3H, s, H-26), 1.24 (1H, m, H-21b), 1.29 (3H, s, H-23), 1.31 (1H, m, H-6b), 1.34 (1H, m, H-19b), 1.38 (1H, m, H-7b), 1.45 (1H, m, H-6a), 1.47 (1H, m, H-1a), 1.59 (1H, m, H-7a), 1.77 (1H, m, H-15b), 1.77 (1H, m, H-9), 1.82 (1H, m, H-2b), 1.83 (3H, s, H-27), 1.88 (3H, s, H-Ac), 1.97 (2H, m, H-11), 2.16 (1H, m, H-22b), 2.24 (1H, dd, J ¼ 13.6, 3.6 Hz, H-2a), 2.35 (1H, m, H-22a), 2.40 (1H, m, H-21a), 2.51 (1H, dd, J ¼ 15.3, 3.1 Hz, H-15a), 2.78 (1H, t, J ¼ 13.6 Hz, H-19a), 3.40 (1H, dd, J ¼ 11.7, 4.3 Hz, H-3), 3.51 (1H, dd, J ¼ 13.6, 4.5 Hz, H-18), 4.00 (1H, m, H-50 ), 4.03 (1H, m, H-20 ), 4.07 (1H, m, H-400 ), 4.09 (1H, m, H-500 ), 4.15 (1H, t, J ¼ 8.4 Hz, H-200 ), 4.20 (1H, m, H-40 ), 4.23 (1H, m, H-300 ), 4.24 (1H, m, H-30 ), 4.40 (1H, dd, J ¼ 11.5, 5.5 Hz, H-60 b), 4.57 (1H, dd, J ¼ 11.5, 2.3 Hz, H-60 a), 4.73 (1H, dd, J ¼ 11.8, 5.3 Hz, H-600 b), 4.82 (1H, dd, J ¼ 11.8, 1.7 Hz, H-600 a), 4.93 (1H, d, J ¼ 7.7 Hz, H-10 ), 5.25 (1H, br s, H-16a), 5.60 (1H, t, J ¼ 3.2 Hz, H-12), 6.25 (1H, d, J ¼ 8.4 Hz, H-100 ). 13C NMR (pyridine-d5, 150 MHz): d: 38.6 (C-1), 26.3 (C-2), 88.6 (C-3), 39.2 (C-4), 55.6 (C-5), 18.2 (C-6), 33.2 (C-7), 39.8 (C-8), 46.9 (C-9), 36.7 (C-10), 23.5 (C-11), 122.5 (C-12), 144.1 (C-13), 41.8 (C-14), 35.8 (C-15), 74.1 (C-16), 48.9 (C-17), 41.0 (C-18), 46.9 (C-19), 30.5 (C-20), 35.6 (C-21), 31.9 (C-22), 27.9 (C-23), 16.8 (C-24), 15.4 (C-25), 17.3 (C-26), 26.9 (C-27), 175.8 (C-28), 32.9 (C-29), 24.3 (C-30), 106.6 (C-10 ), 75.5 (C-20 ), 78.3 (C-30 ), 71.5 (C-40 ), 78.4 (C-50 ), 62.7 (C-60 ), 95.3 (C-100 ), 73.7 (C-200 ), 78.0 (C-300 ), 70.7 (C-400 ), 75.8 (C-500 ), 64.1 (C-600 ), 170.5 (C-Ac), 20.4 (C-Ac). HR-ESI-MS m/z 883.4684 [M þ COOH]2 (calcd for 883.4698, C45H71O17). Supplementary material Supplementary material relating to this article is available online, alongside Table S1 and Figure S1 and 1D, 2D NMR spectra and HR-ESI-MS, IR data of compounds 1– 3. Acknowledgements The work was financially supported by the National Natural Science Foundation of the People’s Republic of China (Grant No. 81274032) and the Scientific Foundation of Shanghai (Grant Nos 11DZ1971301 and 08DZ1971600).
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