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The novel indole glucoalkaloid and secoiridoid glucoside from Tripterospermum chinense
Phytochemistry Letters 35 (2020) 191–196
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The novel indole glucoalkaloid and secoiridoid glucoside from Tripterospermum chinense
T
Tao Zhanga,b,1, Chuan-Jiang Mac,1, Yong-Li Weic, Jin-Guang Sia, Lu Fua, Jun-Xing Dongb,*, Zhong-Mei Zoua,* a
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China Beijing Institute of Radiation Medicine, Beijing 100850, PR China c The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Tripterospermumcin F Tripterospermumcin G Tripterospermum chinense NMR
Tripterospermumcin F (1), a novel indole glucoalkaloid, and tripterospermumcin G (2), a new iridoid tetramer, were isolated from the aerial parts of Tripterospermum chinense. Their structures were determined by analysis of 1D and 2D NMR data, as well as by comparison with model compounds. Their cytotoxicities were tested, but neither of them showed obvious activity.
1. Introduction The genus Tripterospermum (Entianaceae) consists of approximately 17 plant species in Asia. In this genus, approximately 15 species and one variety grow in mainland China (He, 1988). The genus is rich in iridoid glucosides, and xanthones, revealing diverse biological activities, such as antivirus, and anti-hypertension (Zhu et al., 2007a; Fang et al., 2008; Zhu et al., 2007b; Kawa et al., 1988; Calis and Sticher, 1984; Chen et al., 1992; Hsu et al., 1997). Tripterospermum chinense (Migo) H. Smith is widely distributed in South China, and traditionally used for the treatment of cough, haemoptysis, and pulmonary disease (Editorial board of Chinese Materia Medica, 1999). The constituents of this plant have been previously investigated, including iridoid glucosides, flavonoids and xanthones (Zhu et al., 2007a; Fang et al., 2008; Zhu et al., 2007b). Our previous study led to the isolation of seven iridoid glucosides from the aerial parts of T. chinense (Zhang et al., 2012). As a part of our ongoing search for new/novel and bioactive products from the medicinal plants in China, two new compounds, tripterospermumcins F and G (Fig. 1), were isolated from the aerial parts of T. chinense. In this paper, the isolation, structural elucidation and bioactive evaluation of the new compounds are discussed. 2. Results and discussion Compound 1 was isolated as a white amorphous powder. The molecular formula was assigned as C44H57O20N2 on the basis of the
positive-ion HRESIMS ion at m/z 933.3488 [M+H]+, together with its 1 H and 13C NMR data (Table 1). The IR spectrum showed the presence of the hydroxyl/amino groups (3422 cm−1), and α, β-unsaturated ester carbonyl groups (1698 and 1629 cm−1). The 1H and 13C NMR spectra of 1 also showed two α, β-unsaturated ester carbonyl moieties at δH 7.65 (1H, s, H-3a1) and 7.64 (1H, s, H-17a2), δC 152.9 (C-3a1), 110.3 (C4a1), 167.4 (C-11a1), 154.2 (C-17a2), 110.5 (C-16a2) and 168.1 (C22a2); two sets of anomeric signals at δH 5.38 (1H, d, J = 7.8 Hz, H-1 of Glc1) and 5.22 (1H, d, J = 7.8 Hz, H-1 of Glc2), δC 100.7 (C-1 of Glc1) and 100.5 (C-1 of Glc2); two sets of terminal vinyl carbons at δC 134.9 (C-8a1), 119.2 (C-10a1), 135.2 (C-18a2), and 118.5 (C-20a2); two acetal moieties at δH 5.87 (1H, d, J = 6.6 Hz, H-1a1) and 5.71 (1H, d, J = 7.8 Hz, H-21a2), δC 97.0 (C-1a1) and 96.5 (C-21a2); four aromatic protons at δH 7.26 (1H, m, H-9a2), 7.24 (1H, m, H-10a2), 7.74 (1H, m, H-11a2) and 7.47 (1H, m, H-12a2); six aromatic carbons at δC 128.2 (C-8a2), 121.4 (C-9a2), 119.4 (C-10a2), 118.3 (C-11a2), 111.7 (C-12a2), and 137.6 (C-13a2); one carboxyl carbon at δC 175.7 (C-23a2); two methines at δH 4.13 (1H, m, H-5a2) and 4.50 (1H, br d, J = 10.8 Hz, H-3a2), δC 57.6 (C-5a2) and 51.3 (C-3a2). Acid hydrolysis of 1 with 2 M CF3COOH and GCeMS analysis provided D-glucose. The large coupling constants (3J1,2 > 7 Hz) were consistent with the β configuration of the two sugars (Zhu et al., 2007a; Xin et al., 2008; Aquino et al., 1994; Paul et al., 2003; Cardoso et al., 2004, 2003; Berger et al., 2012; Ferrari et al., 1986). These data indicated that the structure of 1 was similar to 5αcarboxystrictosidine except for the unit of tripterospermumcin B in 1 instead of a secoiridoid glucoside moiety (Zhu et al., 2007b; Zhang
Corresponding authors. E-mail addresses: [email protected] (J.-X. Dong), [email protected] (Z.-M. Zou). 1 These authors contributed equally to this study. ⁎
https://doi.org/10.1016/j.phytol.2019.12.005 Received 10 March 2019; Received in revised form 26 November 2019; Accepted 10 December 2019 1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
Phytochemistry Letters 35 (2020) 191–196
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Fig. 1. Structures of compounds 1–2.
et al., 2012; Aquino et al., 1994). This observation was confirmed by analysis of relevant 1H-1H COSY data. In the 1H-1H COSY spectrum, a strong cross-peak between the methine proton δH 4.50 (1H, br d, J = 10.8 Hz, H-3a2) and one of the methylene protons at C-14a2 (δH 2.27, 2.08) suggested that tripterospermumcin B unit was linked to indole monoterpenoid alkaloid (units A and B) (Aquino et al., 1994; Paul et al., 2003). The HMBC correlation from δH 4.50 (H-3a2) to δC 31.9 (C-15a2) was also observed to support the conclusion. The 1H-1H COSY spectrum (Fig. 2) showed two partial structure sequences for aglycone of unit A: CH2(14a2)CH(15a2)CH(19a2)CH(21a2)/CH(18a2)CH2(20a2) and CH2(7a1)CH2(6a1)CH(5a1)CH(9a1)CH(1a1)/CH(8a1)CH2(10a1). The C–C interconnectivity of all fragments was established from the HMBC spectrum (Fig. 2) as correlations of H-14a2 with C-16a2, H-17a2 and H7a1 with C-22a2, H-3a1 and H-12a1 with C-11a1, and H-6a1 with C-4a1. The HMBC correlations between δH 5.38 (H-1 of Glc1) and δC 97.0 (C1a1), and δH 5.22 (H-1 of Glc2) and δC 96.5 (C-21a2) allowed us to
identify linkage sites of D-glucose. On the basis of these data, the planar structure of 1 was established. The relative configuration of 1 was determined by analysis of the NOESY data (Fig. 3), as well as by comparison with known compounds. Comparison of the NMR data of unit A in 1 with those of tripterospermumcin B indicated that they have the same relative configuration (Zhu et al., 2007a, b; Zhang et al., 2012; Tian et al., 2006). The NOESY spectrum confirmed the above conclusion. The NOE correlations of H-6a1/H-1a1, H-8a1/H-1a1, H-14a2/H-21a2 and H-18a2/H-21a2 indicated they were cofacial and were arbitrarily assigned as α-orientations, whereas the correlations of H-5a1/H-9a1 and H-15a2/H-19a2 showed their β-orientations. The similarities between the chemical shifts of C-2a2, C-3a2, C-5a2 and C-6a2 in 1 and 5α-carboxystrictosidine suggested the same configuration for these carbons in the two alkaloids (Xin et al., 2008; Aquino et al., 1994; Paul et al., 2003; Cardoso et al., 2004, 2003; Berger et al., 2012; Ferrari et al., 1986; Aimi et al., 1992;
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Table 1 1 H (600 MHz, δ in ppm, J in Hz) and compound 1 in pyridine-d5.
13
No.
δH
δC
No.
1a1 3a1 4a1 5a1 6a1 7a1 8a1 9a1 10a1
5.87 d (6.6) 7.65 s
97.0 152.9 110.3 30.4 29.4 63.2 134.9 44.4 119.2
2a2 3a2 5a2 6a2 7a2 8a2 9a2 10a2 11a2
3.60 s
167.4 51.2
5.38 d (7.8) 3.97–4.08 o 4.23–4.30 o 4.23–4.30 o 3.97–4.08 o 4.34–4.41 o, 4.54 m
100.7 74.7 78.5 71.5 79.2 62.6
12a2 13a2 14a2 15a2 16a2 17a2 18a2 19a2 20a2
11a1 12a1 Glc1 1 2 3 4 5 6
3.12 m 1.87 m, 2.21 o 4.38 o, 4.28 o 5.83 m 2.76 m 5.13–5.19 o, 5.11 d (10.2)
21a2 22a2 23a2 Glc2 1 2 3 4 5 6 a
spectrum, supporting a β-oriented H-7a2 of 2. Thus, the structure of 2 was defined as shown, named tripterospermumcin G. Compounds 1 and 2 were evaluated for their cytotoxic activity against human cervical (HeLa), colon (LoVo), stomach (BGC-823), and breast cancer (MCF-7) cell lines. But none of them showed any significant activity at the concentration of 40 μM (See Table 1 in the Supporting Information).
C NMR (150 MHz, δ in ppm) data of δH 4.50 br d (10.8) 4.13 m 3.30–3.34 oa, 3.63 m 7.26 m 7.24 m 7.74 m 7.47 m 2.27 o,2.08 m 3.30–3.34 o 7.64 s 5.69 m 2.62 m 5.13–5.19 o, 4.95 d (10.8) 5.71 d (7.8)
5.22 d (7.8) 3.97–4.08 o 4.34–4.41 o 4.23–4.30 o 3.97–4.08 o 4.34–4.41 o, 4.54 m
δC 137.4 51.3 57.6 26.5 108.2 128.2 121.4 119.4 118.3
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Perkin-Elmer 343 polarimeter (PerkinElmer, Waltham, MA, USA). IR spectra were recorded on the Bio-Rad FTS-65A spectrometer (Bio-Rad, Richmond, VA, USA). UV spectra were recorded on Shimadzu UV-2501 PC (Shimadzu, Kyoto, Japan). The NMR spectra were recorded with a Varian UNITYINOVA 600 (Varian, Palo Alto, CA, USA), and the chemical shifts were given on δ (ppm) scale with tetramethylsilane as an internal standard. The HRESIMS was recorded on a 9.4 T Q-FT-MS Apex Qe (Bruker Co. Billerica, MA, USA). ESIMS was recorded on Thermo Finnigan Advantage MAX HPLC-MS (Thermo Electron, Pittsburgh, PA, USA). Macroporous resin AB-8 (Nan Kai College Chemical Inc, Tianjin, China), silica-gel (Qingdao Marine Chemistry Factory, Qingdao, China), Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and ODS silica-gel (120 Å, 50 μm, YMC, Kyoto, Japan) were used for column chromatography. HPLC was performed using Waters 600E system (Waters, Milford, MA, USA): an analytical column, ODS (5 μm, 4.6 × 250 mm, Hanbon Sci.& Tech, Huaian, China); preparative column, a YMC C18 (5 μm, 20.0 × 250 mm, YMC, Kyoto, Japan); detector, Knauer RID (refractive index detector, Knauer, Berlin, Germany) and Alltech ELSD (evaporative light-scattering detector, Alltech, Los Angeles, CA, USA) 2000ES. Gas chromatographic analysis was performed with an Agilent 6890 Series, gas chromatograph equipped with an H2 flameionization detector. The column was an HP-5 capillary column (30 m × 0.25 mm × 0.25 μm) (Agilent, Santa Clara, CA, USA).
111.7 137.6 38.7 31.9 110.5 154.2 135.2 44.9 118.5 96.5 168.1 175.7 100.5 74.7 78.4 71.6 79.0 62.8
Overlapped with other signals.
Brandt et al., 1999).· The NOE correlation of H-3a2/H-5a2 supported the configuration. Thus, the structure of 1 was determined as shown, named tripterospermumcin F. Compound 2, a white amorphous powder, possessed molecular formula of C69H100O39 based on the HRESIMS ion at m/z 1575.5723 [M +Na]+. Its IR spectrum showed hydroxyl (3426 cm−1) and α, β-unsaturated ester carbonyl (1705 and 1627 cm−1) absorptions. The 1H NMR spectrum of 2 indicated the presence of olefinic protons of the enol at δH 7.47 (2H, s), 7.39 (1H, s), and 7.37 (1H, s). The 13C NMR spectrum showed 69 carbon signals, in which the characteristic carbon signals at δC 98.6 × 2, 98.7 and 98.9 were readily assigned. In the 13C NMR spectrum, four α, β-unsaturated ester carbonyl carbons at δC 166.6 × 2, 165.9 × 2, 152.0 × 2, 151.7, 151.5, 109.9, 109.8, and 109.6 × 2, and four sets of terminal vinyl carbons at δC 134.6 × 2, 134.4 × 2, 119.2 × 2, and 118.9 × 2 were characteristic for four secoiridoid glucoside units. Acid hydrolysis of 2 with 2 M CF3COOH and GC–MS analysis gave D-glucose. These data implied that 2 is an iridoid tetramer including two tripterospermumcin B units (units A and B) (Zhu et al., 2007a, b; Zhang et al., 2012; Tian et al., 2006). The HMBC correlation from δ 4.51 (H-7a2) with δ 64.3 (C-6 of Glc4) were observed to support the gross structure as shown. The relative configuration of 2 was elucidated by NOESY analysis. Almost identical NMR data indicate that the units A and B of 2, and tripterospermumcin B share the same relative configuration (Zhu et al., 2007a, b; Zhang et al., 2012; Tian et al., 2006). The NOE correlations of H-7a2/H-12a2, H-7a2/H-6 (Glc4), and H-7a2/H-5a2, indicated that H-7a2 had β-orientations. The correlation between H-12a2 and H-6 of Glc4 was not observed in the NOESY
3.2. Plant material The aerial parts of Tripterospermum chinense were collected from Yongshun region of Hunan Province, China in April 2010. The plant was identified by prof. Bin Li (Beijing Institute of Radiation Medicine) and a voucher specimen (No. 100403) is deposited in the Herbarium of Beijing Institute of Radiation Medicine, Beijing. 3.3. Extraction and isolation The aerial parts of T. chinense (60 kg) were refluxed three times with 60 % aqueous EtOH (3 × 480 L, each for 1.5 h). The combined extract was concentrated under reduced pressure to furnish a dark brown residue (1800 g), which was suspended in H2O and partitioned in turn with CHCl3 and n-BuOH. The n-BuOH extract was evaporated under reduced pressure to yield a residue (386 g). The latter was separated chromatographically on macroporous resin AB-8 (15 × 100 cm) and eluted with a gradient mixture of EtOH–H2O (1:5, 1:1 and 9:1; 60,000 mL each), to give five fractions (A–E). Fraction C (134 g) was separated chromatographically on silica-gel column (12 × 50 cm) with a gradient mixture of CHCl3–MeOH (20:1, 10:1, 5:1, 5:2 and 1:1) as eluent, and total 160 tubes (1000 mL each) were collected. Tubes 70–75 (11 g) were individually subjected to a series of purification steps using Sephadex LH-20 column chromatography (MeOH), ODS silica-gel
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Fig. 2. Key 1H-1H COSY and HMBC correlations of 1–2.
column chromatography (3 × 50 cm; MeOH–H2O, 40: 60 and 70: 30), and finally purified by preparative HPLC (MeOH–H2O, 55: 45, flow rate: 8.0 mL/min) to afford 2 (10 mg, tR: 12.30 min). Tubes 118–127 (4 g) were individually subjected to a series of purification steps using Sephadex LH-20 column chromatography (MeOH), ODS silica-gel column chromatography (3 × 50 cm; MeOH–H2O, 40: 60 and 70: 30), and finally purified by preparative HPLC (MeOH–H2O, 55: 45, flow rate: 8.0 mL/min) to afford 1 (12 mg, tR: 22.23 min). Tripterospermumcin F(1): white amorphous powder (MeOH–H2O, 55:45); [α]D20 −191.3 (c 0.08, MeOH); UV (MeOH) λmax (logε): 235 (3.99), 285 (4.15) nm, IR (KBr) νmax: 3422, 2926, 1698, 1629, 1654, 1289, 1075 cm−1; HRESIMS (pos.): m/z 933.3488 [M+H]+ (calcd for C44H57O20N2, 933.3499); 1H and 13C NMR data, see Table 1. Tripterospermumcin G(2): white amorphous powder (MeOH–H2O, 55:45); [α]D20 −79.3 (c 0.05, MeOH); UV (MeOH) λmax (logε): 234 (3.60) nm, IR (KBr) νmax: 3427, 2929, 1705, 1628, 1654, 1286, 1075 cm−1; HRESIMS (pos.): m/z 1575.5723 [M+Na]+ (calcd for C69H100O39Na, 1575.5734); 1H and 13C NMR data, see Table 2.
3.4. Acid hydrolysis and sugar analysis Compounds 1–2 (2.0 mg each) were individually hydrolyzed in 2 M CF3COOH (2 mL) at 95 °C for 3 h. The reaction mixture was extracted with CH2Cl2 (5 mL) three times. The aqueous layer was repeatedly evaporated to dryness with EtOH until neutral. In the monosaccharide mixture, glucose was detected by TLC on a silica gel [using nBuOH–EtOAc–C5H5N–H2O (6:1:5:4) as development] by comparison with authentic sample: glucose (Rf 0.46). The residue of the sugars was dissolved in anhydrous pyridine (2 mL), 12 mg L-cysteine methyl ester hydrochloride was added, and the mixture was stirred at 60 °C for 1 h. Then, HMDS–TMCS (hexamethyldisilazane- trimethylchlorosilane 2:1) (0.6 mL) was added, and the mixture was kept at 60 °C for 0.5 h (Mitaine-Offer et al., 2010). The supernatant was analyzed by GC under the following conditions: column temperature: 180 °C/250 °C; programmed increase, 15 °C/min; carrier gas: N2 (1 mL/min); injection and detector temperature: 250 °C; injection volume: 4.0 μL, and split ratio: 1/50. The derivative of D-glucose was detected with tR values of 17.93 min.
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Table 2 1 H (600 MHz, δ in ppm, J in Hz) and compound 2 in DMSO-d6.
13
C NMR (150 MHz, δ in ppm) data of
No.
δH
δC
No.
δH
δC
1a1 3a1 4a1 5a1 6a1 7a1 8a1 9a1 10a1 11a1 12a1 Glc1 1 2 3 4 5 6 1b1 3b1 4b1 5b1 6b1 7b1 8b1 9b1 10b1 11b1 12b1
5.48 m 7.47 s
95.6 152.0 109.6 30.2 28.9 62.0 134.6 43.1 118.9 166.6 51.1
1a2 3a2 4a2 5a2 6a2 7a2 8a2 9a2 10a2 11a2 12a2 Glc2 1 2 3 4 5 6 1b2 3b2 4b2 5b2 6b2 7b2 8b2 9b2 10b2 11b2 12b2 13b2 Glc4 1 2 3 4 5 6
5.42 m 7.39 s
95.7 151.7 109.8 27.6 32.3 101.4 134.4 43.1 119.2 165.9 52.0
Glc3 1 2 3 4 5 6 a
2.75–2.78 oa 1.76 o, 1.84 o 4.07 o, 3.99 o 5.70 m 2.57 o 5.22 o, 5.32 o 3.60 s 4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.15–3.18 o 3.68 o, 3.41 o 5.48 m 7.47 s
3.60 s
98.6 73.0 77.3 70.0 76.6 61.1 95.6 152.0 109.6 30.2 28.9 62.0 134.6 43.1 118.9 166.6 51.1
4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.15–3.18 o 3.68 o, 3.41 o
98.6 73.0 77.3 70.0 76.6 61.1
2.75–2.78 o 1.76 o, 1.84 o 4.07 o, 3.99 o 5.70 m 2.57 o 5.22 o, 5.32 o
2.75–2.78 o 1.54 m, 1.90 o 4.51 o 5.62 m 2.57 o 5.22 o, 5.32 o 3.16 br s 4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.15–3.18 o 3.68 o, 3.41 o 5.36 m 7.37 s
98.9 72.8 77.3 69.8 76.6 61.0
3.16 br s 3.16 br s
151.5 109.9 27.6 31.6 101.9 134.4 43.1 119.2 165.9 51.7 53.2
4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.31 o 3.99 o, 3.41 o
98.7 72.9 77.3 69.8 75.4 64.3
2.75–2.78 o 1.44 m, 1.95 o 4.38 m 5.62 m 2.57 o 5.22 o, 5.32 o
Overlapped with other signals.
Compound 1 is the first report of terpenoid indole alkaloids incorporating a secoiridoid dimeric unit. To the best of our knowledge, compound 2 is the fourth-reported iridoid tetramers with four glucosides (Zhang et al., 2012; Ferrari et al., 1986). The planar structures and relative configurations of 1 and 2 were determined by analysis of NMR and NOESY data in the present paper. As compounds 1 and 2 were not suitable functional groups for chemical reactions or suitable crystal for X-ray diffraction, their absolute configurations were not determined and further study was required. Our results not only enrich the chemical diversity of secondary metabolites from the genus Tripterospermum, but also emphasize the importance of chemotaxonomic data to help better understand the morphoanatomical classification of this complex plant family.
Fig. 3. Selected NOESY correlations of 1–2.
3.5. Cytotoxicity assays The Hela, LoVo, BGC-823 and MCF-7 cell lines were obtained from the National Infrastructure of Cell Line Resource (Beijing, China). The assay was run in triplicate. In a 96-well plate, each well was plated with 2 × 104 cells. After cell attachment overnight, the medium was removed, and each well was treated with 100 μL of medium containing 0.1 % DMSO or different concentrations of the test compounds and the positive control cis-platin. The plate was incubated for 4 days at 37 °C in a humidified, 5 % CO2 atmosphere. Cytotoxicity was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Wang et al., 2005). After addition of 10 μL MTT solution (5 mg/mL), cells were incubated at 37 °C for 4 h. After adding 150 μL DMSO, cells were shaken to mix thoroughly. The absorbance of each well was measured at 540 nm in a Multiscan photometer. The IC50 values were calculated by Origin software.
Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements
4. Conclusion
This work was financially supported by the Chinese National S&T Special Project on Major New Drug Innovation (2017ZX09301059), the Natural Sciences Foundation of Beijing (7194297), the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1-010), and
Two new compounds, tripterospermumcin F (1) and tripterospermumcin G (2) were isolated from the aerial parts of T. chinense.
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Peking Union Medical College Discipline Construction Project (201920100901).
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Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.12.005. References Aimi, N., Seki, H., Sakai, S.I., 1992. Synthesis of lyaloside, a prototypal β-carboline glucoindole alkaloid in rubiaceous plants. Chem. Pharm. Bull. 40, 2588–2590. https:// doi.org/10.1248/cpb.40.2588. Aquino, R., Garofalo, L., de Tommasi, N., de Ugaz, O.L., Pizza, C., 1994. Glucoindole alkaloids from bark of two Sickingia species. Phytochemistry. 37, 1471–1475. https:// doi.org/10.1016/S0031-9422(00)90436-9. Berger, A., Fasshuber, H., Schinnerl, J., Brecker, L., Greger, H., 2012. Various types of tryptamine-iridoid alkaloids from Palicourea acuminate (=Psychotria acuminata, Rubiaceae). Phytochem. Lett. 5, 558–562. https://doi.org/10.1016/j.phytol.2012.05. 013. Brandt, V., Tits, M., Geerlings, A., Frederich, M., Penelle, J., Delaude, C., Verpoorte, R., Angenot, L., 1999. β-carboline glucoalkaloids from Strychnos mellodora. Phytochemistry 51, 1171–1176. https://doi.org/10.1016/S0031-9422(99)00129-6. Cardoso, G.L., Gamboa, I.C., Silva, D.H., Furlan, M., Epifanio Rde, A., Pinto Ada, C., de Moraes, R.C., Lima, J.A., Bolzani Vda, S., 2004. Indole Glucoalgaloids from Chimarrhis turbinata and their evaluation as antioxidant agents and acetylcholinesterase inhibitors. J. Nat. Prod. 67, 1882–1885. https://doi.org/10.1021/ np049863m. Cardoso, G.L., Siqueira Silva, D.H., Tomazela, D.M., Verli, H., Young, M.C., Furlan, M., Eberlin, M.N., da Silva Bolzani, V., 2003. Turbinatine, a potential key intermediate in the biosynthesis of corynanthean-type indole alkaloids. J. Nat. Prod. 66, 1017–1021. https://doi.org/10.1021/np020547m. Calis, I., Sticher, O., 1984. Secoiridoid glucosides from Lonicera periclymenum. Phytochemistry. 23, 2539–2540. https://doi.org/10.1016/S0031-9422(00)84094-7. Chen, C.H., Lin, J.Y., Lin, C.N., Hsu, S.Y., 1992. Inhibition of angiotensin-I-converting enzyme by tetrahydroxyxanthones isolated from Tripterospermum lanceolatum. J.
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The novel indole glucoalkaloid and secoiridoid glucoside from Tripterospermum chinense
T
Tao Zhanga,b,1, Chuan-Jiang Mac,1, Yong-Li Weic, Jin-Guang Sia, Lu Fua, Jun-Xing Dongb,*, Zhong-Mei Zoua,* a
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China Beijing Institute of Radiation Medicine, Beijing 100850, PR China c The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Tripterospermumcin F Tripterospermumcin G Tripterospermum chinense NMR
Tripterospermumcin F (1), a novel indole glucoalkaloid, and tripterospermumcin G (2), a new iridoid tetramer, were isolated from the aerial parts of Tripterospermum chinense. Their structures were determined by analysis of 1D and 2D NMR data, as well as by comparison with model compounds. Their cytotoxicities were tested, but neither of them showed obvious activity.
1. Introduction The genus Tripterospermum (Entianaceae) consists of approximately 17 plant species in Asia. In this genus, approximately 15 species and one variety grow in mainland China (He, 1988). The genus is rich in iridoid glucosides, and xanthones, revealing diverse biological activities, such as antivirus, and anti-hypertension (Zhu et al., 2007a; Fang et al., 2008; Zhu et al., 2007b; Kawa et al., 1988; Calis and Sticher, 1984; Chen et al., 1992; Hsu et al., 1997). Tripterospermum chinense (Migo) H. Smith is widely distributed in South China, and traditionally used for the treatment of cough, haemoptysis, and pulmonary disease (Editorial board of Chinese Materia Medica, 1999). The constituents of this plant have been previously investigated, including iridoid glucosides, flavonoids and xanthones (Zhu et al., 2007a; Fang et al., 2008; Zhu et al., 2007b). Our previous study led to the isolation of seven iridoid glucosides from the aerial parts of T. chinense (Zhang et al., 2012). As a part of our ongoing search for new/novel and bioactive products from the medicinal plants in China, two new compounds, tripterospermumcins F and G (Fig. 1), were isolated from the aerial parts of T. chinense. In this paper, the isolation, structural elucidation and bioactive evaluation of the new compounds are discussed. 2. Results and discussion Compound 1 was isolated as a white amorphous powder. The molecular formula was assigned as C44H57O20N2 on the basis of the
positive-ion HRESIMS ion at m/z 933.3488 [M+H]+, together with its 1 H and 13C NMR data (Table 1). The IR spectrum showed the presence of the hydroxyl/amino groups (3422 cm−1), and α, β-unsaturated ester carbonyl groups (1698 and 1629 cm−1). The 1H and 13C NMR spectra of 1 also showed two α, β-unsaturated ester carbonyl moieties at δH 7.65 (1H, s, H-3a1) and 7.64 (1H, s, H-17a2), δC 152.9 (C-3a1), 110.3 (C4a1), 167.4 (C-11a1), 154.2 (C-17a2), 110.5 (C-16a2) and 168.1 (C22a2); two sets of anomeric signals at δH 5.38 (1H, d, J = 7.8 Hz, H-1 of Glc1) and 5.22 (1H, d, J = 7.8 Hz, H-1 of Glc2), δC 100.7 (C-1 of Glc1) and 100.5 (C-1 of Glc2); two sets of terminal vinyl carbons at δC 134.9 (C-8a1), 119.2 (C-10a1), 135.2 (C-18a2), and 118.5 (C-20a2); two acetal moieties at δH 5.87 (1H, d, J = 6.6 Hz, H-1a1) and 5.71 (1H, d, J = 7.8 Hz, H-21a2), δC 97.0 (C-1a1) and 96.5 (C-21a2); four aromatic protons at δH 7.26 (1H, m, H-9a2), 7.24 (1H, m, H-10a2), 7.74 (1H, m, H-11a2) and 7.47 (1H, m, H-12a2); six aromatic carbons at δC 128.2 (C-8a2), 121.4 (C-9a2), 119.4 (C-10a2), 118.3 (C-11a2), 111.7 (C-12a2), and 137.6 (C-13a2); one carboxyl carbon at δC 175.7 (C-23a2); two methines at δH 4.13 (1H, m, H-5a2) and 4.50 (1H, br d, J = 10.8 Hz, H-3a2), δC 57.6 (C-5a2) and 51.3 (C-3a2). Acid hydrolysis of 1 with 2 M CF3COOH and GCeMS analysis provided D-glucose. The large coupling constants (3J1,2 > 7 Hz) were consistent with the β configuration of the two sugars (Zhu et al., 2007a; Xin et al., 2008; Aquino et al., 1994; Paul et al., 2003; Cardoso et al., 2004, 2003; Berger et al., 2012; Ferrari et al., 1986). These data indicated that the structure of 1 was similar to 5αcarboxystrictosidine except for the unit of tripterospermumcin B in 1 instead of a secoiridoid glucoside moiety (Zhu et al., 2007b; Zhang
Corresponding authors. E-mail addresses: [email protected] (J.-X. Dong), [email protected] (Z.-M. Zou). 1 These authors contributed equally to this study. ⁎
https://doi.org/10.1016/j.phytol.2019.12.005 Received 10 March 2019; Received in revised form 26 November 2019; Accepted 10 December 2019 1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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Fig. 1. Structures of compounds 1–2.
et al., 2012; Aquino et al., 1994). This observation was confirmed by analysis of relevant 1H-1H COSY data. In the 1H-1H COSY spectrum, a strong cross-peak between the methine proton δH 4.50 (1H, br d, J = 10.8 Hz, H-3a2) and one of the methylene protons at C-14a2 (δH 2.27, 2.08) suggested that tripterospermumcin B unit was linked to indole monoterpenoid alkaloid (units A and B) (Aquino et al., 1994; Paul et al., 2003). The HMBC correlation from δH 4.50 (H-3a2) to δC 31.9 (C-15a2) was also observed to support the conclusion. The 1H-1H COSY spectrum (Fig. 2) showed two partial structure sequences for aglycone of unit A: CH2(14a2)CH(15a2)CH(19a2)CH(21a2)/CH(18a2)CH2(20a2) and CH2(7a1)CH2(6a1)CH(5a1)CH(9a1)CH(1a1)/CH(8a1)CH2(10a1). The C–C interconnectivity of all fragments was established from the HMBC spectrum (Fig. 2) as correlations of H-14a2 with C-16a2, H-17a2 and H7a1 with C-22a2, H-3a1 and H-12a1 with C-11a1, and H-6a1 with C-4a1. The HMBC correlations between δH 5.38 (H-1 of Glc1) and δC 97.0 (C1a1), and δH 5.22 (H-1 of Glc2) and δC 96.5 (C-21a2) allowed us to
identify linkage sites of D-glucose. On the basis of these data, the planar structure of 1 was established. The relative configuration of 1 was determined by analysis of the NOESY data (Fig. 3), as well as by comparison with known compounds. Comparison of the NMR data of unit A in 1 with those of tripterospermumcin B indicated that they have the same relative configuration (Zhu et al., 2007a, b; Zhang et al., 2012; Tian et al., 2006). The NOESY spectrum confirmed the above conclusion. The NOE correlations of H-6a1/H-1a1, H-8a1/H-1a1, H-14a2/H-21a2 and H-18a2/H-21a2 indicated they were cofacial and were arbitrarily assigned as α-orientations, whereas the correlations of H-5a1/H-9a1 and H-15a2/H-19a2 showed their β-orientations. The similarities between the chemical shifts of C-2a2, C-3a2, C-5a2 and C-6a2 in 1 and 5α-carboxystrictosidine suggested the same configuration for these carbons in the two alkaloids (Xin et al., 2008; Aquino et al., 1994; Paul et al., 2003; Cardoso et al., 2004, 2003; Berger et al., 2012; Ferrari et al., 1986; Aimi et al., 1992;
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Table 1 1 H (600 MHz, δ in ppm, J in Hz) and compound 1 in pyridine-d5.
13
No.
δH
δC
No.
1a1 3a1 4a1 5a1 6a1 7a1 8a1 9a1 10a1
5.87 d (6.6) 7.65 s
97.0 152.9 110.3 30.4 29.4 63.2 134.9 44.4 119.2
2a2 3a2 5a2 6a2 7a2 8a2 9a2 10a2 11a2
3.60 s
167.4 51.2
5.38 d (7.8) 3.97–4.08 o 4.23–4.30 o 4.23–4.30 o 3.97–4.08 o 4.34–4.41 o, 4.54 m
100.7 74.7 78.5 71.5 79.2 62.6
12a2 13a2 14a2 15a2 16a2 17a2 18a2 19a2 20a2
11a1 12a1 Glc1 1 2 3 4 5 6
3.12 m 1.87 m, 2.21 o 4.38 o, 4.28 o 5.83 m 2.76 m 5.13–5.19 o, 5.11 d (10.2)
21a2 22a2 23a2 Glc2 1 2 3 4 5 6 a
spectrum, supporting a β-oriented H-7a2 of 2. Thus, the structure of 2 was defined as shown, named tripterospermumcin G. Compounds 1 and 2 were evaluated for their cytotoxic activity against human cervical (HeLa), colon (LoVo), stomach (BGC-823), and breast cancer (MCF-7) cell lines. But none of them showed any significant activity at the concentration of 40 μM (See Table 1 in the Supporting Information).
C NMR (150 MHz, δ in ppm) data of δH 4.50 br d (10.8) 4.13 m 3.30–3.34 oa, 3.63 m 7.26 m 7.24 m 7.74 m 7.47 m 2.27 o,2.08 m 3.30–3.34 o 7.64 s 5.69 m 2.62 m 5.13–5.19 o, 4.95 d (10.8) 5.71 d (7.8)
5.22 d (7.8) 3.97–4.08 o 4.34–4.41 o 4.23–4.30 o 3.97–4.08 o 4.34–4.41 o, 4.54 m
δC 137.4 51.3 57.6 26.5 108.2 128.2 121.4 119.4 118.3
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Perkin-Elmer 343 polarimeter (PerkinElmer, Waltham, MA, USA). IR spectra were recorded on the Bio-Rad FTS-65A spectrometer (Bio-Rad, Richmond, VA, USA). UV spectra were recorded on Shimadzu UV-2501 PC (Shimadzu, Kyoto, Japan). The NMR spectra were recorded with a Varian UNITYINOVA 600 (Varian, Palo Alto, CA, USA), and the chemical shifts were given on δ (ppm) scale with tetramethylsilane as an internal standard. The HRESIMS was recorded on a 9.4 T Q-FT-MS Apex Qe (Bruker Co. Billerica, MA, USA). ESIMS was recorded on Thermo Finnigan Advantage MAX HPLC-MS (Thermo Electron, Pittsburgh, PA, USA). Macroporous resin AB-8 (Nan Kai College Chemical Inc, Tianjin, China), silica-gel (Qingdao Marine Chemistry Factory, Qingdao, China), Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and ODS silica-gel (120 Å, 50 μm, YMC, Kyoto, Japan) were used for column chromatography. HPLC was performed using Waters 600E system (Waters, Milford, MA, USA): an analytical column, ODS (5 μm, 4.6 × 250 mm, Hanbon Sci.& Tech, Huaian, China); preparative column, a YMC C18 (5 μm, 20.0 × 250 mm, YMC, Kyoto, Japan); detector, Knauer RID (refractive index detector, Knauer, Berlin, Germany) and Alltech ELSD (evaporative light-scattering detector, Alltech, Los Angeles, CA, USA) 2000ES. Gas chromatographic analysis was performed with an Agilent 6890 Series, gas chromatograph equipped with an H2 flameionization detector. The column was an HP-5 capillary column (30 m × 0.25 mm × 0.25 μm) (Agilent, Santa Clara, CA, USA).
111.7 137.6 38.7 31.9 110.5 154.2 135.2 44.9 118.5 96.5 168.1 175.7 100.5 74.7 78.4 71.6 79.0 62.8
Overlapped with other signals.
Brandt et al., 1999).· The NOE correlation of H-3a2/H-5a2 supported the configuration. Thus, the structure of 1 was determined as shown, named tripterospermumcin F. Compound 2, a white amorphous powder, possessed molecular formula of C69H100O39 based on the HRESIMS ion at m/z 1575.5723 [M +Na]+. Its IR spectrum showed hydroxyl (3426 cm−1) and α, β-unsaturated ester carbonyl (1705 and 1627 cm−1) absorptions. The 1H NMR spectrum of 2 indicated the presence of olefinic protons of the enol at δH 7.47 (2H, s), 7.39 (1H, s), and 7.37 (1H, s). The 13C NMR spectrum showed 69 carbon signals, in which the characteristic carbon signals at δC 98.6 × 2, 98.7 and 98.9 were readily assigned. In the 13C NMR spectrum, four α, β-unsaturated ester carbonyl carbons at δC 166.6 × 2, 165.9 × 2, 152.0 × 2, 151.7, 151.5, 109.9, 109.8, and 109.6 × 2, and four sets of terminal vinyl carbons at δC 134.6 × 2, 134.4 × 2, 119.2 × 2, and 118.9 × 2 were characteristic for four secoiridoid glucoside units. Acid hydrolysis of 2 with 2 M CF3COOH and GC–MS analysis gave D-glucose. These data implied that 2 is an iridoid tetramer including two tripterospermumcin B units (units A and B) (Zhu et al., 2007a, b; Zhang et al., 2012; Tian et al., 2006). The HMBC correlation from δ 4.51 (H-7a2) with δ 64.3 (C-6 of Glc4) were observed to support the gross structure as shown. The relative configuration of 2 was elucidated by NOESY analysis. Almost identical NMR data indicate that the units A and B of 2, and tripterospermumcin B share the same relative configuration (Zhu et al., 2007a, b; Zhang et al., 2012; Tian et al., 2006). The NOE correlations of H-7a2/H-12a2, H-7a2/H-6 (Glc4), and H-7a2/H-5a2, indicated that H-7a2 had β-orientations. The correlation between H-12a2 and H-6 of Glc4 was not observed in the NOESY
3.2. Plant material The aerial parts of Tripterospermum chinense were collected from Yongshun region of Hunan Province, China in April 2010. The plant was identified by prof. Bin Li (Beijing Institute of Radiation Medicine) and a voucher specimen (No. 100403) is deposited in the Herbarium of Beijing Institute of Radiation Medicine, Beijing. 3.3. Extraction and isolation The aerial parts of T. chinense (60 kg) were refluxed three times with 60 % aqueous EtOH (3 × 480 L, each for 1.5 h). The combined extract was concentrated under reduced pressure to furnish a dark brown residue (1800 g), which was suspended in H2O and partitioned in turn with CHCl3 and n-BuOH. The n-BuOH extract was evaporated under reduced pressure to yield a residue (386 g). The latter was separated chromatographically on macroporous resin AB-8 (15 × 100 cm) and eluted with a gradient mixture of EtOH–H2O (1:5, 1:1 and 9:1; 60,000 mL each), to give five fractions (A–E). Fraction C (134 g) was separated chromatographically on silica-gel column (12 × 50 cm) with a gradient mixture of CHCl3–MeOH (20:1, 10:1, 5:1, 5:2 and 1:1) as eluent, and total 160 tubes (1000 mL each) were collected. Tubes 70–75 (11 g) were individually subjected to a series of purification steps using Sephadex LH-20 column chromatography (MeOH), ODS silica-gel
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Fig. 2. Key 1H-1H COSY and HMBC correlations of 1–2.
column chromatography (3 × 50 cm; MeOH–H2O, 40: 60 and 70: 30), and finally purified by preparative HPLC (MeOH–H2O, 55: 45, flow rate: 8.0 mL/min) to afford 2 (10 mg, tR: 12.30 min). Tubes 118–127 (4 g) were individually subjected to a series of purification steps using Sephadex LH-20 column chromatography (MeOH), ODS silica-gel column chromatography (3 × 50 cm; MeOH–H2O, 40: 60 and 70: 30), and finally purified by preparative HPLC (MeOH–H2O, 55: 45, flow rate: 8.0 mL/min) to afford 1 (12 mg, tR: 22.23 min). Tripterospermumcin F(1): white amorphous powder (MeOH–H2O, 55:45); [α]D20 −191.3 (c 0.08, MeOH); UV (MeOH) λmax (logε): 235 (3.99), 285 (4.15) nm, IR (KBr) νmax: 3422, 2926, 1698, 1629, 1654, 1289, 1075 cm−1; HRESIMS (pos.): m/z 933.3488 [M+H]+ (calcd for C44H57O20N2, 933.3499); 1H and 13C NMR data, see Table 1. Tripterospermumcin G(2): white amorphous powder (MeOH–H2O, 55:45); [α]D20 −79.3 (c 0.05, MeOH); UV (MeOH) λmax (logε): 234 (3.60) nm, IR (KBr) νmax: 3427, 2929, 1705, 1628, 1654, 1286, 1075 cm−1; HRESIMS (pos.): m/z 1575.5723 [M+Na]+ (calcd for C69H100O39Na, 1575.5734); 1H and 13C NMR data, see Table 2.
3.4. Acid hydrolysis and sugar analysis Compounds 1–2 (2.0 mg each) were individually hydrolyzed in 2 M CF3COOH (2 mL) at 95 °C for 3 h. The reaction mixture was extracted with CH2Cl2 (5 mL) three times. The aqueous layer was repeatedly evaporated to dryness with EtOH until neutral. In the monosaccharide mixture, glucose was detected by TLC on a silica gel [using nBuOH–EtOAc–C5H5N–H2O (6:1:5:4) as development] by comparison with authentic sample: glucose (Rf 0.46). The residue of the sugars was dissolved in anhydrous pyridine (2 mL), 12 mg L-cysteine methyl ester hydrochloride was added, and the mixture was stirred at 60 °C for 1 h. Then, HMDS–TMCS (hexamethyldisilazane- trimethylchlorosilane 2:1) (0.6 mL) was added, and the mixture was kept at 60 °C for 0.5 h (Mitaine-Offer et al., 2010). The supernatant was analyzed by GC under the following conditions: column temperature: 180 °C/250 °C; programmed increase, 15 °C/min; carrier gas: N2 (1 mL/min); injection and detector temperature: 250 °C; injection volume: 4.0 μL, and split ratio: 1/50. The derivative of D-glucose was detected with tR values of 17.93 min.
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Table 2 1 H (600 MHz, δ in ppm, J in Hz) and compound 2 in DMSO-d6.
13
C NMR (150 MHz, δ in ppm) data of
No.
δH
δC
No.
δH
δC
1a1 3a1 4a1 5a1 6a1 7a1 8a1 9a1 10a1 11a1 12a1 Glc1 1 2 3 4 5 6 1b1 3b1 4b1 5b1 6b1 7b1 8b1 9b1 10b1 11b1 12b1
5.48 m 7.47 s
95.6 152.0 109.6 30.2 28.9 62.0 134.6 43.1 118.9 166.6 51.1
1a2 3a2 4a2 5a2 6a2 7a2 8a2 9a2 10a2 11a2 12a2 Glc2 1 2 3 4 5 6 1b2 3b2 4b2 5b2 6b2 7b2 8b2 9b2 10b2 11b2 12b2 13b2 Glc4 1 2 3 4 5 6
5.42 m 7.39 s
95.7 151.7 109.8 27.6 32.3 101.4 134.4 43.1 119.2 165.9 52.0
Glc3 1 2 3 4 5 6 a
2.75–2.78 oa 1.76 o, 1.84 o 4.07 o, 3.99 o 5.70 m 2.57 o 5.22 o, 5.32 o 3.60 s 4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.15–3.18 o 3.68 o, 3.41 o 5.48 m 7.47 s
3.60 s
98.6 73.0 77.3 70.0 76.6 61.1 95.6 152.0 109.6 30.2 28.9 62.0 134.6 43.1 118.9 166.6 51.1
4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.15–3.18 o 3.68 o, 3.41 o
98.6 73.0 77.3 70.0 76.6 61.1
2.75–2.78 o 1.76 o, 1.84 o 4.07 o, 3.99 o 5.70 m 2.57 o 5.22 o, 5.32 o
2.75–2.78 o 1.54 m, 1.90 o 4.51 o 5.62 m 2.57 o 5.22 o, 5.32 o 3.16 br s 4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.15–3.18 o 3.68 o, 3.41 o 5.36 m 7.37 s
98.9 72.8 77.3 69.8 76.6 61.0
3.16 br s 3.16 br s
151.5 109.9 27.6 31.6 101.9 134.4 43.1 119.2 165.9 51.7 53.2
4.52 o 2.96–3.05 o 3.15–3.18 o 2.96–3.05 o 3.31 o 3.99 o, 3.41 o
98.7 72.9 77.3 69.8 75.4 64.3
2.75–2.78 o 1.44 m, 1.95 o 4.38 m 5.62 m 2.57 o 5.22 o, 5.32 o
Overlapped with other signals.
Compound 1 is the first report of terpenoid indole alkaloids incorporating a secoiridoid dimeric unit. To the best of our knowledge, compound 2 is the fourth-reported iridoid tetramers with four glucosides (Zhang et al., 2012; Ferrari et al., 1986). The planar structures and relative configurations of 1 and 2 were determined by analysis of NMR and NOESY data in the present paper. As compounds 1 and 2 were not suitable functional groups for chemical reactions or suitable crystal for X-ray diffraction, their absolute configurations were not determined and further study was required. Our results not only enrich the chemical diversity of secondary metabolites from the genus Tripterospermum, but also emphasize the importance of chemotaxonomic data to help better understand the morphoanatomical classification of this complex plant family.
Fig. 3. Selected NOESY correlations of 1–2.
3.5. Cytotoxicity assays The Hela, LoVo, BGC-823 and MCF-7 cell lines were obtained from the National Infrastructure of Cell Line Resource (Beijing, China). The assay was run in triplicate. In a 96-well plate, each well was plated with 2 × 104 cells. After cell attachment overnight, the medium was removed, and each well was treated with 100 μL of medium containing 0.1 % DMSO or different concentrations of the test compounds and the positive control cis-platin. The plate was incubated for 4 days at 37 °C in a humidified, 5 % CO2 atmosphere. Cytotoxicity was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Wang et al., 2005). After addition of 10 μL MTT solution (5 mg/mL), cells were incubated at 37 °C for 4 h. After adding 150 μL DMSO, cells were shaken to mix thoroughly. The absorbance of each well was measured at 540 nm in a Multiscan photometer. The IC50 values were calculated by Origin software.
Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements
4. Conclusion
This work was financially supported by the Chinese National S&T Special Project on Major New Drug Innovation (2017ZX09301059), the Natural Sciences Foundation of Beijing (7194297), the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1-010), and
Two new compounds, tripterospermumcin F (1) and tripterospermumcin G (2) were isolated from the aerial parts of T. chinense.
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Peking Union Medical College Discipline Construction Project (201920100901).
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