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Iridoid and phenylethanoid glycosides from Heterophragma sulfureum
Phytochemistry Letters 12 (2015) 277–281
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Iridoid and phenylethanoid glycosides from Heterophragma sulfureum Choosak Kaewkongpan a , Poolsak Sahakitpichan b , Somsak Ruchirawat b,c , Tripetch Kanchanapoom a,b, * a
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand Chulabhorn Research Institute, Kamphaeng Phet 6, Talat Bang Khen, Lak Si, Bangkok 10210, Thailand c Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Thailand b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 January 2015 Received in revised form 22 April 2015 Accepted 24 April 2015 Available online 4 May 2015
An unusual iridoid diglycoside (specioside 60 -O-a-D-galactopyranoside) and a new phenylethanoid triglycoside (heterophragmoside) were isolated from the leaves and branches of Heterophragma sulfureum together with specioside, verminoside, 6-trans-feruloylcatapol, stereospermoside, ()-lyoniresinol 3a-O-b-D-glucopyranoside, (+)-lyoniresinol 3a-O-b-D-glucopyranoside, ()-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside, (+)-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside, and dehydroconiferyl 4-O-b-D-glucopyranoside. The structural elucidations were based on analyses of chemical and spectroscopic data. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Heterophragma sulfureum Bignoniaceae Iridoid glycoside Specioside 6'-O-a-D-galactopyranoside Phenylethanoid glycoside Heterophragmoside
1. Introduction The family Bignoniaceae consists of about 120 genera and 800 species. They are mostly distributed in the tropical and subtropical of the old and new world regions. In Thailand, 12 genera and 24 species have been reported as indigenous plants, and several species are imported and cultivated as ornamental purposes (Santisuk, 1987; Srisanga et al., 2003). As part of our further systematic studies on plants from this family (Kanchanapoom et al., 2001, 2002a,b, 2006; Sinaphet et al., 2006), we investigated the constituents of Heterophragma sulfureum Kurz. (Thai name: Rang-Raeng), being collected from Khon Kaen province, Thailand. H. sulfureum is a tree up to 25 m high, distributed in Thailand, Myanmar, Laos and Cambodia. The flowers of this plant are used in north-eastern part of Thailand as vegetable for cooking purpose. The leaves are used for external treatment of skin diseases. The phytochemical investigation has not been reported on this species. The present paper deals with the isolation of 11 compounds from n-BuOH fraction of the MeOH extract of the leaves and branches of this plant, including an unusual new iridoid diglycoside with a-galactose substitution (2) and a new phenylethanoid triglycoside (6), as well as four known iridoid glucosides
* Corresponding author. Tel.: +66 43 362092; fax: +66 43 202379. E-mail address: [email protected] (T. Kanchanapoom).
(1,3–5), four known lignan glucosides (7–10), and a neolignan glucoside (11). 2. Results and discussion The methanolic extract of H. sulfureum was partitioned with solvents of increasing polarity. The n-BuOH soluble fraction was subjected to highly porous copolymer resin of styrene and divinylbenzene (Diaion HP-20), silica gel, RP-18, preparative HPLC-ODS column chromatography to afford 11 compounds (1– 11), of which compounds 2 and 6 were new. Nine known compounds were assigned as specioside (1) (Compadre et al., 1982), verminoside (3) (Sticher and Afifi-Yazar, 1979), 6-transferuloylcatapol (4) (Stuppner and Wagner, 1989), stereospermoside (5) (Kanchanapoom et al., 2006), ()-lyoniresinol 3a-O-b-Dglucopyranoside (7), (+)-lyoniresinol 3a-O-b-D-glucopyranoside (8), ()-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside (9), (+)-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside (10) (Achenbach et al., 1992), and dehydroconiferyl 4-O-b-D-glucopyranoside (11) (Yoshizawa et al., 1990) by comparison of physical data with literature values and from spectroscopic evidence (Fig. 1). Compound 2, [a]D24 60.5, was isolated as an amorphous powder. Its molecular formula was determined as C30H38O17 by negative HR-FAB mass spectrometric analyses. Inspection of the 1H and 13C NMR spectra indicated that this compound is an iridoid glycoside, closely related to specioside (1). In addition, the signals
http://dx.doi.org/10.1016/j.phytol.2015.04.016 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
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Fig. 1. Structures of compounds 1–4 and 6.
of one sugar unit were observed in the 1H and 13C NMR spectra, deduced from an anomeric proton at dH 5.46 (d, J = 3.6 Hz) and six more carbon atoms at dC 100.2, 72.8, 71.6, 70.8, 70.3 and 62.5 (in C5D5N). The anomeric proton signal appeared as doublet with the
coupling constant, J = 3.6 Hz, suggesting that this sugar moiety was in an a-form. This sugar part could be assigned to be a-Dgalactopyranosyl moiety, based on the detailed analysis of COSY and HMQC experiments as well as the splitting patterns of each
Fig. 2. COSY and HMBC correlations of compound 2.
C. Kaewkongpan et al. / Phytochemistry Letters 12 (2015) 277–281
proton. The COSY spectrum clearly showed the proton connectivity from H-100 (dH 5.46, d, J = 3.6 Hz)/ H-200 (dH 4.72, dd, J = 9.8, 3.6 Hz)/ H-300 (dH 4.61, dd, J = 9.8, 3.2 Hz)/ H-400 (dH 4.80, dd, J = 3.2, 3.2 Hz)/ H-500 (dH 4.48, m)/ and H2-600 (dH 4.49 and 4.59, br d, J = 11.0 Hz), corresponding to the carbon atoms at dC 100.2 (C-100 )/70.3 (C-200 )/ 71.6 (C-300 )/70.8 (C-400 )/72.8 (C-500 )/ and 62.5 (C-600 ), respectively. Also, the chemical shifts were correlated to the reported data for a-D-galactopyranosyl moiety (Abreu et al., 1997). This unit was attached to C-6' of the glucopyranosyl moiety due to the downfield shift of this carbon atom to dC 68.0. The HMBC spectrum provided the further confirmation of the structure from the correlation between H-100 (dH 5.46) and C-6' (dC 68.0) as shown in Fig. 2. Consequently, the structure of compound 2 was determined as specioside 60 -O-a-D-galactopyranoside. Compound 6, [a]D24 70.9, was obtained as an amorphous powder. The molecular formula was determined to be C35H46O19 by negative high-resolution FAB mass spectrometric analyses. Inspection of the 1H and 13C NMR spectroscopic data indicated that this compound is a phenylethanoid triglycoside, related to those of our previous works from the same family (Kanchanapoom et al., 2001, 2002a,b, 2006; Sinaphet et al., 2006). The 1H NMR showed the chemical shifts of a set of 1,3,4-trisubstituted aromatic ring system at dH 7.00 (1H, d, J = 2.0 Hz), 6.74 (1H, dd, J = 8.1, 2.0 Hz) and Table 2 NMR spectroscopic data of 6 and lipedoside A-II (measured in CD3OD). Position 1 2 3 4 5 6 7 8
dC
dC (lipedoside A-II)a
131.9 119.8 146.7 146.3 117.0 125.3 36.5 71.7
131.4 116.4 144.6 146.1 117.1 121.3 36.7 72.3
4.17 (1H, d, J = 7.9 Hz) 3.24 (1H, dd, J = 8.8, 7.9 Hz) 3.42 (1H, dd, J = 9.0, 8.8 Hz) 3.30 (1H)b 3.69 (1H)b 4.35 (1H, dd, J = 12.1, 6.6 Hz) 4.51 (1H, br d, J = 12.1 Hz)
104.1 75.6 84.4 70.1 75.5 64.6
104.4 75.7 84.0 70.4 75.4 64.7
5.11 (1H, br s) 3.92 (1H)b 3.92 (1H)b 3.36 (1H, dd, J = 9.7, 9.5 Hz) 3.95 (1H, m) 1.20 (3H, d, J = 6.2 Hz)
102.6 72.2 72.2 73.9 70.0 17.9
102.7 72.3 72.5 74.0 70.0 17.9
4.70 3.48 3.48 3.28 3.20 3.61 3.81
104.1 74.7 77.3 71.6 77.7 62.6
dH 7.00 (1H, d, J = 2.0 Hz)
6.69 (1H, d, J = 8.1 Hz) 6.74 (1H, dd, J = 8.1, 2.0 Hz) 2.68 (2H, m) 3.35 (1H)b 3.67 (1H)b
Glc 10 20 30 40 50 60
Rha 100 200 300 400 500 600 Glc 1000 2000 3000 4000 5000 6000
b
6.69 (1H, d, J = 8.1 Hz) for the 3,4-dihydroxy-b-phenylethoxyl moiety, a set of 1,4-disubstituted aromatic ring system at dH 7.42 and 6.69 (each 2H, d, J = 8.1 Hz) together with two transolefinic protons at dH 6.34 and 7.60 (each 1H, d, J = 16.0 Hz) for the coumaroyl moiety in addition to three anomeric protons assignable for two glucopyranosyl units at dH 4.17 (1H, d, J = 7.9 Hz) and 4.70 (1H, d, J = 7.8 Hz), and one rhamnopyranosyl unit at dH 5.11 (1H, br s). The 13C NMR spectral data (Table 2) were closely similar to those of lipedoside A-II (He et al., 1994), except for the additional signals arising from one b-D-glucopyranosyl unit. This sugar part was suggested to be located at one hydroxyl group of an aromatic ring of 3,4-dihydroxy-b-phenylethoxyl moiety since the chemical shifts of the aromatic ring were changed. The assignment of the structure was supported by COSY, NOESY, HMQC and HMBC experiments. In the 2D NMR spectra, the HMBC correlation from H1000 (dH 4.70) to C-3 (dC 146.7) along with the NOESY correlation between H-1000 (dH 4.70) and H-2 (dH 7.00) were indicative the connectivity of this additional glucopyranosyl moiety to C-3. Moreover, the results of the HMBC correlations from H-10 (dH 4.17) to C-8 (dC 71.7) and C-30 (dC 84.4), from H2-60 (dH 4.35 and 4.51) to C90000 (dC 169.1), and from H-100 (dH 5.11) to C-30 (dC 84.4) provided the further confirmation of the structure as shown in Fig. 3. Therefore, the structure of compound 6 was elucidated to be 3,4-dihydroxyb-phenylethoxyl 3-O-b-D-glucopyranosyl-8-O-[a-L-rhamnopyranosyl-(1“ ! 3’)]-60 -O-trans-coumaroyl-b-D-glucopyranoside, namely heterophragmoside. Eleven compounds were isolated from H. sulfureum, including five iridoid glycosides (1–5), one phenylethanoid glycosides (6), four lignan glycosides (7–10) and one neolignan glycoside (11). Phenylethanoid glycosides, lignans as well as iridoid glycosides lacking a substituent at C-4 are the common secondary metabolites of the plants from tribe Tecomeae of the family Bignoniaceae (von Poser et al., 2000; Kanchanapoom et al., 2001, 2002a,b, 2006; Sinaphet et al., 2006). This species produces large amounts of iridoid glycosides, especially specioside (1). Its derivative bearing sugar moiety in a-orientation form of galactose (6) is quite unusual to isolate from plant source. The present study provides the further confirmation of the typical profile on the secondary metabolites found in this tribe and might be useful for chemotaxonomic methods of the family Bignoniaceae. 3. Experimental 3.1. General procedures
(1H, d, J = 7.8 Hz) (1H)b (1H)b (1H)b (1H, m) (1H, dd, J = 10.1, 5.4 Hz) (1H, br d, J = 10.1 Hz)
Coumaroyl moiety 100 00 200 00 , 600 00 7.42 (2H, d, J = 8.6 Hz) 30 , 50 6.78 (2H, d, J = 8.6 Hz) 400 00 700 00 7.60 (1H, d, J = 16.0 Hz) 800 00 6.34 (1H, d, J = 16.0 Hz) 00 00 9 a
279
127.1 131.3 116.9 161.1 147.0 114.9 169.1
Data taken from He et al., 1994. Chemical shifts were assigned by COSY and HMQC.
NMR spectra were recorded in CD3OD or C5D5N using a Bruker AV-400 (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometer. MS values were obtained on a JEOL JMS-SX 102 spectrometer. FTIR spectra were recorded on a universal attenuated total reflectance attached (UATR) to a PerkinElmer Spectrum One spectrometer. Optical rotations were measured with
127.1 131.2 116.9 161.2 146.9 114.9 169.1
Fig. 3. HMBC and NOESY correlations of compound 6.
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a Jasco P-1020 digital polarimeter. For column chromatography, silica gel 60 (70–230 mesh, GE-0049, Schalau), RP-18 (50 mm, YMC), and Diaion HP-20 (Mitsubishi Chemical Industries Co., Ltd.) were used. Preparative HPLC was carried out on an ODS column (250 20 mm i.d., YMC) with a Jasco RI-2031 refractive index detector. The flow rate was 6 ml/min. The spraying reagent used for TLC was 10% H2SO4 in 50% EtOH. 3.2. 1 Plant material The leaves and branches of H. sulfureum Kurz was collected in April 2005 from Khon Kaen Province, Thailand. The plant was identified by Mr. Bamrung Thavinchiua, Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher specimen (TK-PSKKU0053) was deposited in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University.
Table 1 NMR Spectroscopic data of compound 2. Position
dH a
1 3 4 5 6 7 8 9 10
5.53 6.45 5.09 2.92 5.40 4.48
Glc 10 20 30 40 50 60
3.3. Extraction and isolation The dried leaves and branches portion of H. sulfureum (12.5 kg) was extracted three times with hot MeOH. The MeOH extract was concentrated in vacuo to give a brownish residue (1.6 kg). This residue was suspended in H2O, and then partitioned with nhexane, EtOAc and n-BuOH, successively. The n-BuOH soluble fraction (639.9 g) was subjected to highly porous synthetic resin column chromatography (Diaion HP-20), and eluted with H2O, 20% aqueous MeOH, 40% aqueous MeOH, 60% aqueous MeOH, 80% aqueous MeOH, MeOH and (CH3)2CO, successively. The residue of the 20% aqueous MeOH eluate (51.2 g) was subjected to silica gel column chromatography with solvent systems: EtOAc (3.0 L), EtOAc–MeOH (9:1, 8.0 L), EtOAc–MeOH–H2O (40:10:1, 9.0 L), EtOAc–MeOH–H2O (70:30:3, 6.0 L) and EtOAc–MeOH–H2O (6:4;1, 4.0 L), respectively to provide eight fractions (Fr. A1–H1). Fraction C1 (29.2 g) was applied to a column of RP-18 with a gradient solvent system, H2O-MeOH (80:20 ! 20:80, v/v) to afford compound 1 (24.5 g). Fraction D1 (7.5 g) was applied to a column of RP-18 a gradient solvent system, H2O-MeOH (90:10 ! 30:70, v/v) to afford nine fractions (Fr. D1-1–D1-9). Fraction D1-4 was purified by preparative HPLC with solvent system 15% aqueous MeCN to obtain compound 7 (83.2 mg). Fraction D1-5 was purified by preparative HPLC-ODS with solvent system 17% aqueous MeCN to give compounds 5 (51.1 mg), 9 (47.6 mg), 10 (23.5 mg), and 11 (12.3 mg). Fraction D1-7 was purified by preparative HPLC-ODS with solvent system 20% aqueous MeCN to give compound 6 (119.1 mg). Similarly, fraction E1 (13.3 g) was applied to a column of RP-18 with a gradient solvent system, H2O-MeOH (90:10 ! 20:80, v/v) to afford 12 fractions (Fr. E1-1–E1-12). Fraction E1-10 was purified by preparative HPLC with solvent system 20% aqueous MeCN to obtain compounds 2 (22.2 mg) and 3 (35.4 mg). The residue of the 40% aqueous MeOH eluate (30.0 g from total 202.0 g) was subjected to silica gel column chromatography with solvent systems: EtOAc (3.0 L), EtOAc–MeOH (9:1, 8.0 L), EtOAc– MeOH–H2O (40:10:1, 9.0 L), EtOAc–MeOH–H2O (70:30:3, 6.0 L) and EtOAc–MeOH–H2O (6:4;1, 4.0 L) to provide eight fractions (Fr. A2– H2). Fraction D2 was provided compound 1 (21.2 g) by crystallization. Fraction F2 (3.0 g) was applied to a column of RP-18 with a gradient solvent system, H2O-MeOH (80:20 ! 20:80, v/v) to afford five fractions (Fr. D2-1–D2-5). Fraction D2-3 was purified by preparative HPLC with solvent system 17% aqueous MeCN to give compound 8 (6.4 mg). The residue of the 60% aqueous MeOH eluate (30.0 g from total 65.2 g) was subjected to silica gel column chromatography with solvent systems: EtOAc–MeOH (9:1, 4.0 L), EtOAc–MeOH–H2O (40:10:1, 7.0 L), and EtOAc–MeOH–H2O (70:30:3, 5.0 L) to provide six fractions (Fr. A3–F3). Fraction E3 (3.9 g) was partially purified by
Gal 100 200 300 400 500 600
dC a
dC b
94.8 141.5 102.3 36.1 80.5 58.9 66.6 43.0 59.6
95.4 142.5 102.9 36.6 81.5 59.7 66.7 43.4 60.9
5.39 (1H, d, J = 7.8 Hz) 4.10 (1H, d, J = 8.8, 7.8 Hz) 4.22 (1H, dd, J = 9.0, 8.8 Hz) 4.00 (1H, dd, J = 9.3, 9.0 Hz) 4.01 (1H, m) 4.30 (1H, br d, J = 9.5 Hz) 4.45 (1H)c
100.0 74.5 78.0 71.5 76.3 68.0
100.0 74.8 77.7 71.7 76.6 68.2
5.46 4.72 4.61 4.80 4.48 4.49 4.59
100.2 70.3 71.6 70.8 72.8 62.5
100.2 70.0 72.0 71.0 72.7 62.8
125.7 130.6 116.7 161.5 145.9 114.1 167.3
127.0 131.4 116.9 161.4 147.5 114.4 169.1
(1H, d, J = 9.3 Hz) (1H, d, J = 5.9 Hz) (1H, dd, J = 5.9, 4.2 Hz) (1H)c (1H, br d, J = 7.3 Hz) (1H)c
2.90 (1H)c 4.48 (1H, d, J = 9.5 Hz) 4.75 (1H, d, J = 9.5 Hz)
(1H, d, J = 3.6 Hz) (1H, dd, J = 9.8, 3.6 Hz) (1H, dd, J = 9.8, 3.2 Hz) (1H, dd, J = 3.2, 3.2 Hz) (1H, m) (1H)c (1H, br d, J = 11.0 Hz)
Coumaroyl moiety 1000 2000 , 6000 7.58 (2H, d, J = 8.3 Hz) 3000 , 5000 7.21 (2H, d, J = 8.3 Hz) 4000 7000 7.88 (1H, d, J = 15.9 Hz) 8000 6.52 (1H, d, J = 15.9 Hz) 9000 a b c
Measured in C5D5N. Measured in CD3OD. Chemical shifts were assigned by COSY and HMQC.
a RP-18 column with a gradient solvent system, H2O-MeOH (90:10 ! 30:70, v/v), and then purified by preparative HPLC-ODS using solvent system 24% aqueous MeCN to afford compound 4 (12.8 mg). 3.4. Specioside 6'-O-a-D-galactopyranoside (2) Amorphous powder, [a]D24 60.5 (MeOH, c 0.77); IR spectrum: nmax = 3339, 2922, 1691, 1631, 1603, 1515, 1262, 1226, 1154, 1013,
832 cm1; 1H (C5D5N) and 13C NMR (CD3OD and C5D5N): Table 1. Negative HR-FAB-MS, m/z: 669.1994 (C30H37O17 required 669.2031). 3.5. Heterophragmoside (6) Amorphous powder, [a]D24 70.9 (MeOH, c 2.34); IR spectrum: nmax = 3364, 2927, 1691, 1604, 1515, 1264, 1167, 1062, 1017,
833 cm1; 1H and 13C NMR (CD3OD): Table 2; negative HR-FABMS, m/z: 769.2574 (C35H45O19 required 769.2555). Acknowledgements
This research work was supported by the grant from the Thailand Research Fund (DBG5480007), Chulabhorn Research Institute, Khon Kaen University; and Center of Excellence on Environmental Health and Toxicology, Science & Technology Postgraduate Education and Research Development Office (PERDO), Ministry of Education.
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Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Iridoid and phenylethanoid glycosides from Heterophragma sulfureum Choosak Kaewkongpan a , Poolsak Sahakitpichan b , Somsak Ruchirawat b,c , Tripetch Kanchanapoom a,b, * a
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand Chulabhorn Research Institute, Kamphaeng Phet 6, Talat Bang Khen, Lak Si, Bangkok 10210, Thailand c Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Thailand b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 January 2015 Received in revised form 22 April 2015 Accepted 24 April 2015 Available online 4 May 2015
An unusual iridoid diglycoside (specioside 60 -O-a-D-galactopyranoside) and a new phenylethanoid triglycoside (heterophragmoside) were isolated from the leaves and branches of Heterophragma sulfureum together with specioside, verminoside, 6-trans-feruloylcatapol, stereospermoside, ()-lyoniresinol 3a-O-b-D-glucopyranoside, (+)-lyoniresinol 3a-O-b-D-glucopyranoside, ()-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside, (+)-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside, and dehydroconiferyl 4-O-b-D-glucopyranoside. The structural elucidations were based on analyses of chemical and spectroscopic data. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Heterophragma sulfureum Bignoniaceae Iridoid glycoside Specioside 6'-O-a-D-galactopyranoside Phenylethanoid glycoside Heterophragmoside
1. Introduction The family Bignoniaceae consists of about 120 genera and 800 species. They are mostly distributed in the tropical and subtropical of the old and new world regions. In Thailand, 12 genera and 24 species have been reported as indigenous plants, and several species are imported and cultivated as ornamental purposes (Santisuk, 1987; Srisanga et al., 2003). As part of our further systematic studies on plants from this family (Kanchanapoom et al., 2001, 2002a,b, 2006; Sinaphet et al., 2006), we investigated the constituents of Heterophragma sulfureum Kurz. (Thai name: Rang-Raeng), being collected from Khon Kaen province, Thailand. H. sulfureum is a tree up to 25 m high, distributed in Thailand, Myanmar, Laos and Cambodia. The flowers of this plant are used in north-eastern part of Thailand as vegetable for cooking purpose. The leaves are used for external treatment of skin diseases. The phytochemical investigation has not been reported on this species. The present paper deals with the isolation of 11 compounds from n-BuOH fraction of the MeOH extract of the leaves and branches of this plant, including an unusual new iridoid diglycoside with a-galactose substitution (2) and a new phenylethanoid triglycoside (6), as well as four known iridoid glucosides
* Corresponding author. Tel.: +66 43 362092; fax: +66 43 202379. E-mail address: [email protected] (T. Kanchanapoom).
(1,3–5), four known lignan glucosides (7–10), and a neolignan glucoside (11). 2. Results and discussion The methanolic extract of H. sulfureum was partitioned with solvents of increasing polarity. The n-BuOH soluble fraction was subjected to highly porous copolymer resin of styrene and divinylbenzene (Diaion HP-20), silica gel, RP-18, preparative HPLC-ODS column chromatography to afford 11 compounds (1– 11), of which compounds 2 and 6 were new. Nine known compounds were assigned as specioside (1) (Compadre et al., 1982), verminoside (3) (Sticher and Afifi-Yazar, 1979), 6-transferuloylcatapol (4) (Stuppner and Wagner, 1989), stereospermoside (5) (Kanchanapoom et al., 2006), ()-lyoniresinol 3a-O-b-Dglucopyranoside (7), (+)-lyoniresinol 3a-O-b-D-glucopyranoside (8), ()-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside (9), (+)-50 -methoxyisolariciresinol 3a-O-b-D-glucopyranoside (10) (Achenbach et al., 1992), and dehydroconiferyl 4-O-b-D-glucopyranoside (11) (Yoshizawa et al., 1990) by comparison of physical data with literature values and from spectroscopic evidence (Fig. 1). Compound 2, [a]D24 60.5, was isolated as an amorphous powder. Its molecular formula was determined as C30H38O17 by negative HR-FAB mass spectrometric analyses. Inspection of the 1H and 13C NMR spectra indicated that this compound is an iridoid glycoside, closely related to specioside (1). In addition, the signals
http://dx.doi.org/10.1016/j.phytol.2015.04.016 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
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Fig. 1. Structures of compounds 1–4 and 6.
of one sugar unit were observed in the 1H and 13C NMR spectra, deduced from an anomeric proton at dH 5.46 (d, J = 3.6 Hz) and six more carbon atoms at dC 100.2, 72.8, 71.6, 70.8, 70.3 and 62.5 (in C5D5N). The anomeric proton signal appeared as doublet with the
coupling constant, J = 3.6 Hz, suggesting that this sugar moiety was in an a-form. This sugar part could be assigned to be a-Dgalactopyranosyl moiety, based on the detailed analysis of COSY and HMQC experiments as well as the splitting patterns of each
Fig. 2. COSY and HMBC correlations of compound 2.
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proton. The COSY spectrum clearly showed the proton connectivity from H-100 (dH 5.46, d, J = 3.6 Hz)/ H-200 (dH 4.72, dd, J = 9.8, 3.6 Hz)/ H-300 (dH 4.61, dd, J = 9.8, 3.2 Hz)/ H-400 (dH 4.80, dd, J = 3.2, 3.2 Hz)/ H-500 (dH 4.48, m)/ and H2-600 (dH 4.49 and 4.59, br d, J = 11.0 Hz), corresponding to the carbon atoms at dC 100.2 (C-100 )/70.3 (C-200 )/ 71.6 (C-300 )/70.8 (C-400 )/72.8 (C-500 )/ and 62.5 (C-600 ), respectively. Also, the chemical shifts were correlated to the reported data for a-D-galactopyranosyl moiety (Abreu et al., 1997). This unit was attached to C-6' of the glucopyranosyl moiety due to the downfield shift of this carbon atom to dC 68.0. The HMBC spectrum provided the further confirmation of the structure from the correlation between H-100 (dH 5.46) and C-6' (dC 68.0) as shown in Fig. 2. Consequently, the structure of compound 2 was determined as specioside 60 -O-a-D-galactopyranoside. Compound 6, [a]D24 70.9, was obtained as an amorphous powder. The molecular formula was determined to be C35H46O19 by negative high-resolution FAB mass spectrometric analyses. Inspection of the 1H and 13C NMR spectroscopic data indicated that this compound is a phenylethanoid triglycoside, related to those of our previous works from the same family (Kanchanapoom et al., 2001, 2002a,b, 2006; Sinaphet et al., 2006). The 1H NMR showed the chemical shifts of a set of 1,3,4-trisubstituted aromatic ring system at dH 7.00 (1H, d, J = 2.0 Hz), 6.74 (1H, dd, J = 8.1, 2.0 Hz) and Table 2 NMR spectroscopic data of 6 and lipedoside A-II (measured in CD3OD). Position 1 2 3 4 5 6 7 8
dC
dC (lipedoside A-II)a
131.9 119.8 146.7 146.3 117.0 125.3 36.5 71.7
131.4 116.4 144.6 146.1 117.1 121.3 36.7 72.3
4.17 (1H, d, J = 7.9 Hz) 3.24 (1H, dd, J = 8.8, 7.9 Hz) 3.42 (1H, dd, J = 9.0, 8.8 Hz) 3.30 (1H)b 3.69 (1H)b 4.35 (1H, dd, J = 12.1, 6.6 Hz) 4.51 (1H, br d, J = 12.1 Hz)
104.1 75.6 84.4 70.1 75.5 64.6
104.4 75.7 84.0 70.4 75.4 64.7
5.11 (1H, br s) 3.92 (1H)b 3.92 (1H)b 3.36 (1H, dd, J = 9.7, 9.5 Hz) 3.95 (1H, m) 1.20 (3H, d, J = 6.2 Hz)
102.6 72.2 72.2 73.9 70.0 17.9
102.7 72.3 72.5 74.0 70.0 17.9
4.70 3.48 3.48 3.28 3.20 3.61 3.81
104.1 74.7 77.3 71.6 77.7 62.6
dH 7.00 (1H, d, J = 2.0 Hz)
6.69 (1H, d, J = 8.1 Hz) 6.74 (1H, dd, J = 8.1, 2.0 Hz) 2.68 (2H, m) 3.35 (1H)b 3.67 (1H)b
Glc 10 20 30 40 50 60
Rha 100 200 300 400 500 600 Glc 1000 2000 3000 4000 5000 6000
b
6.69 (1H, d, J = 8.1 Hz) for the 3,4-dihydroxy-b-phenylethoxyl moiety, a set of 1,4-disubstituted aromatic ring system at dH 7.42 and 6.69 (each 2H, d, J = 8.1 Hz) together with two transolefinic protons at dH 6.34 and 7.60 (each 1H, d, J = 16.0 Hz) for the coumaroyl moiety in addition to three anomeric protons assignable for two glucopyranosyl units at dH 4.17 (1H, d, J = 7.9 Hz) and 4.70 (1H, d, J = 7.8 Hz), and one rhamnopyranosyl unit at dH 5.11 (1H, br s). The 13C NMR spectral data (Table 2) were closely similar to those of lipedoside A-II (He et al., 1994), except for the additional signals arising from one b-D-glucopyranosyl unit. This sugar part was suggested to be located at one hydroxyl group of an aromatic ring of 3,4-dihydroxy-b-phenylethoxyl moiety since the chemical shifts of the aromatic ring were changed. The assignment of the structure was supported by COSY, NOESY, HMQC and HMBC experiments. In the 2D NMR spectra, the HMBC correlation from H1000 (dH 4.70) to C-3 (dC 146.7) along with the NOESY correlation between H-1000 (dH 4.70) and H-2 (dH 7.00) were indicative the connectivity of this additional glucopyranosyl moiety to C-3. Moreover, the results of the HMBC correlations from H-10 (dH 4.17) to C-8 (dC 71.7) and C-30 (dC 84.4), from H2-60 (dH 4.35 and 4.51) to C90000 (dC 169.1), and from H-100 (dH 5.11) to C-30 (dC 84.4) provided the further confirmation of the structure as shown in Fig. 3. Therefore, the structure of compound 6 was elucidated to be 3,4-dihydroxyb-phenylethoxyl 3-O-b-D-glucopyranosyl-8-O-[a-L-rhamnopyranosyl-(1“ ! 3’)]-60 -O-trans-coumaroyl-b-D-glucopyranoside, namely heterophragmoside. Eleven compounds were isolated from H. sulfureum, including five iridoid glycosides (1–5), one phenylethanoid glycosides (6), four lignan glycosides (7–10) and one neolignan glycoside (11). Phenylethanoid glycosides, lignans as well as iridoid glycosides lacking a substituent at C-4 are the common secondary metabolites of the plants from tribe Tecomeae of the family Bignoniaceae (von Poser et al., 2000; Kanchanapoom et al., 2001, 2002a,b, 2006; Sinaphet et al., 2006). This species produces large amounts of iridoid glycosides, especially specioside (1). Its derivative bearing sugar moiety in a-orientation form of galactose (6) is quite unusual to isolate from plant source. The present study provides the further confirmation of the typical profile on the secondary metabolites found in this tribe and might be useful for chemotaxonomic methods of the family Bignoniaceae. 3. Experimental 3.1. General procedures
(1H, d, J = 7.8 Hz) (1H)b (1H)b (1H)b (1H, m) (1H, dd, J = 10.1, 5.4 Hz) (1H, br d, J = 10.1 Hz)
Coumaroyl moiety 100 00 200 00 , 600 00 7.42 (2H, d, J = 8.6 Hz) 30 , 50 6.78 (2H, d, J = 8.6 Hz) 400 00 700 00 7.60 (1H, d, J = 16.0 Hz) 800 00 6.34 (1H, d, J = 16.0 Hz) 00 00 9 a
279
127.1 131.3 116.9 161.1 147.0 114.9 169.1
Data taken from He et al., 1994. Chemical shifts were assigned by COSY and HMQC.
NMR spectra were recorded in CD3OD or C5D5N using a Bruker AV-400 (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometer. MS values were obtained on a JEOL JMS-SX 102 spectrometer. FTIR spectra were recorded on a universal attenuated total reflectance attached (UATR) to a PerkinElmer Spectrum One spectrometer. Optical rotations were measured with
127.1 131.2 116.9 161.2 146.9 114.9 169.1
Fig. 3. HMBC and NOESY correlations of compound 6.
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a Jasco P-1020 digital polarimeter. For column chromatography, silica gel 60 (70–230 mesh, GE-0049, Schalau), RP-18 (50 mm, YMC), and Diaion HP-20 (Mitsubishi Chemical Industries Co., Ltd.) were used. Preparative HPLC was carried out on an ODS column (250 20 mm i.d., YMC) with a Jasco RI-2031 refractive index detector. The flow rate was 6 ml/min. The spraying reagent used for TLC was 10% H2SO4 in 50% EtOH. 3.2. 1 Plant material The leaves and branches of H. sulfureum Kurz was collected in April 2005 from Khon Kaen Province, Thailand. The plant was identified by Mr. Bamrung Thavinchiua, Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher specimen (TK-PSKKU0053) was deposited in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University.
Table 1 NMR Spectroscopic data of compound 2. Position
dH a
1 3 4 5 6 7 8 9 10
5.53 6.45 5.09 2.92 5.40 4.48
Glc 10 20 30 40 50 60
3.3. Extraction and isolation The dried leaves and branches portion of H. sulfureum (12.5 kg) was extracted three times with hot MeOH. The MeOH extract was concentrated in vacuo to give a brownish residue (1.6 kg). This residue was suspended in H2O, and then partitioned with nhexane, EtOAc and n-BuOH, successively. The n-BuOH soluble fraction (639.9 g) was subjected to highly porous synthetic resin column chromatography (Diaion HP-20), and eluted with H2O, 20% aqueous MeOH, 40% aqueous MeOH, 60% aqueous MeOH, 80% aqueous MeOH, MeOH and (CH3)2CO, successively. The residue of the 20% aqueous MeOH eluate (51.2 g) was subjected to silica gel column chromatography with solvent systems: EtOAc (3.0 L), EtOAc–MeOH (9:1, 8.0 L), EtOAc–MeOH–H2O (40:10:1, 9.0 L), EtOAc–MeOH–H2O (70:30:3, 6.0 L) and EtOAc–MeOH–H2O (6:4;1, 4.0 L), respectively to provide eight fractions (Fr. A1–H1). Fraction C1 (29.2 g) was applied to a column of RP-18 with a gradient solvent system, H2O-MeOH (80:20 ! 20:80, v/v) to afford compound 1 (24.5 g). Fraction D1 (7.5 g) was applied to a column of RP-18 a gradient solvent system, H2O-MeOH (90:10 ! 30:70, v/v) to afford nine fractions (Fr. D1-1–D1-9). Fraction D1-4 was purified by preparative HPLC with solvent system 15% aqueous MeCN to obtain compound 7 (83.2 mg). Fraction D1-5 was purified by preparative HPLC-ODS with solvent system 17% aqueous MeCN to give compounds 5 (51.1 mg), 9 (47.6 mg), 10 (23.5 mg), and 11 (12.3 mg). Fraction D1-7 was purified by preparative HPLC-ODS with solvent system 20% aqueous MeCN to give compound 6 (119.1 mg). Similarly, fraction E1 (13.3 g) was applied to a column of RP-18 with a gradient solvent system, H2O-MeOH (90:10 ! 20:80, v/v) to afford 12 fractions (Fr. E1-1–E1-12). Fraction E1-10 was purified by preparative HPLC with solvent system 20% aqueous MeCN to obtain compounds 2 (22.2 mg) and 3 (35.4 mg). The residue of the 40% aqueous MeOH eluate (30.0 g from total 202.0 g) was subjected to silica gel column chromatography with solvent systems: EtOAc (3.0 L), EtOAc–MeOH (9:1, 8.0 L), EtOAc– MeOH–H2O (40:10:1, 9.0 L), EtOAc–MeOH–H2O (70:30:3, 6.0 L) and EtOAc–MeOH–H2O (6:4;1, 4.0 L) to provide eight fractions (Fr. A2– H2). Fraction D2 was provided compound 1 (21.2 g) by crystallization. Fraction F2 (3.0 g) was applied to a column of RP-18 with a gradient solvent system, H2O-MeOH (80:20 ! 20:80, v/v) to afford five fractions (Fr. D2-1–D2-5). Fraction D2-3 was purified by preparative HPLC with solvent system 17% aqueous MeCN to give compound 8 (6.4 mg). The residue of the 60% aqueous MeOH eluate (30.0 g from total 65.2 g) was subjected to silica gel column chromatography with solvent systems: EtOAc–MeOH (9:1, 4.0 L), EtOAc–MeOH–H2O (40:10:1, 7.0 L), and EtOAc–MeOH–H2O (70:30:3, 5.0 L) to provide six fractions (Fr. A3–F3). Fraction E3 (3.9 g) was partially purified by
Gal 100 200 300 400 500 600
dC a
dC b
94.8 141.5 102.3 36.1 80.5 58.9 66.6 43.0 59.6
95.4 142.5 102.9 36.6 81.5 59.7 66.7 43.4 60.9
5.39 (1H, d, J = 7.8 Hz) 4.10 (1H, d, J = 8.8, 7.8 Hz) 4.22 (1H, dd, J = 9.0, 8.8 Hz) 4.00 (1H, dd, J = 9.3, 9.0 Hz) 4.01 (1H, m) 4.30 (1H, br d, J = 9.5 Hz) 4.45 (1H)c
100.0 74.5 78.0 71.5 76.3 68.0
100.0 74.8 77.7 71.7 76.6 68.2
5.46 4.72 4.61 4.80 4.48 4.49 4.59
100.2 70.3 71.6 70.8 72.8 62.5
100.2 70.0 72.0 71.0 72.7 62.8
125.7 130.6 116.7 161.5 145.9 114.1 167.3
127.0 131.4 116.9 161.4 147.5 114.4 169.1
(1H, d, J = 9.3 Hz) (1H, d, J = 5.9 Hz) (1H, dd, J = 5.9, 4.2 Hz) (1H)c (1H, br d, J = 7.3 Hz) (1H)c
2.90 (1H)c 4.48 (1H, d, J = 9.5 Hz) 4.75 (1H, d, J = 9.5 Hz)
(1H, d, J = 3.6 Hz) (1H, dd, J = 9.8, 3.6 Hz) (1H, dd, J = 9.8, 3.2 Hz) (1H, dd, J = 3.2, 3.2 Hz) (1H, m) (1H)c (1H, br d, J = 11.0 Hz)
Coumaroyl moiety 1000 2000 , 6000 7.58 (2H, d, J = 8.3 Hz) 3000 , 5000 7.21 (2H, d, J = 8.3 Hz) 4000 7000 7.88 (1H, d, J = 15.9 Hz) 8000 6.52 (1H, d, J = 15.9 Hz) 9000 a b c
Measured in C5D5N. Measured in CD3OD. Chemical shifts were assigned by COSY and HMQC.
a RP-18 column with a gradient solvent system, H2O-MeOH (90:10 ! 30:70, v/v), and then purified by preparative HPLC-ODS using solvent system 24% aqueous MeCN to afford compound 4 (12.8 mg). 3.4. Specioside 6'-O-a-D-galactopyranoside (2) Amorphous powder, [a]D24 60.5 (MeOH, c 0.77); IR spectrum: nmax = 3339, 2922, 1691, 1631, 1603, 1515, 1262, 1226, 1154, 1013,
832 cm1; 1H (C5D5N) and 13C NMR (CD3OD and C5D5N): Table 1. Negative HR-FAB-MS, m/z: 669.1994 (C30H37O17 required 669.2031). 3.5. Heterophragmoside (6) Amorphous powder, [a]D24 70.9 (MeOH, c 2.34); IR spectrum: nmax = 3364, 2927, 1691, 1604, 1515, 1264, 1167, 1062, 1017,
833 cm1; 1H and 13C NMR (CD3OD): Table 2; negative HR-FABMS, m/z: 769.2574 (C35H45O19 required 769.2555). Acknowledgements
This research work was supported by the grant from the Thailand Research Fund (DBG5480007), Chulabhorn Research Institute, Khon Kaen University; and Center of Excellence on Environmental Health and Toxicology, Science & Technology Postgraduate Education and Research Development Office (PERDO), Ministry of Education.
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