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Two new chalcone glycosides from the stems of Entada phaseoloides
Fitoterapia 82 (2011) 1102–1105
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Two new chalcone glycosides from the stems of Entada phaseoloides Zhong-xiang Zhao a, 1, Jing Jin b, 1, Chao-zhan Lin a, Chen-chen Zhu a,⁎, Yi-ming Liu c, Ai-hua Lin c, Ying-xiang Liu a, Li Zhang a, Hua-feng Luo a a b c
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China Guangdong Provincial Hospital of TCM Affiliated to Guangzhou University of Chinese Medicine, Guangzhou 510120, China
a r t i c l e
i n f o
Article history: Received 25 May 2011 Accepted in revised form 6 July 2011 Available online 20 July 2011 Keywords: Entada phaseoloides Chalcone glycosides Flavonoids
a b s t r a c t Two new chalcone glycosides 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′,4-dihydroxychalcone (1) and 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′-hydroxy-4-methoxychalcone (2) together with one known chalcone glycoside 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone (3) were isolated from the stems of Entada phaseoloides. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis, including HSQC, HMBC, 1H–1H COSY, and chemical evidences. This is the first report of chalcone-type compounds isolated from the genus Entada. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The genus Entada (legume family) comprises approximately 30 species of trees, shrubs and lianas, and there is only one species known in China. Entada phaseoloides (L.) Merr, a giant woody climber, is mainly distributed in the south of China and other tropical countries [1]. Both the stems and seeds of E. phaseoloides are well-known traditional Chinese medicines, and they have been used singly or combined with other herbs to treat various diseases for a long time, but their medicinal properties are quite different. The seeds of E. phaseoloides, called “Ketengzi” in China, have been used to treat hemorrhoids, febrifuge, constipation and stomachache, while the stems of this plant, named “Guoganglong” in China, have been used widely for the treatment of rheumatoid arthritis, tetraplegia and traumatic injury [2,3]. Previous phytochemical investigations on the seeds or barks of E. phaseoloides have reported that the main constituents are thioamides [4–7], phenylacetic acid derivatives [8,9], and saponins [10–12]; however, until recently there have been few studies on the constituents of the stems of this species.
⁎ Corresponding author. Tel./fax: + 86 20 39358047. E-mail address: [email protected] (C. Zhu). 1 Co-first authors. 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.07.005
In the effort to search for novel and bioactive compounds from Chinese herbal medicines, we have now investigated the constituents of E. phaseoloides. Herein, we report on the isolation and structural elucidation of two new chalcone glycosides 4′-O(6″-O-galloyl-β-D-glucopyranosyl)-2′,4-dihydroxychalcone (1) and 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′-hydroxy-4methoxychalcone (2) together with one known chalcone glycoside 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone (3) (Fig. 1). To the best of our knowledge, this is the first report of chalcone-type compounds isolated from the genus Entada. 2. Experimental methods 2.1. General Optical rotations were measured on a PerkinElmer Model 341 polarimeter. UV spectra were recorded on an Agilent 8453 UV–Visible spectrophotometer. IR spectra were determined on a Thermo AVATAR330 FT-IR spectrophotometer. NMR spectra were taken with TMS as internal standard on a Bruker AM-400 spectrometer. HRESIMS were obtained on a WATERS Q-TOF micro mass spectrometer. The GC was performed on an Agilent 6890N gas chromatograph with a HP-5 Phenyl Methyl Siloxane capillary column (30 m × 0.32 mm × 0.25 μm). Column
Z. Zhao et al. / Fitoterapia 82 (2011) 1102–1105
1103
Fig. 1. Structures of Compounds 1–3.
chromatography was carried out with silica gel (Qingdao Marine Chemical Company), ODS (230–400 mesh, Fluka BioChemika), and Sephadex LH 20 (25–100 μm, Fluka BioChemika). 2.2. Plant The stems of E. phaseoloides (L.) Merr were purchased from Guangdong Luofushan SinoPharm Co. Ltd. in October 2008. The stems were air-dried at room temperature. A voucher specimen (ZYXY-EP-2008-001) was deposited at College of Chinese Materia Medica, Guangzhou University of Chinese Medicine. 2.3. Extraction and isolation The air-dried stems (5.4 kg) were ground and extracted with MeOH (50 L) under reflux condition. The MeOH extract was concentrated under vacuum to leave a residue, which was suspended in H2O (3 L) and extracted with petroleum ether (3 × 3 L), EtOAc (3 × 3 L), and n-BuOH (3 × 3 L), sequentially. A part of the EtOAc extract (172 g) was chromatographed on silica gel column (200–300 mesh, 1250 g) eluting with CHCl 3 –MeOH gradient system (50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 2:1 → 1:1) to give fifteen fractions (Fr. A1–A15). Fr. A9 (5.1 g) was separated over a silica gel column eluting with EtOAc–MeOH (50:1 → 3:1) to obtain a target portion (0.9 g), which was further purified by RP C18 column chromatography (200 g) eluted with a MeOH–H2O gradient (1:3 → 2:1) to give 2 (22 mg) and 3 (36 mg). Fr. A10 (3.9 g) was subjected a silica gel column eluting with a CHCl3– MeOH gradient (50:1 → 5:1) to give five fractions (Fr. B1–B5). Fraction B5 was separated by Sephadex LH 20 column chromatography using CHCl3–MeOH (1:1), then purified by RP C18 column chromatography (MeOH-H2O, 1:3 → 1:1), yielding 1 (16 mg).
4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′,4-dihydroxychalcone (1), yellow amorphous powder; [α]D20 −97° (c 0.20, acetone); UV (MeOH) λmax (log ε) 284 (4.28), 369 (4.55) nm; IR (KBr) νmax 3417, 2974, 1693, 1636, 1609, 1547, 1514, 1385, 1220, 1077, 1036 cm-1; HRESI-TOFMS m/z 569.1289 [M−H]– (calculated for C28H25O13, 569.1295). 1H NMR (400 MHz in acetone-d6) and 13C NMR (100 MHz in acetone-d6), see Table 1; 1H–1H COSY and HMBC were reported in Fig. 2. 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′-hydroxy-4methoxychalcone (2), yellow amorphous powder; [α]D20 −125° (c 0.12, acetone); UV (MeOH) λmax (log ε) 286 (4.04), 369 (4.49) nm; IR (KBr) νmax 3419, 2936, 1707, 1631, 1602, 1560, 1510, 1384, 1224, 1070, 1029 cm-1; HRESI-TOFMS m/z 583.1450 [M − H] − (calculated for C29H27O13, 583.1451). 1H NMR (400 MHz in DMSO-d6) and 13C NMR (100 MHz in DMSO-d6), see Table 1. 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone (3), yellow powder; [α]D20 − 52° (c 0.13, acetone); UV (MeOH) λmax (log ε) 286 (4.24), 365 (4.50) nm; IR (KBr) νmax 3381, 2924, 1637, 1604, 1567, 1513, 1384, 1219, 1070, 1017 cm -1; ESIMS m/z 433 [M + H] +. 1H NMR (400 MHz in DMSO-d6) and 13C NMR (100 MHz in DMSO-d6), see Table 1.
2.4. Acidic hydrolysis of 1 Compounds 1–3 (each 3 mg) were hydrolyzed with 9% HCl (1.5 mL) at 90 °C for 6 h, respectively. After being cooled, the reaction mixture was filtered, and then the filtrate was freezedried. The residue was dissolved in dry pyridine, and stirred with L-cysteine methyl ester hydrochloride (0.1 M) and HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane, 2:1) using the same procedures as described previously [13]. After the reactions, the supernatant was analyzed by GC under the following conditions: temperature gradient system for the oven, 150 °C for 1 min and then raised to 280 °C at the rate of 5 °C/min; carrier gas N2 (1 mL/min); split ratio 1:10; injector
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Z. Zhao et al. / Fitoterapia 82 (2011) 1102–1105
Table 1 13 C and 1H NMR data for compounds 1–3 (100 and 400 MHz, δ in ppm, J in Hz). 1
2
Position
δC
α β CO 1 2, 6 3, 5 4 4-OMe 1′ 2′ 3′ 4′ 5′ 6′ 2′-OH 1″ 2″ 3″ 4″ 5″ 6″
118.2 146.0 193.4 127.6 132.1 117.0 161.3
d d s s d d s
116.3 166.9 105.2 164.8 108.9 132.9
s s d s d d
101.2 74.7 77.9 71.2 75.5 64.4
d d d d d t
1‴ 2‴, 6‴ 3‴, 5‴ 4‴ COO
121.9 110.3 146.2 139.1 166.7
s d s s s
3
δH
δC
7.60 (brd, 15.2) 7.73 (brd, 15.2)
118.3 144.6 192.0 127.1 131.2 114.5 161.6 55.4 114.9 165.0 103.7 163.3 107.8 132.5
d d s s d d s q s s d s d d
99.5 73.1 76.2 69.4 73.9 63.1
d d d d d t
119.3 108.8 145.5 138.6 165.7
s d s s s
7.64 (d, 8.4) 6.82 (d, 8.4)
6.47 (d, 2.4) 6.59 (dd, 9.2, 2.4) 8.00 (d, 9.2) 13.40 (brs) 5.06 (d, 7.2) 3.45 (m) 3.50 (m) 3.47 (m) 3.82 (m) 4.55 (dd, 12.0, 2.4) 4.24 (dd, 12.0, 6.4) 7.07 (s)
δH
δC
7.83 (brs)a 7.83 (brs)a
118.5 144.5 192.1 127.2 131.1 114.4 161.6 55.4 114.8 165.1 103.5 163.6 108.2 132.5
d d s s d d s q s s d s d d
99.6 73.1 76.4 69.6 77.2 60.6
d d d d d t
7.90 (d, 8.8) 7.06 (d, 8.8) 3.84 (s)
6.59 (d, 2.4) 6.68 (dd, 8.8, 2.4) 8.22 (d, 8.8) 13.36 (brs) 5.15 (d, 7.2) 3.35 (m) 3.37 (m) 3.36 (m) 3.80 (m) 4.46 (dd, 11.2, 1.6) 4.27 (dd, 11.2, 5.2)
δH 7.83 (brd, 15.6) 7.91 (brd, 15.6)
7.90 (d, 8.8) 7.04 (d, 8.8) 3.84 (s)
6.59 (d, 2.4) 6.65 (dd, 9.2, 2.4) 8.31 (d, 9.2) 13.45 (brs) 5.05 (d, 7.2) 3.26 (m) 3.31 (m) 3.17 (t, 8.8) 3.41 (m) 3.72 (brd, 10.8) 3.47 (dd, 10.8, 5.6)
7.01 (s)
Compounds 2 and 3 were measured in DMSO-d6 and Compound 1 in acetone-d6. Assignments are based on HSQC, 1H–1H COSY, and HMBC spectroscopic data. a Overlapping signals.
temp 230 °C and detector temp 250 °C. The peak corresponding to the D-glucosyl derivative appeared at tR of 22.08 min. 3. Results and discussion The air-dried stems of E. phaseoloides were extracted with MeOH, and then the extract was concentrated under vacuum to leave a residue, which was suspended in H2O and extracted with petroleum ether, EtOAc, and n-BuOH, sequentially. The EtOAc-soluble extract of E. phaseoloides was subjected to column chromatography over silica gel, RP C18 and Sephadex LH-20 to afford three compounds 1–3. Compound 1 (Fig. 1) was isolated as yellow amorphous powder and its molecular formula was established as C28H26O13 from HRESI-TOFMS for the peak at m/z 569.1289 [M − H] – (calculated for C28H25O13, 569.1295) and 13C NMR data analysis. The UV spectrum exhibited maxima at 284 and 369 nm, suggesting the existence of a complex chromogen system. The IR spectrum displayed absorption bands for hydroxyl groups (3417 cm -1), carbonyl groups (1693 and 1636 cm -1) and aromatic rings (1609, 1514 cm -1). The 1H NMR spectrum (Table 1) showed a pair of A2B2 type signals at δH 7.64, 6.82 (2H each, both d, J = 8.4 Hz) attributed to a para-substituted phenyl group, a set of ABX type signals at δH 8.00 (1H, d, J = 9.2 Hz), 6.59 (1H, dd, J = 9.2, 2.4 Hz) and 6.47 (1H, d, J = 2.4 Hz) due to a 1,3,4-trisubstituted aromatic ring, a pair of trans-olefinic proton signals at δH 7.73, 7.60 (1H each, both d, J = 15.2 Hz), an anomeric proton at δH 5.06 (1H, d, J = 7.2 Hz) suggesting a sugar moiety. The 13C NMR spectrum (Table 1) analyzed with the aid of the DEPT and HSQC spectra further supported these
results and revealed the existence of one conjugated ketone (δC 193.4), one double bond (δC 146.0 and 118.2) and a galloyl group (δC 166.7, 146.2, 139.1, 121.9, and 110.3) which was confirmed by the presence of a two-proton singlet at δH 7.07 (2H, s) in the 1H NMR spectrum. On acid hydrolysis of 1, D-glucose was detected by GC. The β-configuration of the glycosidic bond was deduced from the coupling constant of the anomeric proton and the 13C NMR data of the sugar unit (Table 1). The above structural characteristics indicated 1 is a chalcone derivative consisting of a chalcone moiety, a D-glucose moiety and a galloyl group. The 1H and 13C NMR signals of 1 were completely assigned by a combination of HSQC, HMBC, and 1H– 1H COSY experiments (Table 1). The 1H– 1H COSY correlation (Fig. 2) between H-α (δH 7.60) and H-β (δH 7.73), and the HMBC correlations (Fig. 2) of H-α to C = O (δC 193.4), C-β (δC 146.0) and C-1 (δC 127.6), H-β to C = O (δC 193.4), C-α (δC 118.2), C-1 and C-2/6 (δC 132.1), H-2/6 (δH 7.64) to C-β, C-3/5 (δC 117.0) and C-4 (δC 161.3), and H-6′ (δH 8.00) to C = O (δC 193.4), C-1′ (δC 116.3), C-2′ (δC 166.9), C-4′ (δC 164.8) and C-5′ (δC 108.9) established the structure of aglycone as 2′,4,4′-trihydroxychalcone. Both C-2′ and C-4′ of the aglycone are oxygenated aromatic carbons, which have very close chemical shifts at δC 166.9 or 164.8, and they were distinguished through long-range coupling to H-5′ (δ H 6.59). The signal at δ C 164.8 was assigned to C-4′ on the basis of an HMBC correlation to H-5′ (Fig. 2), and the other signal at δC 166.9 was thus assigned to C-2′. The linkage of the glucose unit to the aglycone was determined by 1H NMR and HMBC spectral analysis. The 1H NMR spectrum of 1 showed the presence of an extreme
Z. Zhao et al. / Fitoterapia 82 (2011) 1102–1105
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Fig. 2. Key HMBC and 1H–1H COSY correlations of compound 1.
downfield characteristic signal for 2′-OH (δ H 13.40) due to the intramolecular hydrogen bonding with C = O (δ C 193.4) [14,15], and this suggested that C-2′ was not the sugar binding site. The attachment of the D -glucose to C-4′ of the aglycone was finally established by the HMBC correlation between H-1″ (δ H 5.06) and C-4′ (δC 164.8) (Fig. 2). The above substitution pattern of the aglycone was further verified by the comparison of the NMR data of 1 with those of 4′-O-glucopyranosyl-2′,4-dihydroxy-3methoxy-chalcone [16], which is a known compound with similar aglycone structure. An HMBC correlation of H2 -6″ (δ H 4.55 and 4.24) to carbonyl carbon (δ C 166.7) indicated that the galloyl group was located at C-6″ of the glucose, which was also supported by the chemical shift of C-6″ of the glucose at lower field (δ C 64.4, Δδ 3.8) compared with that of 3. Therefore, compound 1 was unambiguously characterized as 4′-O-(6″-O-galloyl-β-D glucopyranosyl)-2′,4-dihydroxychalcone. Compound 2 was isolated as yellow amorphous powder and had the molecular formula C29H28O13 determined by HRESITOFMS (m/z 583.1450 [M− H]−, calculated for C29H27O13, 583.1451) and 13C NMR. The 1H and 13C NMR data (Table 1) of 2 were very close to those of 1. The only difference between these two compounds was the appearance of a methoxyl group in 2, and an HMBC correlation of OMe-4 (δH 3.84, s) to C-4 (δC 161.3) established the location of the methoxy at C-4. The detailed assignments of the 1H and 13C NMR signals (Table 1) were performed by HSQC, 1H–1H COSY and HMBC experiments. Thus, the structure of 2 was assigned as 4′-O-(6″-O-galloyl-β-Dglucopyranosyl)-2′-hydroxy-4-methoxychalcone. The known compound 3 was isolated from natural resources for the first time and its spectroscopic data have not been previously reported. The structure of 3 was elucidated as 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone on the basis of extensive spectroscopic analysis, including 1H and 13C NMR, HMBC, 1H– 1H COSY and chemical evidence.
References [1] Delectis florae Reipublicae Popularis Sinicae agendae Academiae Sinicae edita. Flora Reipublicae Popularis Sinicae, Vol. 39. Beijing: Science Press; 1988. p. 13. [2] Chinese Materia Medica Editorial Committee. Chinese Materia Medica. Shanghai: Shanghai Scientific and Technical Press; 1999. p. 465. [3] Nanjing University of Traditional Chinese Medicine. Dictionary of Chinese Materia Medica. Shanghai: Shanghai Science and Technology Publishing House; 2006. p. 3563. [4] Xiong H, Xiao E, Zhao YH, Yang GZ, Mei ZN. Sulfur-containing amides from Entada phaseoloides. Acta Pharm Sin 2010;45:624–6. [5] Ikegami F, Sekine T, Duangteraprecha S, Matsushita N, Matsuda N, Ruangrungsi N, et al. Entadamide C, a sulfur-containing amide from Entada phaseoloides. Phytochemistry 1989;28:881–2. [6] Ikegami F, Shibasaki I, Ohmiya S, Ruangrungsi N, Murakoshi I. Entadamide A, a new sulfur-containing amide from Entada phaseoloides seeds. Chem Pharm Bull 1985;33:5153–4. [7] Ikegami F, Ohmiya S, Ruangrungsi N, Sakai S, Murakoshi I. Entadamide B, a second new sulfur-containing amide from Entada phaseoloides. Phytochemistry 1987;26:1525–6. [8] Dai J, Kardono LBS, Tsauri S, Padmawinata K, Pezzuto JM, Kinghorn AD. Studies on Indonesian medicinal plants. Part 3. phenylacetic acid derivatives and a thioamide glycoside from Entada phaseoloides. Phytochemistry 1991;30:3749–52. [9] Barua AK, Chakrabarty M, Datta PK, Ray S. Phaseoloidin, a homogentisic acid glucoside from Entada phaseoloides. Phytochemistry 1988;27:3259–61. [10] Liu WC, Kugelman M, Wilson RA, Rao KV. Crystalline saponin with antitumor activity from Entada phaseoloides. Phytochemistry 1972;11:171–3. [11] Okada Y, Shibata S, Javellana Ana MJ, Kamo O. Entada saponins (ES) II and IV from the bark of Entada phaseollides. Chem Pharm Bull 1988;36:1264–9. [12] Okada Y, Shibata S, Ikekawa T, Javellana AMJ, Kamo O. Entada saponin III, a saponin isolated from the bark of Entada phaseoloides. Phytochemistry 1987;26:2789–96. [13] Zhao ZX, Ruan JL, Jin J, Zou J, Zhou DN, Fang W, et al. Flavan-4-ol glycosides from the rhizomes of Abacopteris penangiana. J Nat Prod 2006;69:265–8. [14] Exarchou V, Troganis A, Gerothanassis IP, Tsimidou M, Boskou D. Do strong intramolecular hydrogen bonds persist in aqueous solution? Variable temperature gradient 1H, 1H-13C GE-HSQC and GE-HMBC NMR studies of flavonols and flavones in organic and aqueous mixtures. Tetrahedron 2002;58:7423–9. [15] Ngameni B, Patnam R, Sonna P, Ngadjui BT, Roy R, Abegaz BM. Hemisynthesis and spectroscopic characterization of three glycosylated 4-hydroxylonchocarpins from Dorstenia barteri Bureau. Arkivoc 2008;6: 152–9. [16] Calanasan CA, Macleod JK. A diterpenoid sulfate and flavonoids from Wedelia asperrima. Phytochemistry 1998;47:1093–9.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Two new chalcone glycosides from the stems of Entada phaseoloides Zhong-xiang Zhao a, 1, Jing Jin b, 1, Chao-zhan Lin a, Chen-chen Zhu a,⁎, Yi-ming Liu c, Ai-hua Lin c, Ying-xiang Liu a, Li Zhang a, Hua-feng Luo a a b c
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China Guangdong Provincial Hospital of TCM Affiliated to Guangzhou University of Chinese Medicine, Guangzhou 510120, China
a r t i c l e
i n f o
Article history: Received 25 May 2011 Accepted in revised form 6 July 2011 Available online 20 July 2011 Keywords: Entada phaseoloides Chalcone glycosides Flavonoids
a b s t r a c t Two new chalcone glycosides 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′,4-dihydroxychalcone (1) and 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′-hydroxy-4-methoxychalcone (2) together with one known chalcone glycoside 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone (3) were isolated from the stems of Entada phaseoloides. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis, including HSQC, HMBC, 1H–1H COSY, and chemical evidences. This is the first report of chalcone-type compounds isolated from the genus Entada. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The genus Entada (legume family) comprises approximately 30 species of trees, shrubs and lianas, and there is only one species known in China. Entada phaseoloides (L.) Merr, a giant woody climber, is mainly distributed in the south of China and other tropical countries [1]. Both the stems and seeds of E. phaseoloides are well-known traditional Chinese medicines, and they have been used singly or combined with other herbs to treat various diseases for a long time, but their medicinal properties are quite different. The seeds of E. phaseoloides, called “Ketengzi” in China, have been used to treat hemorrhoids, febrifuge, constipation and stomachache, while the stems of this plant, named “Guoganglong” in China, have been used widely for the treatment of rheumatoid arthritis, tetraplegia and traumatic injury [2,3]. Previous phytochemical investigations on the seeds or barks of E. phaseoloides have reported that the main constituents are thioamides [4–7], phenylacetic acid derivatives [8,9], and saponins [10–12]; however, until recently there have been few studies on the constituents of the stems of this species.
⁎ Corresponding author. Tel./fax: + 86 20 39358047. E-mail address: [email protected] (C. Zhu). 1 Co-first authors. 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.07.005
In the effort to search for novel and bioactive compounds from Chinese herbal medicines, we have now investigated the constituents of E. phaseoloides. Herein, we report on the isolation and structural elucidation of two new chalcone glycosides 4′-O(6″-O-galloyl-β-D-glucopyranosyl)-2′,4-dihydroxychalcone (1) and 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′-hydroxy-4methoxychalcone (2) together with one known chalcone glycoside 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone (3) (Fig. 1). To the best of our knowledge, this is the first report of chalcone-type compounds isolated from the genus Entada. 2. Experimental methods 2.1. General Optical rotations were measured on a PerkinElmer Model 341 polarimeter. UV spectra were recorded on an Agilent 8453 UV–Visible spectrophotometer. IR spectra were determined on a Thermo AVATAR330 FT-IR spectrophotometer. NMR spectra were taken with TMS as internal standard on a Bruker AM-400 spectrometer. HRESIMS were obtained on a WATERS Q-TOF micro mass spectrometer. The GC was performed on an Agilent 6890N gas chromatograph with a HP-5 Phenyl Methyl Siloxane capillary column (30 m × 0.32 mm × 0.25 μm). Column
Z. Zhao et al. / Fitoterapia 82 (2011) 1102–1105
1103
Fig. 1. Structures of Compounds 1–3.
chromatography was carried out with silica gel (Qingdao Marine Chemical Company), ODS (230–400 mesh, Fluka BioChemika), and Sephadex LH 20 (25–100 μm, Fluka BioChemika). 2.2. Plant The stems of E. phaseoloides (L.) Merr were purchased from Guangdong Luofushan SinoPharm Co. Ltd. in October 2008. The stems were air-dried at room temperature. A voucher specimen (ZYXY-EP-2008-001) was deposited at College of Chinese Materia Medica, Guangzhou University of Chinese Medicine. 2.3. Extraction and isolation The air-dried stems (5.4 kg) were ground and extracted with MeOH (50 L) under reflux condition. The MeOH extract was concentrated under vacuum to leave a residue, which was suspended in H2O (3 L) and extracted with petroleum ether (3 × 3 L), EtOAc (3 × 3 L), and n-BuOH (3 × 3 L), sequentially. A part of the EtOAc extract (172 g) was chromatographed on silica gel column (200–300 mesh, 1250 g) eluting with CHCl 3 –MeOH gradient system (50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 2:1 → 1:1) to give fifteen fractions (Fr. A1–A15). Fr. A9 (5.1 g) was separated over a silica gel column eluting with EtOAc–MeOH (50:1 → 3:1) to obtain a target portion (0.9 g), which was further purified by RP C18 column chromatography (200 g) eluted with a MeOH–H2O gradient (1:3 → 2:1) to give 2 (22 mg) and 3 (36 mg). Fr. A10 (3.9 g) was subjected a silica gel column eluting with a CHCl3– MeOH gradient (50:1 → 5:1) to give five fractions (Fr. B1–B5). Fraction B5 was separated by Sephadex LH 20 column chromatography using CHCl3–MeOH (1:1), then purified by RP C18 column chromatography (MeOH-H2O, 1:3 → 1:1), yielding 1 (16 mg).
4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′,4-dihydroxychalcone (1), yellow amorphous powder; [α]D20 −97° (c 0.20, acetone); UV (MeOH) λmax (log ε) 284 (4.28), 369 (4.55) nm; IR (KBr) νmax 3417, 2974, 1693, 1636, 1609, 1547, 1514, 1385, 1220, 1077, 1036 cm-1; HRESI-TOFMS m/z 569.1289 [M−H]– (calculated for C28H25O13, 569.1295). 1H NMR (400 MHz in acetone-d6) and 13C NMR (100 MHz in acetone-d6), see Table 1; 1H–1H COSY and HMBC were reported in Fig. 2. 4′-O-(6″-O-galloyl-β-D-glucopyranosyl)-2′-hydroxy-4methoxychalcone (2), yellow amorphous powder; [α]D20 −125° (c 0.12, acetone); UV (MeOH) λmax (log ε) 286 (4.04), 369 (4.49) nm; IR (KBr) νmax 3419, 2936, 1707, 1631, 1602, 1560, 1510, 1384, 1224, 1070, 1029 cm-1; HRESI-TOFMS m/z 583.1450 [M − H] − (calculated for C29H27O13, 583.1451). 1H NMR (400 MHz in DMSO-d6) and 13C NMR (100 MHz in DMSO-d6), see Table 1. 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone (3), yellow powder; [α]D20 − 52° (c 0.13, acetone); UV (MeOH) λmax (log ε) 286 (4.24), 365 (4.50) nm; IR (KBr) νmax 3381, 2924, 1637, 1604, 1567, 1513, 1384, 1219, 1070, 1017 cm -1; ESIMS m/z 433 [M + H] +. 1H NMR (400 MHz in DMSO-d6) and 13C NMR (100 MHz in DMSO-d6), see Table 1.
2.4. Acidic hydrolysis of 1 Compounds 1–3 (each 3 mg) were hydrolyzed with 9% HCl (1.5 mL) at 90 °C for 6 h, respectively. After being cooled, the reaction mixture was filtered, and then the filtrate was freezedried. The residue was dissolved in dry pyridine, and stirred with L-cysteine methyl ester hydrochloride (0.1 M) and HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane, 2:1) using the same procedures as described previously [13]. After the reactions, the supernatant was analyzed by GC under the following conditions: temperature gradient system for the oven, 150 °C for 1 min and then raised to 280 °C at the rate of 5 °C/min; carrier gas N2 (1 mL/min); split ratio 1:10; injector
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Z. Zhao et al. / Fitoterapia 82 (2011) 1102–1105
Table 1 13 C and 1H NMR data for compounds 1–3 (100 and 400 MHz, δ in ppm, J in Hz). 1
2
Position
δC
α β CO 1 2, 6 3, 5 4 4-OMe 1′ 2′ 3′ 4′ 5′ 6′ 2′-OH 1″ 2″ 3″ 4″ 5″ 6″
118.2 146.0 193.4 127.6 132.1 117.0 161.3
d d s s d d s
116.3 166.9 105.2 164.8 108.9 132.9
s s d s d d
101.2 74.7 77.9 71.2 75.5 64.4
d d d d d t
1‴ 2‴, 6‴ 3‴, 5‴ 4‴ COO
121.9 110.3 146.2 139.1 166.7
s d s s s
3
δH
δC
7.60 (brd, 15.2) 7.73 (brd, 15.2)
118.3 144.6 192.0 127.1 131.2 114.5 161.6 55.4 114.9 165.0 103.7 163.3 107.8 132.5
d d s s d d s q s s d s d d
99.5 73.1 76.2 69.4 73.9 63.1
d d d d d t
119.3 108.8 145.5 138.6 165.7
s d s s s
7.64 (d, 8.4) 6.82 (d, 8.4)
6.47 (d, 2.4) 6.59 (dd, 9.2, 2.4) 8.00 (d, 9.2) 13.40 (brs) 5.06 (d, 7.2) 3.45 (m) 3.50 (m) 3.47 (m) 3.82 (m) 4.55 (dd, 12.0, 2.4) 4.24 (dd, 12.0, 6.4) 7.07 (s)
δH
δC
7.83 (brs)a 7.83 (brs)a
118.5 144.5 192.1 127.2 131.1 114.4 161.6 55.4 114.8 165.1 103.5 163.6 108.2 132.5
d d s s d d s q s s d s d d
99.6 73.1 76.4 69.6 77.2 60.6
d d d d d t
7.90 (d, 8.8) 7.06 (d, 8.8) 3.84 (s)
6.59 (d, 2.4) 6.68 (dd, 8.8, 2.4) 8.22 (d, 8.8) 13.36 (brs) 5.15 (d, 7.2) 3.35 (m) 3.37 (m) 3.36 (m) 3.80 (m) 4.46 (dd, 11.2, 1.6) 4.27 (dd, 11.2, 5.2)
δH 7.83 (brd, 15.6) 7.91 (brd, 15.6)
7.90 (d, 8.8) 7.04 (d, 8.8) 3.84 (s)
6.59 (d, 2.4) 6.65 (dd, 9.2, 2.4) 8.31 (d, 9.2) 13.45 (brs) 5.05 (d, 7.2) 3.26 (m) 3.31 (m) 3.17 (t, 8.8) 3.41 (m) 3.72 (brd, 10.8) 3.47 (dd, 10.8, 5.6)
7.01 (s)
Compounds 2 and 3 were measured in DMSO-d6 and Compound 1 in acetone-d6. Assignments are based on HSQC, 1H–1H COSY, and HMBC spectroscopic data. a Overlapping signals.
temp 230 °C and detector temp 250 °C. The peak corresponding to the D-glucosyl derivative appeared at tR of 22.08 min. 3. Results and discussion The air-dried stems of E. phaseoloides were extracted with MeOH, and then the extract was concentrated under vacuum to leave a residue, which was suspended in H2O and extracted with petroleum ether, EtOAc, and n-BuOH, sequentially. The EtOAc-soluble extract of E. phaseoloides was subjected to column chromatography over silica gel, RP C18 and Sephadex LH-20 to afford three compounds 1–3. Compound 1 (Fig. 1) was isolated as yellow amorphous powder and its molecular formula was established as C28H26O13 from HRESI-TOFMS for the peak at m/z 569.1289 [M − H] – (calculated for C28H25O13, 569.1295) and 13C NMR data analysis. The UV spectrum exhibited maxima at 284 and 369 nm, suggesting the existence of a complex chromogen system. The IR spectrum displayed absorption bands for hydroxyl groups (3417 cm -1), carbonyl groups (1693 and 1636 cm -1) and aromatic rings (1609, 1514 cm -1). The 1H NMR spectrum (Table 1) showed a pair of A2B2 type signals at δH 7.64, 6.82 (2H each, both d, J = 8.4 Hz) attributed to a para-substituted phenyl group, a set of ABX type signals at δH 8.00 (1H, d, J = 9.2 Hz), 6.59 (1H, dd, J = 9.2, 2.4 Hz) and 6.47 (1H, d, J = 2.4 Hz) due to a 1,3,4-trisubstituted aromatic ring, a pair of trans-olefinic proton signals at δH 7.73, 7.60 (1H each, both d, J = 15.2 Hz), an anomeric proton at δH 5.06 (1H, d, J = 7.2 Hz) suggesting a sugar moiety. The 13C NMR spectrum (Table 1) analyzed with the aid of the DEPT and HSQC spectra further supported these
results and revealed the existence of one conjugated ketone (δC 193.4), one double bond (δC 146.0 and 118.2) and a galloyl group (δC 166.7, 146.2, 139.1, 121.9, and 110.3) which was confirmed by the presence of a two-proton singlet at δH 7.07 (2H, s) in the 1H NMR spectrum. On acid hydrolysis of 1, D-glucose was detected by GC. The β-configuration of the glycosidic bond was deduced from the coupling constant of the anomeric proton and the 13C NMR data of the sugar unit (Table 1). The above structural characteristics indicated 1 is a chalcone derivative consisting of a chalcone moiety, a D-glucose moiety and a galloyl group. The 1H and 13C NMR signals of 1 were completely assigned by a combination of HSQC, HMBC, and 1H– 1H COSY experiments (Table 1). The 1H– 1H COSY correlation (Fig. 2) between H-α (δH 7.60) and H-β (δH 7.73), and the HMBC correlations (Fig. 2) of H-α to C = O (δC 193.4), C-β (δC 146.0) and C-1 (δC 127.6), H-β to C = O (δC 193.4), C-α (δC 118.2), C-1 and C-2/6 (δC 132.1), H-2/6 (δH 7.64) to C-β, C-3/5 (δC 117.0) and C-4 (δC 161.3), and H-6′ (δH 8.00) to C = O (δC 193.4), C-1′ (δC 116.3), C-2′ (δC 166.9), C-4′ (δC 164.8) and C-5′ (δC 108.9) established the structure of aglycone as 2′,4,4′-trihydroxychalcone. Both C-2′ and C-4′ of the aglycone are oxygenated aromatic carbons, which have very close chemical shifts at δC 166.9 or 164.8, and they were distinguished through long-range coupling to H-5′ (δ H 6.59). The signal at δ C 164.8 was assigned to C-4′ on the basis of an HMBC correlation to H-5′ (Fig. 2), and the other signal at δC 166.9 was thus assigned to C-2′. The linkage of the glucose unit to the aglycone was determined by 1H NMR and HMBC spectral analysis. The 1H NMR spectrum of 1 showed the presence of an extreme
Z. Zhao et al. / Fitoterapia 82 (2011) 1102–1105
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Fig. 2. Key HMBC and 1H–1H COSY correlations of compound 1.
downfield characteristic signal for 2′-OH (δ H 13.40) due to the intramolecular hydrogen bonding with C = O (δ C 193.4) [14,15], and this suggested that C-2′ was not the sugar binding site. The attachment of the D -glucose to C-4′ of the aglycone was finally established by the HMBC correlation between H-1″ (δ H 5.06) and C-4′ (δC 164.8) (Fig. 2). The above substitution pattern of the aglycone was further verified by the comparison of the NMR data of 1 with those of 4′-O-glucopyranosyl-2′,4-dihydroxy-3methoxy-chalcone [16], which is a known compound with similar aglycone structure. An HMBC correlation of H2 -6″ (δ H 4.55 and 4.24) to carbonyl carbon (δ C 166.7) indicated that the galloyl group was located at C-6″ of the glucose, which was also supported by the chemical shift of C-6″ of the glucose at lower field (δ C 64.4, Δδ 3.8) compared with that of 3. Therefore, compound 1 was unambiguously characterized as 4′-O-(6″-O-galloyl-β-D glucopyranosyl)-2′,4-dihydroxychalcone. Compound 2 was isolated as yellow amorphous powder and had the molecular formula C29H28O13 determined by HRESITOFMS (m/z 583.1450 [M− H]−, calculated for C29H27O13, 583.1451) and 13C NMR. The 1H and 13C NMR data (Table 1) of 2 were very close to those of 1. The only difference between these two compounds was the appearance of a methoxyl group in 2, and an HMBC correlation of OMe-4 (δH 3.84, s) to C-4 (δC 161.3) established the location of the methoxy at C-4. The detailed assignments of the 1H and 13C NMR signals (Table 1) were performed by HSQC, 1H–1H COSY and HMBC experiments. Thus, the structure of 2 was assigned as 4′-O-(6″-O-galloyl-β-Dglucopyranosyl)-2′-hydroxy-4-methoxychalcone. The known compound 3 was isolated from natural resources for the first time and its spectroscopic data have not been previously reported. The structure of 3 was elucidated as 4′-O-β-D-glucopyranosyl-2′-hydroxy-4-methoxychalcone on the basis of extensive spectroscopic analysis, including 1H and 13C NMR, HMBC, 1H– 1H COSY and chemical evidence.
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