Fitoterapia 80 (2009) 358–360
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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
A new iridoid from Adenosma caeruleum R. Br. Maryan Bruzual De Abreu a, Nicola Malafronte b, Phan Van Kiem c, Alessandra Braca a,⁎ a b c
Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Nghiado, Caugiay, Hanoi, Viet Nam
a r t i c l e
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Article history: Received 22 January 2009 Accepted in revised form 30 April 2009 Available online 11 May 2009 Keywords: Adenosma caeruleum Scrophulariaceae Iridoid Phenylpropanoids
a b s t r a c t A new iridoid glycoside, adenosmoside (1), together with five known phenylpropanoids, crenatoside, verbascoside, cistanoside F, campneoside I, and campneoside II and two known flavonoids, apigenin 7-O-β-D-glucuronopyranoside and apigenin 7-O-β-D-glucopyranoside, were isolated from the aerial parts of Adenosma caeruleum R. Br. Their structures were elucidated by spectral evidence. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
The genus Adenosma (Scrophulariaceae family) counts of about 15 species widespread in South and South East Asia, China, and Pacific Islands. All its species are herbs, erect or creeping, blackened when dry, and frequently aromatic [1]. Adenosma caeruleum R. Br., known in Vietnam as “nhan tran” is a perennial herb, 30–100 cm tall, with erect stems, widely distributed in the mountains of North Vietnam where its dried aerial parts are used in folk medicine for the treatment of jaundice, bile, and liver diseases as well as illness of women after child birth [2]. Previous phytochemical studies on the chloroform extract of A. caeruleum aerial parts, reported the isolation of a monoterpene peroxide [3], betulinic acid, arbutin, aucubin, β-sitosterol, stigmasterol, and campesterol [4], but a literature survey indicates that its polar extracts have not yet been investigated. In continuation of our studies on natural products from Vietnamese medicinal plants [5,6], we isolated and characterized a new iridoid glycoside, together with five known phenylpropanoids and two known flavonoids from A. caeruleum aerial parts methanol extract.
2.1. Generals Optical rotations: Perkin-Elmer 241 polarimeter equipped with a sodium lamp (589 nm) and a 1 dm microcell. HPLC separations: Shimadzu LC-8A series pumping system (Shimadzu, Kyoto, Japan) equipped with a Waters R401 refractive index detector (Waters Corporation, Milford, MA, USA) and Shimadzu injector using a μ-Bondapack C18 semipreparative column (300 × 7.8 mm; 5 μm); HRMS: Q-TOF Premier instrument (Waters, Milford, MA), equipped with a nanoelectrospray ion source; ESIMS analyses: LCQ Advantage ThermoFinnigan spectrometer (ThermoFinnigan, San Jose, CA, USA), equipped with a Xcalibur software. NMR spectra: Bruker DRX-600 spectrometer using the UXNMR software package. TLC: precoated Kieselgel 60 F254 plates (Merck, Darmstadt, Germany); detection Ce(SO4)2/H2SO4 and NTS/ PEG. Column chromatographies: Sephadex LH-20 (Pharmacia, Sweden). HPCPC: Everseiko CPC240 chromatograph (Everseiko Corporation, Tokyo, Japan). 2.2. Plant material
⁎ Corresponding author. Tel.: +39 050 2219688; fax: +39 050 221660. E-mail address:
[email protected] (A. Braca). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.05.003
A. caeruleum R. Br. (Scrophulariaceae), aerial parts were collected in Tamdao National Park, Vinh Phuc province,
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Vietnam, in May 2004. A voucher specimen (no. 7536 Adenosma caeruleum/1) has been deposited at the Herbarium Hortii Botanici Pisani, Pisa, Italy. 2.3. Extraction and isolation The dried aerial parts of A. caeruleum (750 g) were macerated at room temperature with solvents at increasing polarity: n-hexane, chloroform, chloroform–methanol (9:1), and methanol. The methanol extract was partitioned between n-BuOH and H2O. The n-BuOH soluble fraction (5.3 g) was separated by column chromatography using Sephadex LH-20 with methanol as eluent. Six major fractions (A–F) were collected and pooled after TLC analysis with n-BuOH–AcOH– H2O (60:15:25) and CHCl3–MeOH–H2O (40:9:1). Fraction B (1.26 g) was purified by HPCPC with CHCl3–MeOH–H2O (7:13:8) in which the stationary phase consisted of the upper phase (descending mode, flow rate 3.0 ml/min). Compound 1 (15 mg) was isolated from fraction 3 of HPCPC by further purification with RP-HPLC eluting with MeOH–H2O (3:7) (flow rate: 2.0 ml/min). Fractions D (250 mg) and E (270 mg) were further purified by RP-HPLC eluting with MeOH–H2O (35:65) (flow rate: 2.0 ml/min) to obtain cistanoside F (15 mg), campneoside II (15 mg), campneoside I (16.5 mg), and verbascoside (19 mg) from fraction D and verbascoside (25 mg) and crenatoside (75 mg) from fraction E, respectively. Fraction F (200 mg) was subjected to Si-gel CC eluting with a gradient of CHCl3–MeOH to give apigenin 7-O-β-Dglucopyranoside (19 mg) and apigenin 7-O-β-D-glucuronopyranoside (18.3 mg). Adenosmoside (1). Colorless amorphous powder, [α]25 D : −28°(c 0.1, MeOH); 1H and 13C NMR (600 MHz, CD3OD): see Table 1; 1H NMR of the aglycon (600 MHz, DMSO-d6): δ 1.02 (3H, d, J = 6.0 Hz, Me-10), 1.11 (3H, s, Me-11), 1.78 (1H, m, H6b), 2.08 (1H, m, H-6a), 2.14 (2H, m, H-3), 2.28 (1H, m, H-8), 2.82 (1H, m, H-9), 3.22 (1H, m, H-5), 4.52 (1H, m, H-7), 5.37
Table 1 1 H and 13C NMR data of compound 1 (600 MHz, CD3OD).a Position
δH
δc
1 3 4 5 6a 6b 7 8 9 10 11 Glucose 1′ 2′ 3′ 4′ 5′ 6′a 6′b
5.49 d (2.0) 2.26 m
95.3 52.3 80.0 31.8 39.1
a
3.16 m 2.27 m 1.97 m 4.92 m 2.38 m 2.60 m 1.11 d (6.0) 1.33 s 4.70 d (7.8) 3.23 dd (9.0, 7.8) 3.33 t (9.0) 3.27 t (9.0) 3.40 m 3.93 dd (12.0, 2.5) 3.67 dd (12.0, 5.0)
82.3 43.1 43.3 14.6 24.9 99.6 74.7 78.3 71.6 77.9 62.6
HMBC (H to C)
ROESY (H to H)
1, 4, 5
10 5 3, 8
1, 4, 5
7, 8 3, 4, 5
10 5, 9 8 1, 7
1 1′
5′
Assignments were confirmed by 1D-TOCSY, DQF-COSY, HSQC, and HMBC experiments.
Fig. 1. The structure of compound 1.
(1H, d, J = 2.0 Hz, H-1); ESI-MS: m/z 387 [M + Na]+; HRMS: m/z 365.1708 [M + H]+ calcd. for C16H28O9 364.1733. 3. Results and discussion The phytochemical study of the methanol extract obtained from the aerial parts of A. caeruleum afforded the new iridoid glycoside adenosmide (1) (Fig. 1). Glycoside adenosmoside (1) was obtained as a colorless amorphous powder. Its molecular formula was determined to be C16H28O9 by the [M + H]+ quasiion peak at m/z 365.1708 (calcd. for C16H28O9 364.1733) in the HRMS. The 1H NMR spectrum of 1 showed the presence of an iridoid structure with one acetal proton [δ 5.49 (d, J = 2.0 Hz)], one oxygenated methine proton [δ 4.92 (m)], two oxygenated methylene protons [δ 2.26 (m)], two methylene protons [δ 2.27 (m),1.97 (m)], three methine protons [δ 3.16 (m), 2.60 (m), 2.38 (m)], two methyls [δ 1.33 (s), 1.11 (d, J = 6.0 Hz)], and protons ascribable to a monosaccharide group (Table 1). The 13C NMR of 1 (Table 1) revealed the presence of two methyls [δ 24.9, 14.6], two methylenes [δ 52.3, 39.1], three methines [δ 43.3, 43.1, 31.8], one oxygenated methine [δ 82.3], one quaternary carbon [δ 80.0], one acetal carbon [δ 95.3], and characteristic signals of a β-glucopyranosyl group [δ 99.6, 78.3, 77.9, 74.7, 71.6, 62.6]. Assignments of all chemical shifts of protons and carbons were ascertained from a combination of 1D-TOCSY, DQF-COSY, and HSQC analysis. Direct evidence of the substituent sites was derived from the HSQC and HMBC data (Table 1): HMBC correlations between H-3—C-1, H-3—C-4, H-3 —C-5; H-6—C-1, H-6—C-4, H-6—C-5; Me-11—C-3, Me-11—C-4, Me-11—C-5; Me-10—C-7, Me-10—C-8 substantiated the presence of hydroxy groups at C-4 and C-7, respectively, while correlation between H-1′—C-1 confirmed the position of glucopyranosyl moiety. The relative stereochemistry of 1 was ascertained by 2D-ROESY experiment. ROE correlations were observed between H-7 and Me-10, H-1 and Me-10, H-8 and H5, H-8 and H-9, H-3 and H-5. No ROE correlations for Me-11 were observed. To fully elucidate the structure of 1, 2D-ROESY spectrum was recorded again in DMSO-d6 and a ROE correlation was observed between Me-11 and H-1. On the basis of these data, the structure of adenosmoside (1) was determined to be that shown in Fig. 1. Five known phenylpropanoids, crenatoside, verbascoside, cistanoside F, campneoside I, and campneoside II and two
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known flavonoids, apigenin 7-O-β-D-glucuronopyranoside and apigenin 7-O-β-D-glucopyranoside were also isolated and identified by spectral analysis and comparison of data with those reported in the literature [7–12]. To our knowledge this is the first time that phenylpropanoids and iridoids are reported in the genus Adenosma. Acknowledgement This work was supported by the Third Vietnamese-Italian Program of Cooperation in Science and Technology (2006– 2008) (project n. 15 MH 3). References [1] Deyuan H, Hanbi Y, Cunli J, Holmgren NH. In: Wu ZY, Raven PH, editors. Scrophulariaceae. Flora of China, Beijing: Science Press; 1998. p. 25.
[2] Do Tat Loi. Glossary of Vietnamese medicinal plants and drugs. Hanoi: Publishing House for Science and Technics; 1997. p. 633. [3] Adam G, Porzel A, Sung T, Schmidt J. Phytochemistry 1992;31:2885. [4] Tran VS. Tap Chi Hoa Hoc 1997;35:7. [5] Nguyen Thi HV, Chau VM, De Leo M, Siciliano T, Braca A. Biochem Syst Ecol 2006;34:882. [6] Braca A, Phan VK, Pham HY, Nguyen XN, Tran HQ, Nguyen XC, et al. Fitoterapia 2008;79:117. [7] Afifi MS, Lahloub MF, El-Khayaat SA, Anklin CG, Ruegger H, Sticher O. Planta Med 1993;59:359. [8] Liu Y, Wagner H, Bauer R. Phytochemistry 1998;48:339. [9] Nicoletti M, Galeffi C, Messana I, Marini-Bettolo G, Garbarino J, Gambaro V. Phytochemistry 1988;27:639. [10] Imakura Y, Kobayashi, Mima A. Phytochemistry 1985;24:139. [11] Flamini G, Antognoli E, Morelli I. Phytochemistry 2001;57:559. [12] Harborne JB. The flavonoids advances in research since 1986. London: Chapman & Hall; 1994.
