Fitoterapia 79 (2008) 117–120www.elsevier.com/locate/fitote

New monoterpene glycosides from Paeonia lactiflora

Alessandra Braca a,⁎, Phan Van Kiem b, Pham Hai Yen b, Nguyen Xuan Nhiem b,Tran Hong Quang b, Nguyen Xuan Cuong b, Chau Van Minh b

a Dipartimento di Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italyb Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology; 18 Hoang Quoc Viet,

Nghiado, Caugiay, Hanoi, Vietnam

Received 21 March 2007; accepted 13 November 2007Available online 16 January 2008

Abstract

Three newmonoterpene glycosides named 4-O-methyl-paeoniflorin (1), isopaeoniflorin (2), and isobenzoylpaeoniflorin (3), togetherwith two known monoterpene glycosides, paeoniflorin (4) and benzoylpaeoniflorin (5), were isolated from the roots of Paeonialactiflora. Their structures were established on the basis of spectral and chemical evidence.© 2007 Elsevier B.V. All rights reserved.

Keywords: Paeonial lactiflora; Monoterpene glycosides; 4-O-Methyl-paeoniflorin

1. Introduction

Paeonia ca. 33 species, which are distributed in Pakistan, India, Afghanistan, Japan, China, Korea, and Vietnam [1,2].Paeonia Radix is one of the most important crude drugs in Chinese, Korean, Japanese, and Vietnamese traditionalmedicine, used as an anticoagulant, antiinflammatory, analgesic, and sedative agent. The Paeonia spp. are a rich source ofmonoterpenes possessing a “cage-like” pinane skeleton, which are established as the main biologically active constituents.Among this, P. lactiflora is an important and popular species, which exhibited various biological activities such asimprovement of the memory and blood circulation, hepato-protection, antimutagenic properties, and platelet aggregationinhibition [3–6]. As part of our current investigations on bioactive compounds from Vietnamese medicinal plants,this paper deals with the isolation and structural elucidation of three new monoterpene glycosides, 4-O-methyl-paeoniflorin (1), isopaeoniflorin (2), and isobenzoylpaeoniflorin (3), and two known monoterpene glycosides,paeoniflorin (4) and benzoylpaeoniflorin (5), from P. lactiflora growing in Vietnam.

2. Experimental

2.1. General

Melting points: Electrothermal IA-9200 instrument. IR: Hitachi 270-30. Optical rotations: JASCO DIP-1000 KUYpolarimeter equipped with a sodium lamp (589 nm) and a 1 dm microcell. ESIMS: Agilent 1100 LC-MSD Trap.

⁎ Corresponding author. Tel.: +39 050 2219688; fax: +39 050 2219660.E-mail address: braca@farm.unipi.it (A. Braca).

0367-326X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2007.11.001

Fig. 1. Structure of compounds 1–5.

118 A. Braca et al. / Fitoterapia 79 (2008) 117–120

HRESIMS: Q-TOF premier spectrometer equipped with a nanospray source. 1H-NMR (500 MHz) and 13C-NMR(125 MHz): Bruker AM500.

2.2. Plant material

P. lactiflora Pall. (Ranunculaceae), roots collected in Tamdao, Vinh Phuc province, Vietnam in January 2006 WASidentified by Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, VASTof Vietnam. An authentic samplewas deposited at the Institute of Natural Products Chemistry, VAST, Vietnam (No 20060108).

2.3. Extraction and isolation

Dried and powdered roots of P. lactiflora (5.0 kg) were extracted with MeOH. The extract (145 g), was suspended inwater and partitioned with CH2Cl2 to give 70.0 g of residue which was subjected to Si-gel CC, using CHCl3-MeOH

Table 11H NMR data for 1, 2, and 3 (500 MHz, CD3OD, J in Hz and δ in ppm) a

C 1 2 3

δH HMBC (H to C) δH HMBC (H to C) δH HMBC (H to C)

3 2.15 d (13.0)1.95 br d (13.0)

1, 2, 4, 5 2.08 dd (13.0, 1.5)2.29 d (13.0)

2, 4 2.08 dd (13.0, 1.5)2.29 d (13.0)

2, 4

5 2.80 dd (6.5, 1.5) 8 3.24⁎ 8, 3.24⁎

6 1.91 d (10.5)2.51 dd (10.5, 6.5)

5, 7, 2, 4 1.93 d (11.0)2.64 dd (11.0, 7.0)

5, 7, 2, 4 1.93 d (11.0)2.64 dd (11.0, 7.0)

5, 7, 2, 4

8 4.79 s 7′′, 1, 5, 7, 9 4.74⁎ 7′′, 1, 5, 7, 9 4.75⁎ 7′′, 1, 5, 7, 99 5.47 s 8, 7, 2, 4 5.57 s 8, 7, 2, 4 5.57 s 8, 7, 2, 410 1.38 s 2, 1, 3, 4 1.31 s 2, 1, 3, 4 1.31 s 2, 1, 3, 41′ 4.55 d (8.0) 1 4.58 d (8.0) 1 4.58 d (8.0) 12′ 3.22 dd (9.5, 8.0) 3.27 dd (9.5, 8.0) 3.27 dd (9.5, 8.0)3′ 3.26 a 3.40 a 3.40 a

4′ 3.27 a 3.40 a 3.40 a

5′ 3.33 m 3.62 m 3.62 m6′ 3.64 dd (11.0, 5.0)

3.87 dd (11.0, 2.0)5′ 4.51 dd (12.0, 6.0)

4.65 dd (12.0, 2.0)5′ 4.51 dd (12.0, 5.0)

4.65 dd (12.0, 2.0)5′, 7′′′

2′′/6′′ 8.07 dd (7.5, 1.5) 8.05 dd (7.5, 1.5) 8.05 dd (7.5, 1.5) 7′′, 4′′3′′/5′′ 7.48 t (7.5) 7.50 t (7.5) 7.50 t (7.5)4′′ 7.62 t (7.5) 7.61 t (7.5) 7.61 t (7.5)OCH3 3.39 s 42′′′/6′′′ 8.05 dd (7.5, 1.5)3′′′/5′′′ 7.50 t (7.5)4′′′ 7.61 t (7.5)

⁎Overlapped signals.a Assignments were confirmed by 1H–1H COSY, 1D-TOCSY, HSQC, and HMBC experiments.

Table 213C NMR data for 1–5 (125 MHz, CD3OD)

a

C 1 2 3 4 5

1 89.3 89.3 89.4 89.3 89.32 87.2 88.6 88.5 87.2 87.03 42.3 39.4 40.0 44.5 44.44 109.5 95.6 95.5 106.4 106.25 41.1 43.3 43.2 44.0 43.86 23.4 24.2 23.9 23.4 23.07 71.8 72.8 72.6 72.2 72.08 61.7 61.6 61.5 61.8 61.69 102.5 105.5 105.4 102.3 102.210 19.6 19.8 19.8 19.6 19.51′ 100.2 100.2 100.0 100.2 100.02′ 75.0 75.0 75.0 75.0 74.93′ 78.0 77.9 77.9 77.9 77.84′ 71.9 71.7 71.9 71.7 71.95′ 78.1 78.0 75.3 78.0 75.26′ 62.9 62.8 65.3 62.9 65.11′′ 131.2 131.2 131.2 131.2 131.72′′/6′′ 130.8 130.7 130.7 130.8 130.63′′/5′′ 129.6 129.6 129.8 129.6 129.74′′ 134.4 134.3 134.5 134.4 134.47′′ 168.0 168.0 168.0 168.0 167.61′′′ 131.2 131.22′′′/6′′′ 130.4 130.53′′′/5′′′ 129.6 129.64′′′ 134.3 134.47′′′ 167.8 167.9OCH3 51.4a Assignments were confirmed by 1H–1H COSY, 1D-TOCSY, HSQC, and HMBC experiments.

119A. Braca et al. / Fitoterapia 79 (2008) 117–120

(from 50:1 to 5:1) as eluents to give fractions F1 (12.0 g), F2 (8.5 g), F3 (18.5 g), F4 (7.8 g), and F5 (12.0 g). FractionF3 (18.5 g) was Si-gel CC using CHCl3-MeOH-H2O (90:10:1) as eluent to give subfractions F3A (4.5 g), F3B (3.4 g),F3C (5.0 g), and F3D (4.7 g). Subfraction F3A (4.5 g) was subjected to YMC RP-18 column using MeOH-H2O (8:2)as eluent to yield 5 (1.5 g) as white amorphous powder. Subfraction F3B (3.4 g) was CC on a YMC RP-18 columnusing MeOH-H2O (8:2) as eluent to give 2 (800 mg) as white amorphous powder. Fraction F4 (7.8 g) was CC on aYMC RP-18 column using MeOH-H2O (7:3) as eluent to give 3 (100 mg) as white amorphous powder. Compounds 1(37.5 mg) and 4 (3.2 g) were obtained as white amorphous powder from fraction F5 (12.0 g) after chromatographyon a YMC RP-18 column using MeOH-H2O (7:3) as eluent.

4-O-Methyl-paeoniflorin (1, Fig. 1). White amorphous powder; mp 159-160 oC; [α]D25-37 (c 1.00, MeOH); IR (KBr):

3425 (OH), 1698 (CfO), 1046 (C–O–C) cm−1; 1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z: 517.2 [M+Na]+;HRESIMS m/z: 517.1891 [M+Na]+, calcd. 517.1686 for C24H30O11Na.

Isopaeoniflorin (2). White amorphous powder; mp 157–158 oC; [α]D25 +17.0 (c 1.00, MeOH); IR (KBr): 3433 (OH),

1701 (CfO), 1050 (C–O–C) cm−1; 1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z: 481.1 [M+H]+, 503.1 [M+Na]+, HRESIMS m/z: 503.1291 [M+Na]+, calcd. 503.1529 for C23H28O11Na.

Isobenzoylpaeoniflorin (3). White amorphous powder; mp 131–132 oC; a½ �25D +21.0 (c 1.00, MeOH); IR (KBr):3433 (OH), 1701 (CfO), 1050 (C–O–C) cm−1; 1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z: 585.1 [M+H]+,549.1 [M-2H2O+H]+; HRESIMS m/z: 607.1478, calc. 607.1580 for C30H32O12Na.

3. Results and discussion

The methanol extract obtained from the dried roots of P. lactiflora yielded three new (1, 2, 3) and two known (4, 5)monoterpene derivatives (Fig. 1). Compounds 4 and 5 were identified as paeoniflorin (4) and benzoylpaeoniflorin (5),respectively, by spectral analysis and comparison of data with those reported in the literature. They have been isolatedfrom P. suffruticosa [7] and P. lactiflora [8].

120 A. Braca et al. / Fitoterapia 79 (2008) 117–120

Compound 1 was isolated as white amorphous powder. Its molecular formula was established as C24H30O11 bymeans of HRESIMS (m/z 517.1891 [M+Na]+). Its NMR data were almost superimposable to those of 4 except foradditional signals due to the presence of a methoxyl group (δH 3.39 and δC 51.4, Tables 1 and 2). This suggested that 1was amethoxyl derivative of 4. The changes of the carbon chemical shifts at C-3, C-4, and C-5 between 1 and 4 (Table 2)suggested that the methoxyl group should be linked to C-4; this hypothesis was further confirmed by the H–C long-range correlation between the methoxyl protons at δH 3.39 and carbon C-4 at δC 109.5 in the heteronuclear multiple-bond correlation (HMBC) spectrum. All the NMR data of 1 were carefully deduced from the 1H–1H correlationspectroscopy (1H–1H COSY), the heteronuclear multiple quantum coherence (HMQC) spectrum, and the HMBCspectra. On the basis of these data, the structure of 1 was proposed to be 4-O-methyl-paeoniflorin, isolated now for thefirst time from plant source but obtained by synthesis from peaoniflorin [9].

The ESI-MS spectra of 2 showed the quasi molecular ion peaks at m/z 481.1 [M+H]+and 503.1 [M+Na]+,corresponding to the molecular formula of C23H28O11. The

1H and 13C NMR spectra of 2 (Tables 1 and 2) were verysimilar to those of 4 [7,8], including the signals for the monoterpene unit, the sugar, and the benzoyl unit, suggestingthat 2 was a close derivative of 4. Differences were observed between the carbon chemical shifts of C-3, C-4, and C-5(Table 2), suggesting the α orientation of the hydroxyl group at C-4 of 2. Consequently, compound 2 was proposed tobe isopaeoniflorin.

Themolecular formula of 3was deduced to be C30H32O12 from the quasi molecular ion peaks atm/z 585.1 [M+H]+and549.1 [M-2H2O+H]+in the positive ESIMS spectrum, which was further confirmed by the HRESIMS spectrum(foundm/z: 607.1478). The structure of 3was determined by the comparison of its NMR data with those of 5 [7] (Tables 1and 2), and confirmed by the 2D NMR experiments, including the 1H–1H COSY, HMQC, and HMBC spectra. TheirNMR spectra showed close similarities except for C-3, C-4, and C-5 chemical shifts that differed in 3 in respect of 5(Table 2), suggesting the α orientation of the hydroxyl group. Key HMBC correlations between H-8 (δH 4.75) and C-7′′(δC 168.0), H-1′ (δH 4.58) and C-1 (δC 89.4), H-6′ (δH 4.51/4.65) and C-7′′′ (δC 167.8) established that the first benzoylgroup was linked to C-8, the sugar unit was attached to C-1, and the second benzoyl moiety was linked to C-6′ of theglucopyranosyl unit, respectively. The detailed analysis of the HMBC spectrum is summarized in Table 1. Thus, thestructure of 3 was proposed to be the new natural product isobenzoylpaeoniflorin.

Acknowledgements

The authors would like to thank Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, VietnameseAcademy of Science and Technology for the plant identification and Dr. Fabrizio Dal Piaz, Dipartimento di ScienzeFarmaceutiche, Università di Salerno for registering the HRESIMS spectra.

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