evaluated by measurement of LDL susceptibility to oxidation.

grey hair of young man in the traditional Chinese medicine [4]. The only constituent reported was gallic acid [3]. The

spectrometer; H and C NMR: Bruker DRX 500 NMR; HRESIMS: Bruker APEX II. Column chromatography wascarried out with silica gel (100300 mesh) (Tsingtao Marine Chemistry Co. Ltd.), ODS (100200 mesh) (Fuji Silysia

Fitoterapia 77 (2006) 534537www.elsevier.com/locate/fitotepresent paper deals with the isolation and the structure elucidation of a new compound, named pomegranatate (1)(Fig. 1) and six known compounds, ellagic acid, 3,3,4-tri-O-methylellagic acid, ethyl brevifolincarboxylate, urolicand, maslinic acids, and daucosterol.

2. Experimental

2.1. General

UV: Varian Cary Eclipse 300 spectrometer using methanol as solvent; IR: Thermo Nicolet Nexus 470 FT-IR1 13 2006 Elsevier B.V. All rights reserved.

Keywords: Punica granatum; Pomegranatate; Maslinic acid; Antioxidant activity

1. Introduction

Punica granatum L. (Punicaceae) is a shrub distributed originally in Iran and Afghanistan, introduced into China inthe second century BC [1,2]. The flower of this plant was reported as an astringent, haemostatic, and as a remedy fordiabetes in Unani and Ayurvedic medicine [3]. This flower was also used for the treatment of injuries from falls andConstituents of the flowers of Punica granatum

Rufeng Wang a,b, Wei Wang a, Liang Wang a, Ruining Liu a, Yi Ding a, Lijun Du a,

a Laboratory of Pharmaceutical Sciences, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, Chinab Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing, 100083, China

Received 31 March 2006; accepted 19 June 2006Available online 7 July 2006

Abstract

A new polyphenol compound named pomegranatate (1), together with, ellagic acid, 3,3,4-tri-O-methylellagic acid, ethylbrevifolincarboxylate, urolic and maslinic acids, and daucosterol were isolated from the ethanolic extract of the flowers of Punicagranatum. The structure of compound 1 was determined by spectroscopic analysis. Maslinic acid exhibited antioxidant activity, Corresponding author. Fax: +86 10 62773630.E-mail address: lijundu@mail.tsinghua.edu.cn (L. Du).

0367-326X/$ - see front matter 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2006.06.011

Chemical Co. Ltd.) and Sephadex LH-20 (18110 m) (Pharmacia Co. Ltd.). Laboratory animals were obtained fromthe Laboratory Animal Institute, Chinese Academy of Medical Science, Beijing, China.

2.2. Plant

Fig. 1. Structure and HMBC correlations of pomegranatate (1).

535R. Wang et al. / Fitoterapia 77 (2006) 534537The flowers, collected in October 2004 from Zaozhuang City in Shandong Province of China were authenticated byDr. R. F. Wang. A voucher specimen (No. 041005) has been deposited in the Herbarium of the Laboratory ofPharmaceutical Sciences, Department of Biological Sciences and Biotechnology, Tsinghua University.

2.3. Extraction and isolation

The flowers (3 kg) were extracted with 95% EtOH under reflux to obtain 540 g of crude extract, treated successivelywith petroleum ether, EtOAc, and acetone in a Soxhlet extractor. The EtOAc extract (110 g) was subjected to Si-gel CCwith CHCl3MeOH (30:12:1) as eluents to afford 5 fractions (Fr. AFr. E). Fr. B was further subjected to Si-gel CC

Table 11H NMR and 13C NMR spectral data of compounds 1 and 2 (DMSO-d6)

a

C 1 2H (J in Hz) C H (J in Hz) C

1 159.8 163.82 110.3 112.23 109.9 112.74 149.6 150.95 138.3 139.56 139.4 140.97 7.45 (1H, s) 112.7 7.38 (1H, s) 114.28 160.9 162.49 117.3 114.810 137.9 143.611 6.77 (1H, s) 74.9 5.54 (2H, s) 68.012 3.81 (1H, d, 17.5); 3.74 (1H d, 17.5) 32.7 3.47 (2H, s) 32.313 167.1 173.114 4.18 (1H, m); 4.09 (1H, m) 60.615 1.22 (3H, t, 7.5) 14.016 169.017 4.14 (1H, m); 4.00 (1H, m) 63.018 1.13 (3H, t, 7.5) 13.5

a Assignments were confirmed by 1D and 2D NMR.

536 R. Wang et al. / Fitoterapia 77 (2006) 534537with CHCl3MeOH (20:12:1) as eluents to afford 6 fractions (Fr. B-1Fr. B-6), of which Fr. B-2 was washed withacetone to obtain urolic acid (500 mg). Fr. C was treated with the same method to provide daucosterol (500 mg). Fr. Dwas Si-gel CC isolated with CHCl3MeOH (15:11:1) as eluents to provide 4 fractions (Fr. D-1Fr. D-4), of whichFr. D-2 and Fr. D-4 were Si-gel CC further purified with CHCl3MeOH (15:11:1 and Sephadex LH-20 CC withMeOH to get 3,3,4-tri-O-methylellagic acid (12 mg) and maslinic acid (31 mg), respectively. The acetone extract(220 g) was subjected to macroporous resin D101 column, using water, and 20%, 50%, 70%, and 95% EtOH as eluentsto provide 5 fractions. The 20% EtOH part (31 g) was subjected to ODS CC employing MeOHH2O mixtures (from15% to 60%) as eluents to provide 6 fractions (Fr. FFr. J). Fr. H (2 g) was isolated by Sephadex LH-20 CC usingMeOH as eluent to obtain ellagic acid (250 mg). The 50% EtOH part (31 g) was purified with Si-gel CC using CHCl3MeOH (25:12:1) as eluents to get 5 fractions (Fr. KFr. O), of which Fr. O was purified on Sephadex LH-20 CCeluted with MeOH to provide 1 (25 mg). Ethyl brevifolincarboxylate (1.2 g) was isolated from the 20% EtOH part byrepeated CC over Si-gel and Sephadex LH-20.

2.3.1. Pomegranatate (1)Colorless needle crystal, mp 182184 C; []D

20-20 (c 0.50, MeOH); UV max (H2O): 267 (2.51) nm; IR (KBr):3439, 2992, 1760, 1731, 1695, 1594, 1519, 1488, 1413, 1371, 1330, 1278, 1214, 1189, 1105, 956, 889, 854, 768, 732,653, 566 cm1; HRESIMS m/z (negative mode): 391.0667 [M-1] (calcd. for C18H16O10: 391.0671).

1H NMR (500 MHz, DMSO-d6) and13C NMR (125 MHz, DMSO-d6), see Table 1.

2.4. Measurement of LDL susceptibility to oxidation

Fig. 2. Phyllanthusin E (2).Isolation and analysis of LDL from the plasma of New Zealand White rabbits were performed as prescribedpreviously with a slight modification according to Beaudeux et al. [5]. The dialysis of LDL was conducted against abuffer (NaCl, 140 mmol/l; KCl 3 mmol/l; Na2HPO4 8 mmol/l; NaH2PO4 2 mmol/l; pH 7.4) for 4 h at 4 C in the dark(four changes). Protein content was measured by the method of Lowry et al. [6] using bovine serum albumin as thestandard. LDL was diluted in dialysis buffer to a final concentration of 0.25 mg LDL/ml. The isolated compounds(with three different final concentrations: 50, 5 and 0.5 g/ml), and positive (freshly prepared vitamin C, final

Table 2Effect of maslinic acid on conjugated diene (CD) production LDL in rat plasmaa

Concentration (g/ml) Increase CD (%)SDb

Control 31.42.1Vitamin C 50 1.60.2

Maslinic acid 50 10.61.3

5 39.71.80.5 57.82.7

aFor the protocols used, see the Experimental section. bn=3, Pb0.01; Pb0.05.

concentration 50 g/ml) were added to buffer containing LDL for incubation 30 min at 20 C, respectively. Thecontrol was treated with the same volume of the vehicle solution of the solvent, saline. Subsequently, oxidation wasinitiated by addition of a freshly prepared aqueous CuSO4 solution (final concentration, 5.0 mol/l). Incubation wasperformed at 37 C for 4 h. The LDL oxidation was monitored by the change in 234 nm absorbance at 37 C for 4 h in

537R. Wang et al. / Fitoterapia 77 (2006) 534537a spectrophotometer. The percentage increased of conjugated diene between 0 h and 4 h was determined by theformula: (Absorbance4 hAbsorbance0 h) /Absorbance0 h100%.

3. Results and discussion

Compound 1, C18H16O10, exhibited an aromatic UV absorption at 267 nm and IR absorptions at 3439 cm1 (OH),

aromatic bands at 1594, 1519, and 1488 cm1, and CO absorptions at 1760 cm1 and at 1731 cm1. The 1H NMRspectrum of 1 exhibited two one-proton singlets at 7.45 and 6.77, a methylene group and two ethyl residues(Table 1).

The 13C NMR and DEPT spectra demonstrated the presence of 2 methyls, 3 methylenes, 2 methines, and 11quaternary carbons. In general, both 1H and 13C NMR spectral data of 1were similar to those of phyllanthusiin E (2) [7](Fig. 2), the major difference between the two compounds being the presence in 1 of an ethoxy and a CH3CH2O(C_O)CH2-groups. The ethoxy group was found to be connected to C-13 ( 167.1) and the CH3CH2O(C_O)CH2-grouplinked to C-9 ( 117.3) as supported by a HMBC correlation experiment (Fig. 1). On the basis of the above evidence, thestructure of compound 1 was elucidated as ethyl 3-[(ethoxycarbonyl)methyl]-4,6-dihydro-8,9-dihydroxy-2,6-dioxo-2H-pyrano [3,4,5-de]chromene-4-carboxylate and named as pomegranatate.

Six known compounds were identified by comparing their spectral data with the values reported in the literature asellagic acid, 3,3,4-tri-O-methylellagic acid, ethyl brevifolincarboxylate, urolic acid, maslinic acid, and daucosterol[813].

The antioxidant activities of the isolated compounds were evaluated by measurement of low-density lipoprotein(LDL) susceptibility to oxidation in the rat brain tissue in vitro. The results (Table 2) showed that maslinic acid stronglydecreased conjugated diene (CD) production of LDL in rat plasma in vitro.

Acknowledgements

Wewould like express our thinks to Prof. X. W. Yang, School of Pharmaceutical Sciences, Peking University, for hisvaluable suggestions. This study was supported by National Natural Science Foundation of China (30572340).

References

[1] Flora Respublicae Popularis Sinicae, Tomus 52. Beijing: Science Press; 1983. p. 120.[2] The flora of Chinese drugs, Vol. 3. Beijing: Peoples Health Press; 1961. p. 288.[3] Tom HW, Huang GP, Bhavani PK. Toxicol Appl Pharmacol 2005;207:160.[4] Li SZ. Bencao Gangmu. Beijing: People's Health Press; 1982. p. 504.[5] Beaudeux JL, Guillausseau PJ, Peynet J, Flourie F. Clin Chim Acta 1995;239:131.[6] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. J Biol Chem 1951;193:265.[7] Yoshida T, Itoh H, Matsunaga S, Tanaka R, Okuda T. Chem Pharm Bull 1992;40:53.[8] Nawwar MAM, Hussein SAM, Merfort I. Phytochemistry 1994;36:793.[9] Wang RF, Xie WD, Zhang Z, Xing DM, Ding Y, Wang W, et al. J Nat Prod 2004;67:209.[10] Sha DX, Liu YH, Wang LS. J Shenyang Pharm Univ 2000;17:176.[11] Zhao H, Wang BZ, Feng BR. J Chin Pharm 1994;29:523.[12] Bilia AR, Mendez J, Morelli I. Pharm Acta Helv 1996;71:191.[13] Kojima H, Sato N, Hatano A. Phytochemistry 1990;29:2351.

Constituents of the flowers of Punica granatumIntroductionExperimentalGeneralPlantExtraction and isolationPomegranatate (1)

Measurement of LDL susceptibility to oxidation

Results and discussionAcknowledgementsReferences