Fitoterapia 81 (2010) 480–484

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Fitoterapia

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Ursane triterpenoids from the bark of Terminalia arjuna

Wei Wang a, Zulfiqar Ali a, Yunheng Shen a, Xing-Cong Li a, Ikhlas A. Khan a,b,⁎a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USAb Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA

a r t i c l e i n f o

⁎ Corresponding author. National Center for NaturResearch Institute of Pharmaceutical Sciences, UnivUniversity, MS 38677, USA. Tel.: +1 662 915 7821; fa

E-mail address: ikhan@olemiss.edu (I.A. Khan).

0367-326X/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.fitote.2010.01.006

a b s t r a c t

Article history:Received 3 September 2009Accepted in revised form 5 January 2010Available online 15 January 2010

Five ursane type triterpene glucosyl esters including a new one, 2α,3β-dihydroxyurs-12,18-dien-28-oic acid 28-O-β-D-glucopyranosyl ester (1) were isolated from the bark of Terminalia arjuna,alongwith two knownphenolic compounds. It is thefirst report of ursane type triterpenoids fromthis species.

© 2010 Elsevier B.V. All rights reserved.

Keywords:CombretaceaeTerminalia arjunaUrsane triterpenoidTriterpene glucosyl ester

1. Introduction

Terminalia arjuna Wight & Arn (Combretaceae) is a20–30 m tall tree distributed in India, Burma, and Sri Lanka.The bark of T. arjuna is a famous Indian folk medicinal plantused as a cardio-tonic in heart failure, ischaemic cardiomy-opathy, atherosclerosis and myocardium necrosis [1–5]. Also,it is an essential ingredient of many Ayurvedic preparationswhich are sold as cardio-tonics [6]. It was reported thatseveral species of Terminalia have been used in traditionaltreatment of cancer [7]. Previous phytochemical investiga-tions showed triterpenoids, tannins and flavonoids wereisolated from the bark of T. arjuna [8]. Many oleanane typetriterpenoids have been reported from the title plant, someof which possess antitumoral, antioxidant, antiallergic, anti-asthmatic, antifeedant and cardioprotective activities [9–13].

In continuing to search for new chemical and bio-markersfrom themedicinal plant T. arjuna for the quality control study ofthe related dietary supplements, one new ursane triterpeneglucosyl ester, 2α,3β-dihydroxyurs-12,18-dien-28-oic acid28-O-

al Products Researchersity of Mississippix: +1 662 915 7989.

All rights reserved.

,,

β-D-glucopyranosyl ester (1) and four known ursane triterpeneglycosyl esters (Fig. 1), namely, 2α,3β,23-trihydroxyurs-12,18-dien-28-oic acid 28-O-β-D-glucopyranosyl ester (2) [14],quadranoside VIII (3) [15], kajiichigoside F1 (4) and 2α,3β,23-trihydroxyurs-12,19-dien-28-oic acid 28-O-β-D-glucopyrano-syl ester (5) [14,16], aswell as twoknownphenolic compounds,3-O-methylellagic acid 4′-O-α-L-rhamnopyranoside and (−)-epicatechinwere isolated [17,18].Hereinwe report the isolationand structure elucidation of the new metabolite. Its structurewas identified on the basis of extensive NMR experiments .

2. Experimental

2.1. General

Optical rotation: Rudolph ResearchAutoPol IV. UV: Hewlett–Packard 8453. IR: Bruker Tensor 27 FT-IR andMIRacle ATRFT-IR.HRESIMS:Agilent Series 1100 SL. NMR spectra: AmericanVarianMercury plus 400 (1H 400 MHz, 13C 100 MHz). HPLC:Waters LCModule I. HPLC column: Phenomenex Gemini C18 5 μ ODScolumn (10×250 mm).

2.2. Plant material

The bark of T. arjuna Wight & Arn. was purchased fromGarry & Sun (Reno, Nevada, USA), and was authenticated by

Fig. 1. Structures of 1–5.

481W. Wang et al. / Fitoterapia 81 (2010) 480–484

Dr. Vaishali C. Joshi (University of Mississippi). A voucherspecimen (#3799) was deposited at the National Center forNatural Products Research, Research Institute of Pharmaceuti-cal Sciences, University of Mississippi, USA.

2.3. Extraction and isolation

The air-dried and powdered bark of T. arjuna (2 kg) wasextracted by sonicating with MeOH (4×2 L) at room temper-ature for 2 h. The pooled MeOH solution was evaporated invacuo to give a residue (645 g). The MeOH extract wassuspended in H2O (1.5 L) and then partitioned with hexanes(3×4 L), Et2O (3×4 L) and EtOAc (3×4 L). The EtOAc layerafforded a residue (11 g),whichwas separated into 11 fractionsby column chromatography (CC) on silica gel with a gradientelution of CHCl3–MeOH (20:1–1:1). Fr. 7 was subjected to CCover reversed-phase C18 silica and eluted with MeOH/H2O(2:3, v/v) to give (−)-epicatechin (42.5 mg). Fr. 9 wasseparated by reversed-phase C18 silica gel CC eluted withMeOH/H2O (3:7, v/v) and further purified by semipreparativeHPLC with MeCN/H2O (33:67) as mobile phase (flow rate6.0 mL/min) to afford 1 (5.2 mg), 4 (26.2 mg) and 3-O-methylellagic acid 4′-O-α-L-rhamnopyranoside (21.4 mg). Fr.10 was treated in a similar manner to that of Fr. 9 to yield 2(14.4 mg) and 5 (7.7 mg). Fr. 11 was subjected to a reversed-phase C18 silica gel column eluted with MeOH/H2O (2:3, v/v)

and purified by semipreparative HPLCwithMeOH/H2O (64:36)as mobile phase (flow rate 4.0 mL/min) to yield 3 (5.8 mg).

2α,3β-dihydroxyurs-12,18-dien-28-oic acid 28-O-β-D-glu-copyranosyl ester (1): colorless gum; [α]D20: +59.2 (c 0.3,MeOH); UV,λmax (MeOH) nm (logε): 250 (3.60) nm; IR ( KBr ),νmax cm−1: 3386, 2932, 2872, 1714, 1453, 1368, 1225, 1175,1071, and 892; HR-ESI-MS m/z 655.3827 [M+Na]+ (calcd forC36H56O9Na, 655.3822); For 1H and 13C NMR data: see Table 1.

Acid hydrolysis of 1 and determination of D-glucose werecarried out according to the method reported previously [19].

3. Results and discussion

Compound 1was isolated as a colorless gum. Themolecularformula of C36H56O9 (positivemodem/z=655.3827 [M+Na]+

calcd. for C36H56O9Na: 655.3822) was established by HR-ESI-MS and 13C NMR spectra. The IR spectrum showed the bands at3386 and 1714 cm−1 corresponding to the absorptions of thehydroxy and the carbonyl groups, respectively. The 1H NMRspectrum (Table 1) showed six tertiary methyl [δH 1.04, 1.09(×2), 1.18, 1.28 and 1.79], one secondary methyl (δH 1.03, d,J=4.0 Hz) and the anomeric proton of a β-glucopyranosyl unit(δH−1′ 6.35, d, J=8.0 Hz). The 13C NMR and DEPT (Table 1)spectra indicated the presence of 36 carbons, including onecarboxyl (δC 175.1), fourolefinic [δC 139.1 (s), 136.4 (s), 134.1 (s)and126.9 (d)] and twooxymethine (δC 84.1 and69.0) carbons inthe low-field region and seven methyl, eight methylene, three

Table 11H (400 MHz) and 13C (100 MHz) NMR data for 1.

No. δH (mult.) δC (mult.) No. δH (mult.) δC (mult.)

1 1.32 (1H, m) 48.8 (t) 19 136.4 (s)2.34 (1H, br d, J=12.0) 20 2.14 (1H, m) 34.9 (d)

2 4.14 (1H, m) 69.0 (d) 21 1.18 (1H, m) 27.1 (t)3 3.42 (1H, d, J=9.6) 84.1 (d) 2.12 (1H, m)4 40.2 (s) 22 1.60 (1H, m) 35.6 (t)5 1.01 (1H, m) 56.4 (d) 2.60 (1H, br d, J=14.0)6 1.58 (1H, m) 19.0 (t) 23 1.28 (1H, s) 29.7 (q)7 1.48 (1H, m) 35.4 (t) 24 1.09 (1H, s) 18.1 (q)

1.50 (1H, m) 25 1.04 (1H, s) 19.0 (q)8 39.8 (s) 26 1.18 (1H, s) 19.0 (q)9 1.61 (1H, m) 48.7 (d) 27 1.09 (1H, s) 22.4 (q)10 38.7 (s) 28 175.1 (s)11 0.88 (1H, d, J=12.8) 23.9 (t) 29 1.79 (1H, s) 19.9 (q)

2.01 (1H, m) 30 1.03 (1H, d, J=4.0) 18.0 (q)12 5.70 (1H, br s) 126.9 (d) 1′ 6.35 (1H, d, J=8.0) 96.2 (d)13 139.1 (s) 2′ 4.20 (1H, m) 74.5 (d)14 45.3 (s) 3′ 4.30 (1H, m) 79.2 (d)15 1.41 (1H, m) 29.4 (t) 4′ 4.40 (1H, m) 71.4 (d)16 1.70 (1H, m) 31.3 (t) 5′ 4.00 (1H, m) 79.5 (d)

2.20 (1H, m) 6′ 4.38 (1H, m) 62.6 (t)17 50.3 (s) 4.40 (1H, m)18 134.1 (s)

In pyridine-d5; δ in ppm, J in Hz.

482 W. Wang et al. / Fitoterapia 81 (2010) 480–484

methine and five quaternary carbons in the high-field region,along with a set of resonances for a β-glucopyranosyl unit (δC96.2, 79.5, 79.2, 74.5, 71.2 and 62.6). These data are consistentwith the HR-MS empirical formula suggesting that compound 1belongs to an ursane triterpene glucoside [14]. The twoconjugated double bonds were identified at C-12―C-13 andC-18―C-19 by the HMBC cross-peaks (Fig. 2) of H-12 with C-9,C-14 and C-18, and of H3-29with C-18, C-19 and C-20, aswell asby H-12/H-11 1H–1H COSY correlation and H-12/H3-29 ROESYcorrelation (Fig. 2). Twohydroxy groupswere located at C-2 andC-3, based on the correlations of H-3/C-2, C-4, C-23 and C-24 in

Fig. 2. Selected COSY, HMBC and

theHMBC experiment and H-2/H2-1 andH-3 correlations in the1H–1H COSY experiment. The interglycosidic linkage of theβ-glucopyranosyl unit to the aglycone was established at thecarboxylic position due to the HMBC correlation between H-1′and C-28 (δC 175.1). The ROESY correlations of H-2/H3-24 andH-3/H-5,H3-23disclosed that thehydroxy groups at C-2 andC-3have a 2α,3β-orientation. The D-glucose was determined by GCanalysis of its acetylated thiazolidine derivatives after acidhydrolysis [19]. Thus, the structure of 1 was established as2α,3β-dihydroxyurs-12,18-dien-28-oic acid 28-O-β-D-gluco-pyranosyl ester.

ROESY correlations of 1.

Scheme 1. Biogenetic pathyway proposed for compounds 1–3 and 5.

483W. Wang et al. / Fitoterapia 81 (2010) 480–484

The in vitro cytotoxicity of 1 was evaluated against foursolid tumor cell lines: malignant melanoma (SK-MEL), oralepidermal carcinoma (KB), breast ductal carcinoma (BT549)and ovary carcinoma (SK-OV3) cells by Neutral Red assay[20]. It did not show any cytotoxicity up to 10 µg/mL.

It is well known that many oleanane type triterpenoidsexist in the bark of T. arjuna [8–13], but compounds 1–5belong to ursane type triterpene glycosyl ester which isreported in the title species for the first time. Interestingly,compounds 1–3 and 5might be generated from kajiichigosideF1 (4) via dehydration and oxidation for biogenetic consid-eration as shown in Scheme 1.

Acknowledgements

This workwas supported by the United States Departmentof Agriculture Specific Cooperative Research AgreementNumber 58-6408-6-067 and the FDA/CFSAN grant entitled“Science Based Authentication of Dietary Supplements”Number 2 U01 FD 002071-07. Authors thank Dr. Bharathi

Avula for conducting the HR-ESI-MS spectra and Dr. ShabanaKhan for cytotoxicity testing.

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