Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation

Fitoterapia 113 (2016) 74–79

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Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation Md. Abdul Muhit a,b, Kaoru Umehara a,⁎, Hiroshi Noguchi a a b

School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka 1000, Bangladesh

a r t i c l e

i n f o

Article history: Received 12 May 2016 Received in revised form 2 July 2016 Accepted 13 July 2016 Available online 15 July 2016 Keywords: Terminalia citrina Combretaceae Lignan Furofuranone Dioxabicyclooctyl Antiestrogenic activity Haritaki

a b s t r a c t Five new polyalkoxylated furofuranone lignan glucosides, terminalosides L–P (1–5), were isolated from EtOAc extracts of the leaves of Terminalia citrina, a Bangladeshi medicinal plant. The structures of the isolates were deduced primarily by NMR spectroscopy, and four of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. The absolute configurations and conformations of the furofuranone ring were confirmed by ECD spectroscopy. All of the isolates were evaluated for their estrogenic and/or antiestrogenic properties using two estrogen responsive breast cancer cell lines, T47D and MCF-7. At a concentration of 10 nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common. This is the first report of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Terminalia species (Combretaceae) have been traditionally used for a wide range of medicinal purposes worldwide [1]. T. arjuna, and T. chebula which are listed in Ayurvedic materia medica, are particularly well documented species being native in Asia [2,3]. Recent reports have also revealed the medicinal potential of species from Africa and Australia [4,5]. Terminalia citrina is commonly found in tropical countries such as Bangladesh, Myanmar, and India. The plant is traditionally known as haritaki in Bangladesh, and various parts of the plant have been used for menstrual pain, bleeding piles, heart disease, dysentery, and constipation [6]. Previous research by our collaborators has shown that its leaf extract has significant anti-nociceptive, anti-inflammatory, anxiolytic, and anthelmintic activity [7,8]. We have also previously reported 13 furofuran lignan glucosides isolated from the leaves of T. citrina, and their significant antiestrogenic effect on breast cancer cells [9]. In addition, tannin compounds with antimicrobial properties from the fruit of the plant have been reported [10]. In another of our studies of estrogenic/antiestrogenic phytochemicals from T. citrina, EtOAc extract from the leaves was evaluated, and afforded five new polyalkoxylated furofuranone lignan monoglucosides (1–5). The furofuranones, a rare and small class of naturally occurring lignan, have drawn great interest due to their bizarre structural ⁎ Corresponding author. E-mail address: [email protected] (K. Umehara).

http://dx.doi.org/10.1016/j.fitote.2016.07.004 0367-326X/© 2016 Elsevier B.V. All rights reserved.

conformation [11] and promising biological properties, e.g. vasodilation of rabbit aorta [12], antioxidation [13,14], insulin secretagogic activity and sensitization [15], and phosphodiesterase inhibition [16]. Other studies have reported the occurrence in nature of furofuranone lignans such as styraxin [17], aptosimon [18,19], 4-ketopinoresinol [20], mayuenolide [14], graminone [12], and a glycoside named styraxlignolide B [13]. In addition, several synthetic studies of furofuranone lignans and furofurandione lignans have been reported as candidates for anti-inflammatory agents [21,22]. This paper reports on the isolation and characterization of five furofuranone glucosides, and evaluates their antiestrogenic activity using the estrogen responsive breast cancer cell lines MCF-7 and T47D. This is the first report of the occurrence of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity. This study also demonstrates the chemotaxonomic significance of the plants of the Combretaceae family. 2. Experimental section 2.1. General experimental procedures 1H NMR (500 MHz), 13C NMR (125 MHz) and 2D-NMR spectra were recorded on a JEOL ECX-500. Chemical shifts are presented in δ (ppm) using tetramethylsilane (TMS) as an internal standard, and coupling constants (J) are expressed in hertz. Inverse-detected heteronuclear correlations were measured using HMQC (optimized for 1JC-H = 145 Hz) and HMBC (optimized for 3JC-H = 8 Hz) pulse

M.A. Muhit et al. / Fitoterapia 113 (2016) 74–79

sequences with a pulsed field gradient. Optical rotation was measured with a JASCO DIP-360 digital polarimeter. A Hitachi U2010 UV/VIS spectrophotometer was used to determine the UV spectra. ECD spectra were recorded with a JASCO J-720 WI spectropolarimeter. HRFABMS data was recorded with a JEOL JMS 700 spectrometer using an m-nitrobenzyl alcohol matrix. Column chromatography was carried out with powdered silica gel (Keiselgel 60, 230–400 mesh, Merck KGaA, Darmstadt, Germany) and styrene-divinylbenzene (Diaion HP-20, 250–800 μm particle size, Mitsubishi Chemical Co., Ltd., Japan). Precoated glass plates of silica gel (Keiselgel 60, F254, Merck Co., Ltd., Japan) and RP-18 (F254S, Merck KGaA) were used for TLC analysis. The TLC spots were investigated under UV light at a wavelength of 254 nm, and the plate was sprayed with dilute H2SO4 followed by heating. Repeated HPLC was carried out primarily with a JASCO model 887-PU pump, and isolates were detected with an 875-UV variable-wavelength detector at 205 nm. Separation and purification of the isolates was carried out using reversed-phase columns for preparative separation (Tosoh TSK gel ODS-80Ts, 5 μm, 6 × 60 × 2 cm; Tosoh Chemicals Co. Ltd., Tokyo, Japan; flow rate 45 mL/min with detection at 205 nm) and semi-preparative separation (Cosmosil Cholester, 5 μm, 2 × 25 cm, Nacalai Co. Ltd., Kyoto, Japan; YMC-Pack R&D ODS, 5 μm, 2 × 25 cm, YMC Co. Ltd., Kyoto, Japan; flow rate 9 mL/min with detection at 205 nm).

Table 1 1H NMR (500 MHz) spectroscopic data for compounds 1–5a (MeOH-d4, J in Hz). Position 1

2

3

4

1 2 4 5

3.30, m 5.40, d (3.5)

3.30, m 5.48, d (3.5)

3.47, m 5.79, d (2.5)

3.50, m 3.25, m 5.82, d (3.0) 5.50, d (3.5)

3.60, dd (9.5, 3.5) 5.19, d (3.5) 4.31, dd (9.5, 7.0) 4.05, dd (9.5, 4.5)

3.58, dd (10.0, 4.5) 5.16, d (4.5) 4.31, dd (10.0, 7.5) 4.06, dd (10.0, 4.5)

3.54, dd (9.0, 3.5) 5.16, d (3.5) 4.39, dd (9.0, 7.5) 4.03, dd (9.0, 5.0)

3.62, dd (9.0, 3.0) 5.18, d (3.0) 4.36, dd (10.0, 7.5) 4.08, dd (10.0, 5.0)

3.57, dd (9.0, 4.0) 5.14, d (4.0) 4.29, dd (9.0, 7.5) 4.05, dd (9.0, 5.5)

6.75, d (1.5)

6.73, s

6.50, s

6.63, s

6.89, s

6.64, d (1.5)

6.66, d (1.5)

6.64, d (1.5)

6.64, d (2.0) 6.64, d (2.0)

6.59, d (1.5) 5.91, s 3.88, s 3.86, s 3.82, s

6.60, d (1.5) 5.92, s 3.89, s 3.89, s 3.88, s 3.84, s 5.10, d (7.5) 3.48, m 3.42, m 3.36, m 3.22, m 3.75, dd (12.0, 2.0) 3.65, dd (12.0, 5.0)

6.59, d (1.5) 5.92, s 3.89, s 3.88, s 3.85, s

6.60, d (2.0) 5.92, s 3.89, s 3.88, s 3.85, s 3.82, s 5.15, d (8.0) 3.44, m 3.40, m 3.34, m 3.23, m 3.80, dd (12.0, 2.5) 3.60, dd (12.0, 6.0)

6 8a 8b 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ OCH2O OMe

2.2. Plant material The leaves of the plant were collected from Rangamati District in the Hill Tracts Region of Bangladesh in May 2013, and were identified by Mr. Sardar Nasir Uddin, Senior Scientific Officer, National Herbarium, Dhaka, Bangladesh. A voucher specimen has been deposited in the Herbarium for further reference (DACB accession no. 38094).

1‴ 2‴ 3‴ 4‴ 5‴ 6‴

2.3. Extraction and isolation The leaves were collected in fresh condition, and the stalks were removed immediately. The air-dried leaves were ground into coarse powder (3.4 kg approx.) and then extracted four times with hot methanol (4 × 15 L) by refluxing for 3 h each to give a viscous mass of 608 g. Part of the methanolic crude extracts (220 g) was suspended in 2 L of water and partitioned with EtOAc (2 L × 3). The EtOAc extract (93 g) was subjected to silica gel column chromatography, which was eluted with a chloroform-MeOH gradient solvent system with increasing polarities (100:0, 99:1, 98:2, 95:5, 90:10, 67:33, 50:50). All the fractions were collected and pooled by analyzing their TLC to afford 16 combined fractions. From these fractions, fraction 11 [1.5 g: eluted with chloroformMeOH (9:1)] was subjected to preparative HPLC using MeCN-water (1:3) as the mobile phase, followed by semi-preparative HPLC to afford 1 [7.4 mg; tR 130 min, YMC ODS pack column with solvent MeCN/H2O (22:78)], 2 [1.7 mg; tR 160 min, YMC ODS pack column with solvent MeCN/H2O (22:78)], 3 [7.0 mg; tR 74 min, YMC ODS pack column with solvent MeCN/H2O (20:80)], 4 [4.5 mg; tR 152 min, YMC ODS pack column with solvent MeCN/H2O (22:78)], and 5 [2.0 mg; tR 144 min, Cosmosil Cholester column with solvent MeCN/H2O (25:75)]. Terminaloside L (1): Pale yellow, amorphous powder; [α]25D +42.0 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 216 (4.46), 272 (3.47) nm; ECD (c 0.2 mM, MeOH) 215 (Δε +12.4), 245 (Δε +4.0), 280 (Δε +0.8) nm; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRFABMS m/z 593.1877 [M + H]+ (calcd for C28H33O14, 593.1870). Terminaloside M (2): Colorless, amorphous powder; [α]25D +52.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.48), 280 (3.65) nm; ECD (c 0.1 mM, MeOH) 220 (Δε + 10.2), 240 (Δε + 4.3), 285 (Δε + 0.8) nm; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRFABMS m/z 645.1813 [M + Na]+ (calcd for C29H34O15 Na, 645.1795). Terminaloside N (3): Pale yellow, amorphous powder; [α]25D +26.7 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 216 (4.41), 281 (3.66) nm; ECD

75

5

6.84, d (1.5)

4.91, d (7.5) 3.52, m 3.44, m 3.35, m 3.40, m 3.87, overlapped 3.65, dd (12.0, 6.0)

5.09, d (7.5) 3.44, m 3.41, m 3.34, m 3.24, m 3.79, dd (12.0, 2.0) 3.61, dd (12.0, 6.0)

6.59, d (2.0) 5.91, s 3.89, s 3.88, s 3.88, s 3.85, s 4.84, d (8.0) 3.40, m 3.38, m 3.35, m 3.27, m 3.86, overlapped 3.65, dd (12.0, 5.5)

a, Methylene proton signal observed in lower field; b, methylene proton signal observed in higher field a Assignments were based on HMQC and HMBC experiments.

Table 2 13C NMR (125 MHz) spectroscopic data for compounds 1–5a in MeOH-d4. Position

1

2

3

4

5

1 2 4 5 6 8 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ OCH2O OMe

51.0 86.4 179.4 54.4 85.0 74.1 137.2 108.1 152.7 140.2 155.2 105.8 136.7 107.3 145.2 136.3 150.8 100.9 102.8 61.7 57.5 56.9

49.9 82.6 180.3 54.3 85.4 75.2 130.3 140.7 147.7 143.3 148.8 108.7 136.7 107.5 145.2 136.4 150.8 101.1 102.8 62.0 61.4 57.5

1‴ 2‴ 3‴ 4‴ 5‴ 6‴

102.8 75.1 78.2 71.6 78.5 62.7

50.7 85.2 179.7 55.4 85.0 74.8 129.2 146.3 144.8 145.0 151.2 107.8 136.6 107.4 145.2 136.5 150.9 100.9 102.8 62.6 61.9 57.5 57.0 104.5 75.8 78.1 71.6 78.5 62.7

49.8 83.0 180.3 54.5 85.4 75.2 129.8 142.1 147.8 145.0 151.8 106.0 136.7 107.5 145.2 136.4 150.8 101.1 102.8 62.2 61.6 57.5 57.1 104.7 75.8 78.1 71.8 78.7 62.7

50.5 84.1 179.6 55.0 85.0 74.6 129.0 147.3 148.4 145.7 148.5 111.1 136.5 107.3 145.1 136.4 150.7 100.9 102.7 62.0 61.7 61.6 57.4 103.1 75.0 78.2 71.5 78.4 62.6

a

104.8 75.8 78.1 71.8 78.7 62.9

Assignments were based on HMQC and HMBC experiments.

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(c 0.2 mM, MeOH) 215 (Δε +10.5), 245 (Δε +2.2), 290 (Δε −0.6) nm; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRFABMS m/z 631.1635 [M + Na]+ (calcd for C28H32O15 Na, 631.1639). Terminaloside O (4): Pale yellow, amorphous powder; [α]25D +70.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.52), 280 (3.66) nm; ECD (c 0.1 mM, MeOH) 215 (Δε +14.8), 245 (Δε +3.4), 290 (Δε −0.4) nm; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRFABMS m/z 645.1791 [M + Na]+ (calcd for C29H34O15 Na, 645.1795). Terminaloside P (5): Pale yellow, amorphous powder; [α]25D +12.6 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 225 (4.11), 278 (3.53) nm; ECD (c 0.2 mM, MeOH) 215 (Δε +8.7), 245 (Δε +2.8), 285 (Δε +1.4) nm; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRFABMS m/z 645.1819 [M + Na]+ (calcd for C29H34O15 Na, 645.1795). 2.4. Cell culture and cell proliferation assay Estrogen responsive human breast cancer cells (MCF-7 and T47D) were purchased from the American Type Culture Collection (Manassas, VA) and cultured as described in a previous report [23]. The estrogenic activity of the isolates (cell proliferation assay) was determined by following the protocol described in the same report. 2.5. Antiestrogenic assay All of the isolates were tested for antiestrogenic activity following the procedure described in a previous report [24]. MCF-7 and T47D cells were seeded at a density of 1.0 × 104 cells/well in 96-well plates in 90 μL of 5% DCC-treated, FBS-supplemented RPMI phenol red-free medium. After 3 h incubation, 5 μL of each test compound at four different concentrations ranging from 0.01 to 10 μM was added to each well along with 5 μL of estradiol (E2) at a concentration of 20 nM to make a final volume 100 μL in each well. Finally, the plates were incubated in a CO2 incubator for 96 h. 5 μL of serially diluted tamoxifen at concentrations ranging from 0.01 to 10 μM was used as a positive control. The result was calculated from the cell populations, and the iEqE values of each sample (iEqE50, iEqE10, and iEqE1) were determined for the concentration required to inhibit the E2 effect (iEqE50, iEqE10, and iEqE1, with a concentration suppressing the E2 effect to the equivalent level of 50, 10, and 1 pM, respectively). From the results of the concentrations tested, samples were categorized as strong (S) if they suppressed E2 activity to a level of b 10 pM, and mild (M) if they suppressed the activity level to between 10 and 50 pM. 2.6. Acid hydrolysis and sugar identification Acid hydrolysis in presence of 50% TFA was carried out to determine the absolute configuration of the sugar moiety in all of the isolates [25]. A portion of each isolate (0.5–1.0 mg) was hydrolyzed (50 μL of 50% TFA) in a hot water bath at 100 °C for 1 h. The air-dried reaction mixtures were diluted with H2O and extracted with EtOAc. The H2O layers were concentrated under a vacuum evaporator and mixed with L-cysteine methyl ester hydrochloride in pyridine (20 mg/mL, 50 μL) at 60 °C for 1 h. o-Tolyl isothiocyanate (5 μL) was added to the mixtures and heated at 60 °C for 1 h. The reaction mixtures were then air-dried and concentrated under a vacuum evaporator. A few drops of MeOH were added to each sample before HPLC analysis [column: YMC-pack R&D ODS, 4.6 × 300 mm, MeCN-H2O (22:78) solvents used as mobile phase, flow rate 1 mL/min, UV detection at 205 nm] and D-glucose (tR 35 min) and L-glucose (tR 32.5 min) were identified by comparison with standard samples. 2.7. Data and statistical analysis Statistical differences for antiestrogenic activity were determined by analysis of variance followed by Dunnett's multiple comparison tests. Statistical significance was expressed at the p b 0.05 level.

3. Results and discussion Silica-gel column chromatography of the EtOAc soluble fractions followed by repeated reverse-phase HPLC analysis yielded five new (1–5) lignan glycosides containing 4-oxofurofuran moieties (Fig. 1). The structures of the isolates were established on the basis of a variety of spectroscopic data. Compound 1, a pale yellowish amorphous solid, had the molecular formula C28H32O14 based on the protonated ion peak [M + H]+ at m/z 593.1877 (calcd 593.1870) in the HRFABMS data, indicating 13 indices of unsaturation. The UV spectrum revealed the absorption band of aromatic rings (272 and 216 nm) in its structure, which was supported by twelve aromatic carbon resonances in 13C NMR spectra. 1H NMR showed the characteristic signals of a furofuran ring, such as two benzylic oxymethine protons [δH 5.40 (d, J = 3.5 Hz, H-2) and 5.19 (d, J = 3.5 Hz, H-6)], two methines [δH 3.30 (m, H-1) and 3.60 (dd, J = 9.5, 3.5 Hz, H-5)], and one oxymethylene [δH 4.31 (dd, J = 9.5, 7.0 Hz, H-8a) and 4.05 (dd, J = 9.5, 4.5 Hz, H-8b)] (Table 1). However, a carbonyl carbon at δC 179.4 (C-4) was found in the 13C NMR spectra, instead of the expected oxymethylene (Table 2). In accordance with the 1H–1H COSY spectra, two sets of partial structures,\\O\\CH\\CH\\CH2\\and \\O\\CH\\CH\\CO\\, were assigned to the positions C-2, 1, 8 and C-6, 5, 4, respectively. In the HMBC spectra, two oxymethines (H-2 and 6) showed clear correlations to the carbonyl group (δC 179.4), which indicated the partial structure of a 4-oxo-3,7-dioxabicyclo[3.3.0]octane moiety [26]. Two sets of meta-coupled aromatic proton resonances [δH 6.84 (d, J = 1.5, H-2′) and 6.75 (d, J = 1.5 Hz, H-6′); δH 6.64 (d, J = 1.5, H-2″) and 6.59 (d, J = 1.5 Hz, H-6″)] indicated the presence of 1′,3′,4′,5′-tetrasubstituted aromatic moieties. The HMBC spectrum showed the connectivity of four partial structures: (i) dioxymethylene protons (δ 5.91) and one of the meta-coupled aromatic proton signals [δ 6.59 (H-6″)] to two oxygenated aromatic carbons [δ 136.3 (C-4″) and 150.8 (C-5″)] in ring B, (ii) the same meta-coupled proton signal (H-6″) to an oxymethine carbon signal [δ 85.0 (C-6)], (iii) another doublet signal of second aromatic ring [δ 6.84 (H-2′)] and an anomeric proton signal [δ 4.91 (H-1‴)] to an oxygenated aromatic carbon [δ 152.7 (C-3′)] in ring A, and (iv) the second doublet proton signal (H2′) to another oxymethine carbon signal [δ 86.4 (C-2)] (Fig. 2). Ring B was recognized to be attached at C-6 based on the HMBC correlation of characteristic methine resonance [δH 3.60 (dd, J = 9.5, 3.5 Hz, H5)], which was observed in a series of isolates, 1–5. The HMBC spectrum also suggested that the attachment positions of the three methoxy groups based on the correlated signals [δH 3.82 (MeO)/δC 140.2 (C-4′), δH 3.86 (MeO)/δC 155.2 (C-5′), and δH 3.88 (MeO)/δC 145.2 (C-3″)]. Based on the small coupling constants (J = 3.5 Hz) of the oxymethines (H-2 and 6) and the chemical shift of the oxymethylene protons, two sets of protons (H-2/H-1 and H-6/H-5) were indicated as being trans oriented. The ECD spectra showed positive Cotton effects at 245 (Δε + 4.0) nm, which was the opposite of (-)-styraxlignolide B [13]. Hence, the absolute configuration of the 4-oxofurofuran nucleus was determined as 1R, 2S, 5S, and 6S. Acid hydrolysis of 1–5 afforded a sugar moiety, which was identified as D-glucose by comparing it with an authentic sample by HPLC. On the basis of the coupling constant of the anomeric proton resonance [δH 4.91 (d, J = 7.5 Hz, H-1‴), the βconfiguration of the glucose unit was confirmed. As part of the characterization of furofuran lignan glycosides from T. citrina [9], 1 (terminaloside L) was identified as (1R,2S,5S,6S)-2-(3′-hydroxy-4′,5′dimethoxyphenyl)-6-(3″-methoxy-4″,5″-methylenedioxyphenyl)-4oxo-3,7-dioxabicyclo[3.3.0]octane 3′-O-β-D-glucopyranoside. Compound 2, [α]25D + 52.4, was obtained as a colorless solid, and the molecular formula was assigned as C29H34O15 based on the sodiated ion peak [M + Na]+ at m/z 645.1813 (calcd 645.1795) that appeared in the HRFABMS data. The NMR spectra of 2 were very similar to those of 1, and exhibited the characteristic resonances of a 4-oxofurofuran, a glucose moiety, and a 1″, 3″, 4″, 5″-substituted phenyl moiety as partial structures (Tables 1 and 2). In the HMBC spectrum of 2, a meta-coupled

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77

Fig. 1. New furofuranone lignan glycosides (1–5) from T. citrina.

aromatic proton signal [δH 6.60 (d, J = 1.5 Hz, H-6″)] showed a correlation to an oxymethine carbon [δC 85.0 (C-6)], which also shared correlations to two oxygenated carbons [δC 136.5 (C-4″) and 150.9 (C-5″)] with a dioxymethylene proton signal (δH 5.92). The paired meta-coupled aromatic proton signal [δH 6.66 (d, J = 1.5 Hz, H-2″)] presented a correlation to the oxygenated carbon [δC 145.2 (C-3″)] in common with an oxymethyl proton [δH 3.89 (MeO-3″)]. Meanwhile, a singlet aromatic proton resonance [δH 6.73 (H-6′)] exhibited correlations to three oxygenated carbons [δC 146.3 (C-2′), 145.0 (C-4′) and 151.2 (C-5′)] and an oxymethine carbon [δC 85.2 (C-2)]. The attachment position of the sugar moiety was also confirmed from the HMBC spectrum, which showed a correlation from the anomeric proton [δH 5.10 (d, J = 7.5 Hz, H-1‴)] to the oxygenated aromatic carbon [δC 144.8 (C-3′)]. Thus, this aryl unit was identified as a 3′-glucosyloxy-2′,4′,5′trimethoxyphenyl moiety. The ECD spectrum of 2 displayed positive Cotton effects at 240 (Δε + 4.3) nm. Hence, 2 (terminaloside M) was distinguished as (1R,2S,5S,6S)-2-(3′-hydroxy-2′,4′,5′trimethoxyphenyl)-6-(3″-methoxy-4″,5″-methylenedioxyphenyl)-4oxo-3,7-dioxabicyclo[3.3.0]octane 3′-O-β-D-glucopyranoside. Compounds 3 and 4 were isolated as pale yellow amorphous powders. Their molecular formula were determined to be C28H32O15 and C29H34O15 for 3 and 4 based on their respective sodiated ion peaks at m/z 631.1635 [M + Na]+ (calcd 631.1639) and 645.1791 [M + Na]+ (calcd 645.1795) in the HRFABMS data. Their spectroscopic features were very similar to one another and shared many features with those

of 2. The 1H NMR spectra of 3 and 4 showed a pair of oxymethine proton signals [3: δ 5.79 (d, J = 2.5 Hz, H-2), 5.16 (d, J = 3.5 Hz, H-6); 4: δ 5.82 (d, J = 3.0 Hz, H-2), 5.18 (d, J = 3.0 Hz, H-6)], which indicated the presence of a furofuran ring system in their structures. However, both were lower field shifted by approximately 0.3 ppm when compared with 2 and showed common correlations to the oxygenated carbon signal [3: δC 140.7 (C-2′); 4: 142.1 (C-2′)] with the anomeric proton signal [3: δ 5.09 (d, J = 7.5 Hz, H-1‴); 4: δ 5.15 (d, J = 8.0 Hz, H-1‴)] in their HMBC spectra. In the HMBC spectra, two oxygenated aromatic carbons [3: δC 136.4 (C-4″) and 150.8 (C-5″); 4: 136.4 (C-4″) and 150.8 (C-5″)] were recognized as having correlations to both a dioxymethylene signal [3: δ 5.92 (s); 4: δ 5.92 (s)] and a meta-coupled aromatic proton signal [3: δ 6.59 (d, J = 1.5 Hz, H-6″); 4: δ 6.60 (d, J = 2.0 Hz, H-6″)], which also showed a correlation to another oxymethine [3: δH 5.16/δC 85.4; 4: δH 5.18/δC 85.4]. The shared correlation between the paired metacoupled aromatic proton signals [3: δ 6.64 (d, J = 1.5 Hz, H-2″); 4: δ 6.64 (d, J = 2.0 Hz, H-2″)] and the oxymethyl proton to an oxygenated carbon [3: δC 145.2 (C-3″); 4: 145.2 (C-3″)] suggested that these compounds have the same aromatic moiety as 1 and 2. From the above spectroscopic data, the methoxy groups of 3 were assigned as being attached at C-3′ and C-4′, as these groups were deduced to have substituted groups at both of their ortho positions based on their chemical shifts [δ 61.4 and 62.6] in the 13C NMR spectrum. In the NMR spectrum of 4, an additional methoxy signal (δH 3.81/δC 57.1) was observed when compared with 3. The attachment positions of the methoxy groups

Fig. 2. Selected HMBC, COSY (bold line), and NOE (dotted arrow) correlations of 1.

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were indicated by their chemical shifts and were further confirmed from the HMBC spectrum. Based on their ECD spectra, 3 (terminaloside N) was identified as (1R,2S,5S,6S)-2-(2′,5′-dihydroxy-3′,4′dimethoxyphenyl)-6-(3″-methoxy-4″,5″-methylenedioxyphenyl)-4oxo-3,7-dioxabicyclo[3.3.0]octane 2′-O-β-D-glucopyranoside, and 4 (terminaloside O) was shown to be (1R,2S,5S,6S)-2-(2′-hydroxy3′,4′,5′-trimethoxyphenyl)-6-(3″-methoxy-4″,5″methylenedioxyphenyl)-4-oxo-3,7-dioxabicyclo[3.3.0]octane 2′-O-βD-glucopyranoside. Compound 5, a pale yellow amorphous powder, was assigned the identical molecular formula as 2 and 4, C29H34O15, based on the sodiated ion peak observed at m/z 645.1819 [M + Na]+ (calcd 645.1795). The NMR spectra of 5 exhibited similar characteristics to those of 2 and 4, indicating the presence of a 4-oxofurofuran ring system, a glucose moiety, and ring B in the form of a 3″-methoxy-4″,5″-methylenedioxyphenyl. The NMR data also revealed the same number of methoxy groups presenting on ring A, which possessed a glucose unit. Based on the chemical shifts of the methoxy groups of 5 in the 13C NMR spectrum, these groups were assigned as being attached at C-2′, C-3′, and C-4′. This assignment is further supported by the glycosylation shifts of the aromatic proton/carbon signals located at α and β positions [Δ +0.3 ppm at H-6′; Δ −3 ppm at C-5′ and Δ +5 ppm at C-6′] (Table 2). On the basis of the ECD data, 5 (terminaloside P) was identified as (1R,2S,5S,6S)-2-(5′-hydroxy-2′,3′,4′-trimethoxyphenyl)-6-(3″-methoxy-4″,5″methylenedioxyphenyl)-4-oxo-3,7-dioxabicyclo[3.3.0]octane 5′-O-βD-glucopyranoside. In a screening of the estrogenic property of the isolates based on estrogen responsive breast cancer cell lines MCF-7 and T47D, none of the compounds showed cell proliferation stimulatory activity (data not shown). In order to evaluate the antiestrogenic properties of the isolates, cell proliferation was stimulated with 100 pM of E2, followed by cotreatment of the isolates at concentrations of 0.01, 0.1, 1.0 and 10 μM. Tamoxifen was used as a positive control. At a concentration of 10 nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common (Table 3). We have previously isolated analogous furofuran lignan glucosides, terminalosides C–F, and reported their antiestrogenic activity [16]. Although terminalosides L (1), N (3), and O (4) share the same aromatic moieties, their antiestrogenic activity was the same or lower than that of their corresponding furofuran lignans, terminalosides C–F. This suggests that the oxofurofuran ring moiety does not enhance antiestrogenic activity. To our knowledge, this is the first report of the antiestrogenic activity of furofuranone lignans. As high levels of estradiol increase the risk of breast cancer in postmenopausal women, this

Table 3 Inhibitory activity of compounds 1–5 against E2-enhanced cell proliferation. MCF-7

T47D

Compound

iEqE50a

iEqE10a

iEqE1a

ILb

iEqE50a

iEqE10a

iEqE1a

ILb

1 2 3 4 5 Tamoxifene

b0.01 b0.01 b0.1 0.5 b0.1 0.1

– b0.01 10 1.8 – 0.5

– – – – – 5.0

Mc Sd Sd Sd

b0.01 b0.01 8.4 8.6 b0.1 0.1

b0.01 – – – – 0.8

– – – – – 7.9

Sd Mc

a iEqE50, iEqE10, iEqE1 represent the concentrations of the compounds (μM) that decrease cell proliferation (enhanced by 100 pM of E2) to equivalent levels of those induced by 50 pM, 10 pM, and 1 pM of E2 treatment, respectively. The values were calculated by linear regression analysis using four different concentrations. b IL inhibitory level of the compound. c Mild inhibition (M), N50% inhibition with the concentrations tested. d Strong inhibition (S), N90% inhibition with the concentrations tested. e Positive control.

study suggests that terminaloside L (1) and terminaloside M (2) have potential as lead compounds for future drug development [27]. However, detailed studies are required to establish their efficacy in humans. 4. Conclusion In conclusion, our extensive phytochemical investigation of EtOAc extracts of T. citrina has revealed five new furofuranone lignan monoglucosides, terminalosides L (1)–P (5). All of the isolates were tested for their estrogenic and/or antiestrogenic properties using estrogen responsive breast cancer cell lines T47D and MCF-7. All of the compounds showed at least 50% inhibition in both cell lines. Among them, terminalosides L (1) and M (2) inhibited cell growth by up to 90% in the T47D and MCF-7 lines, respectively, at a minimum concentration of 10 nM. Acknowledgements This work was supported by a MEXT scholarship to M.A.M. from the Ministry of Education, Culture, Sports, Science and Technology, Japan. It was also supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 25460126 to K.U. The authors are thankful to Mr. Narhari Das, Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Bangladesh for his generous support of sample collections, Mr. S. Makita, Institute for Molecular Science, National Institute of Natural Sciences, Japan for his support in measuring the ECD spectra of isolates, and Mr. P. Hawke, Scientific English Program, University of Shizuoka for his comments on the English in the manuscript. Appendix A. Supplementary data 1H and 13C spectra of 1–5 (PDF). Supplementary data associated with this article can be found in the online version, at doi: http://dx. doi.org/10.1016/j.fitote.2016.07.004. References [1] I.E. Cock, The medicinal properties and phytochemistry of plants of the genus Terminalia (Combretaceae), Inflammopharmacol. 23 (2015) 203–229. [2] P.M. Paarakh, Terminalia arjuna (Roxb.) Wt. and Arn.: a review, Int. J. Pharm. 6 (2010) 513–534. [3] R.R. Chattopadhyay, S.K. Bhattacharyya, Terminalia chebula: an update, Pharmacogn. Rev. 1 (2007) 151–156. [4] J.N. Eloff, D.R. Katerere, L.J. McGaw, The biological activity and chemistry of the southern African Combretaceae, J. Ethnopharmacol. 119 (2008) 686–699. [5] A.C. Tan, D.X. Hou, I. Konczak, S. Tanigawa, I. Ramzan, D.M.Y. Sze, Native Australian fruit polyphenols inhibit COX-2 and iNOS expression in LPS-activated murine macrophages, Food Res. Int. 44 (2011) (2662–2367). [6] M. Yusuf, J. Begum, M.N. Hoque, J.U. Chowdhury, Medicinal Plants of Bangladesh, second ed. Bangladesh Council of Scientific and Industrial Research Laboratories, Chittagong, 2009 625. [7] N. Das, D. Goshwami, M.S. Hasan, S.Z. Raihan, N.K. Subedi, Phytochemical screening and anthelmintic activity of methanol extract of Terminalia citrina leaves, Asian Pac J. Trop. Dis. 5 (Suppl. 1) (2015) 166–168. [8] N. Das, D. Goshwami, M.S. Hasan, Z.A. Mahmud, S.Z. Raihan, M.Z. Sultan, Evaluation of antinociceptive, anti-inflammatory and anxiolytic activities of methanolic extract of Terminalia citrina leaves, Asian Pac J. Trop. Dis. 5 (Suppl. 1) (2015) 137–141. [9] M.A. Muhit, K. Umehara, H. Noguchi, Furofuran lignan glucosides with estrogen-inhibitory properties from the Bangladeshi medicinal plant Terminalia citrina, J. Nat. Prod. 79 (2016) 1298–1307. [10] S. Burapadaja, A. Bunchoo, Antimicrobial activity of tannins from Terminalia citrina, Planta Med. 61 (1995) 365–366. [11] R.S. Ward, Lignans, neolignans and related compounds, Nat. Prod. Rep. 16 (1999) 75–96. [12] K. Matsunaga, M. Shibuya, Y. Ohizumi, Graminone B, a novel lignan with vasodilative activity from Imperata cylindrica, J. Nat. Prod. 57 (1994) 1734–1736. [13] B.S. Min, M.K. Na, S.R. Oh, K.S. Ahn, G.S. Jeong, G. Li, S.K. Lee, H. Joung, H.K. Lee, New furofuran and butyrolactone lignans with antioxidant activity from the stem bark of Styrax japonica, J. Nat. Prod. 67 (2004) 1980–1984. [14] C.C. Kuo, W. Chiang, G.P. Liu, Y.L. Chien, J.Y. Chang, C.K. Lee, J.M. Lo, S.L. Huang, M.C. Shih, Y.H. Kuo, 2,2-Diphenyl-1-picrylhydrazyl radical-scavenging active components from adlay (Coix lachrymal-jobi var. ma-yuen. Stapf) hulls, J. Agric. Food Chem. 50 (2002) 5850–5855.

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