Hydrolyzable Tannins of Tamaricaceous Plants ... - ACS Publications

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Hydrolyzable Tannins of Tamaricaceous Plants. 7.1 Structures and Cytotoxic Properties of Oligomeric Ellagitannins from Leaves of Tamarix nilotica and Cultured Tissues of Tamarix tetrandra Mohamed A. A. Orabi,† Shoko Taniguchi,‡ Hiroshi Sakagami,§ Morio Yoshimura,⊥ Yoshiaki Amakura,⊥ and Tsutomu Hatano*,‡ †

Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan § Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, Meikai University, School of Dentistry, Sakado, Saitama 350-0283, Japan ⊥ College of Pharmaceutical Sciences, Matsuyama University, Bunkyo-cho, Matsuyama 790-8578, Japan ‡

S Supporting Information *

ABSTRACT: Partially unacylated new oligomeric hydrolyzable tannins, nilotinin T2 (1, trimer) and nilotinin Q1 (2, tetramer), together with four known trimers, nilotinin T1 (3) and hirtellins T1−T3 (4−6), and a dimer, tamarixinin B (7), were isolated from the aqueous acetone extracts of leaves of Tamarix nilotica. Among them, the new trimer 1 and the known trimers 4 and 6, in addition to the partially unacylated new trimer nilotinin T3 (8), the known dimers nilotinin D3 (9) and tamarixinin C (10), and the monomer tellimagrandin I (11), were isolated from the cultured shoots of Tamarix tetrandra. The structures of the new hydrolyzable tannins were established by chromatographic analyses and extensive 1D and 2D NMR, HRESI-TOFMS, and ECD spectroscopic experiments. Among the new oligomeric tannins, the particular unacylated position of a glucose core is attributed to a possible biosynthetic route. Isolation of the same oligomeric tannins from cultured shoots of T. tetrandra emphasizes the unique biogenetic ability of the obtained cultures on production of the structurally and biologically characteristic tamaricaceous tannins commonly produced by the intact Tamarix plants. Additionally, tannins obtained in the present study together with gemin D (12) and 1,3-di-O-galloyl-4,6-O-(aS)-hexahydroxydiphenoyl-β-Dglucose (13), from our previous investigation of the leaves of T. nilotica, exhibited variable tumor-specific cytotoxic effects. The ellagitannin trimers 4, 6, and 8 and the dimer 9 exerted predominant tumor-selective cytotoxic effects with high specificity toward human promyelocytic leukemia cells. including volatile oils, sterols, triterpenes, flavonoids, phenolics, and occasionally sulfated congeners of these metabolites are the phytoconstituents often reported from the leaves, roots, and flowers of T. nilotica.3a,12 In previous reports several hydrolyzable tannins from leaves of T. nilotica and cultured shoots of T. tetrandra Pall. were reported.13 Repeating chromatographic isolation on gels especially for the fractions containing oligomeric molecules from extracts of the leaves of T. nilotica and cultured shoots of T. tetrandra led to purification of a new ellagitannin trimer, nilotinin T2 (1), a tetramer, nilotinin Q1 (2), four known trimers, nilotinin T1 (3) and hirtellins T1−T3 (4−6), and a dimer, tamarixinin B (7), from the former species. The new trimers nilotinins T2 (1) and T3 (8), the known trimers hirtellins T1 (4) and T3 (6), the dimers nilotinin D3 (9) and tamarixinin C (10), and the monomer tellimagrandin I (11) were obtained from the latter species.

Tamarix, also known as Salt Cedar, is the largest genus in the family Tamaricaceae.1,2 The common traditional uses shown in various reports for plant species of the genus are as a diaphoretic, diuretic, and hepatotonic and to treat liver disorders, relieve headache, ease prolonged or difficult labor, reduce spleen edema, and cure sores and wounds besides being an astringent and employed for tanning and dyeing purposes.2,3 Modern pharmacological investigations revealed that an extract of T. gallica had a chemopreventive effect and suppressed thioacetamide-mediated hepatic oxidative stress, toxicity, and tumor promotion response in rats. 4 Antioxidant and hepatoprotective activities of T. nilotica5 and T. gallica,4 as well as antioxidant and/or antimicrobial activities of T. ramosissima,6 T. hispida,7 T. boveana,8 T. aphylla,9,10 T. nilotica,3a and T. gallica,11 were also reported. Among Tamarix plants, T. nilotica (Ehrenb.) Bunge is a native plant in Egypt with a long history. Its leaves and young branches are used for spleen edema, and it is mixed with ginger for uterus infections, while an aqueous decoction of its bark with vinegar is used as a licicidal lotion.2 Secondary metabolites © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 2, 2015

A

DOI: 10.1021/acs.jnatprod.5b01065 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR Spectroscopic Data (δH, J in Hz) of the Glucose Protons of 1, 2, and 8 (600 MHz, Acetone-d6−D2O, 9:1, v/v) 8

8 1 glucose-1 1 5.37 d (J = 7.8) 2 5.52 dd (J = 7.8, 9.6) 3 5.63 t (J = 9.6) 4 5.12 t (J = 9.6) 5 4.23 dd (J = 6.6, 9.6) 6 5.27 dd (J = 6.6, 12.6) 3.82 d (J = 12.6) glucose-2 1 5.85 d (J = 7.8) 2 5.55 dd (J = 7.8, 9.6) 3 5.26 t (J = 9.6) 4 5.09 t (J = 9.6) 5 4.30 dd (J = 6.6, 9.6) 6 5.27 dd (J = 6.6, 12.6) 3.93 d (J = 12.6) glucose-3

α-anomer 5.33 d (J = 3.6) 5.04 dd (J = 3.6, 10.2) 5.79 t (J = 10.2) 5.06 t (J = 10.2) 4.59 dd (J = 6.6, 10.2) 5.18 dd (J = 6.5, 13.2) 3.74 d (J = 13.2.)

β-anomer

1

2

4.52 d (J = 7.8) 5.14 dd (J = 7.8, 10.2) 5.49 t (J = 10.2) 5.04 t (J = 10.2) 4.07 dd (J = 6.6, 10.2) 5.20 dd (J = 6.6, 13.2) 3.78 d (J = 13.2)


5.55 d 5.61 d (J = 7.8) (J = 7.8) 5.44 dd 5.46 dd (J = 7.8, 10.2) (J = 7.8, 10.2) 5.51 t (J = 10.2) 5.08 t (J = 10.2) 4.23 dd (J = 6.6, 10.2) 5.22 dd (J = 6, 12.6) 3.81 d (J = 12.6)


1 5.84 (J = 7.8) 2 3.98 dd (J = 7.8, 9.6) 3 5.46 t (J = 9.6) 4 5.00 t (J = 9.6) 5 4.28 dd (J = 6.6, 9.6) 6 5.22 dd (J = 6.6, 12.6) 3.77 d (J = 12.6) glucose-4 1 2 3 4 5 6

α-anomer 6.11a d (J = 8.4)

β-anomer

6.10a d (J = 8.4) 5.55 dd (J = 8.4, 10.2) 5.76b t 5.75b t (J = 10.2) (J = 10.2) 5.19c t 5.18c t (J = 10.2) (J = 10.2) 4.50d dd 4.48d dd (J = 6, 10.2) (J = 6, 10.2) 5.31 dd (J = 6, 12.6) 3.82e d 3.80e d (J = 12.6) (J = 12.6)

2 5.80 br d (J = 7.8) 5.49 dd (J = 7.8, 9.6) 5.48 t (J = 9.6) 5.13 t (J = 9.6) 4.32 dd (J = 6.6, 9.6) 5.27 dd (J = 6.6, 12.6) 3.92 d (J = 12.6) 5.83 d (J = 8.4) 3.99 dd (J = 8.4, 9.6) 5.46 t (J = 9.6) 5.00 t (J = 9.6) 4.28 dd (J = 6.6, 9.6) 5.22 dd (J = 6.6, 12.6) 3.79 d (J = 12.6)

a−e

Exchangeable.

■

RESULTS AND DISCUSSION An aqueous acetone homogenate of the leaves of T. nilotica was fractionated over a Diaion HP-20 column. The H2O−MeOH (4:6, v/v) eluate was refractionated over Toyopearl HW-40C gel. The crude eluates, especially those containing oligomeric tannins, were alternatively fractionated over Sephadex LH-20 and MCI-gel CHP-20P gels. The crude ellagitannins were purified from the different eluates by preparative RP-HPLC. An aqueous acetone extract of dark-grown cultured tissues of T. tetrandra was similarly chromatographed over the same type of gels. As a result, two new ellagitannin trimers (1 and 8), a new tetramer (2), and eight known tannins (3−7 and 9−11) including dimeric and trimeric ones were obtained in high purity. The purity of the compounds was ascertained from showing single homogeneous peaks in different HPLC analyses and the absence of significant proton or carbon signals from contaminants in the NMR spectra (Supporting Information). The known tannins were identified as an ellagitannin monomer, tellimagrandin I (11),13c the dimers tamarixinin B (8),13e nilotinin D3 (9),13a and tamarixinin C (10),13e and a trimer, nilotinin T1 (3),13e based on the comparisons of their 1H NMR and ESIMS data with those of the authentic samples. The known trimers (4−6) were identified independently by extensive spectroscopic experiments, and their data corresponded with those of the known hirtellins T1−T3, respectively.15

It should be noted that in a previous study it was observed that hydrolyzable tannin oligomers with large molecular sizes are exceptional inhibitors of tumor growth in mice, among the more than a 100 monomeric and oligomeric tannins and simple phenolics screened.14 In the previous investigations, hydrolyzable tannin monomers and dimers from T. nilotica also exhibited strong structural-dependent cytotoxic effects against human oral squamous cell carcinoma (OSCC) (HSC-2, HSC3, and HSC-4) and human promyelocytic leukemia (HL-60) cell lines.13c,e Since the cytotoxic effects by Tamarix tannins depend on the structures, and the effect of the large oligomers was not sufficiently studied, the hydrolyzable tannin trimers 3, 4, 6, and 8 in comparison with the dimers 9 and 10 and the monomer 11, together with gemin D (12) and 1,3-di-O-galloyl4,6-O-(aS)-hexahydroxydiphenoyl-β-D-glucose (13), which were previously purified from the same Tamarix species, were tested for their cytotoxic activities against OSCC (HSC-2, HSC-3, HSC-4) compared to their effects on human normal oral cells, gingival fibroblasts (HGF), human periodontal ligament fibroblasts (HPLF), and human pulp cells (HPC). The aim of this report is to elucidate the structures of the new oligomeric ellagitannins 1, 2, and 8, suggest possible biosynthetic routes for the new isolates, and examine the cytotoxicity of numerous ellagitannins with various molecular weight (Mr). B



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Table 2. 13C NMR Spectroscopic Data of the Glucose Carbons of 1, 2, and 8 (151 MHz, Acetone-d6−D2O, 9:1, v/ v)

Structural Determination of the New Ellagitannins. Structure of Nilotinin T2. Nilotinin T2 (1) was isolated as an off-white, amorphous powder, and its trimeric nature was demonstrated by the long retention time (tR = 14.96 min) upon NP-HPLC analysis.16 The molecular formula of 1 was determined to be C116H82O74 by HRESIMS and from the following spectroscopic features. The 1H NMR spectrum of 1 exhibited a pair of one-proton singlets (δH 7.07 and 7.06) and two pairs of meta-coupled doublets [δH (7.35, 6.52) and (7.19, 6.42); each 1H, d, J = 1.8 Hz] characteristic of the presence of two dehydrodigalloyl (DHDG) moieties that commonly occur among tannins of the family Tamaricaceae.13a−c,15,17 The spectrum also showed four two-proton singlets (δH 7.03, 7.00, 6.95, and 6.86) accounting for the presence of four galloyl units.18 The presence of six one-proton singlets, half of them with relatively downfield shifts (δH 6.62, 6.602, and 6.598) and the other with relatively upfield shifts (δH 6.51, 6.49, and 6.46), are diagnostic for the presence of three hexahydroxydiphenoyl (HHDP) units.19 The spectrum also showed aliphatic proton signals (Table 1) assignable to three 4C1 glucopyranose cores. The chemical shifts of the glucose proton signals (Table 1) indicated that two of the glucose cores (glucose-1 and -2) are fully acylated, while the hydroxy group at C-2 of glucose-3 is unacylated, as evidenced from the shielding of the resonance at δH 3.99 (dd, J = 7.8, 9.6 Hz) of H-2 of glucose-3. The acyl units at the anomeric centers of the glucose residues are in the βconfiguration, as demonstrated by the large coupling constant (J = 7.8 Hz) of their anomeric proton signals. The aromatic acyl moieties as well as the glucose cores in 1 were further confirmed by aliphatic, aromatic (oxygenated and nonoxygenated), and carbonyl carbon resonances listed in Tables 2 and 3. The large differences in the chemical shifts of the C-6 diastereotopic protons of glucose-1 (δH 5.27, 3.82), glucose-2 (δH 5.27, 3.93), and glucose-3 (δH 5.22, 3.77) indicate bridging of the HHDP units at O-4/O-6 of the respective glucose cores.19 This was confirmed by the HMBC correlations among the H-3′ signals of the HHDP groups (δH 6.51, 6.49, and 6.46) and glucose cores H-4 (δH 5.12, 5.09, and 5.00) via the common carbonyl carbons (δC 167.7, 167.8, and 167.9) and similar correlations of the H-3 signals of the HHDP groups (δH 6.62, 6.602, and 6.60) and the C-6 diastereotopic protons of glucose-1 (δH 5.27, 3.82), glucose-3 (δH 5.22, 3.77), and glucose-2 (δH 5.27, 3.93) through the common carbonyl carbons (δC 168.33, 168.35, and 168.41). The HMBC spectrum also showed correlations of meta-coupled doublets of a DHDG moiety (δH 7.35, 6.52) with H-1 (δH 5.84) of glucose-3 through the ester carbonyl carbon (δC 165.1) and correlations of the doublets (δH 7.19, 6.42) of the other DHDG moiety with H-1 (δH 5.85) of glucose-2 through the ester carbonyl carbon (δC 164.6). Likewise, two one-proton singlets (δH 7.07, 7.06) of the DHDG moieties showed HMBC correlations with H-2 (δH 5.55) of glucose-2 and H-2 (δH 5.52) of glucose-1 through the ester carbonyl carbons (δC 164.53, 164.56). These correlations correspond to the bridging of the DHDG moieties between the glucose cores, where the galloyl parts of the DHDG moieties showing one-proton singlets are esterified with O-2 of glucose1 and with O-2 of glucose-2, while the other galloyl parts with two meta-coupled doublets are esterifying O-1 of glucose-2 and O-1 of glucose-3. The galloyl units are located at O-1 of glucose-1 and O-3 of each of the glucoses 1−3, as confirmed by the HMBC correlations of the galloyl proton signals (δH 7.00, 6.95, 6.86, and 7.03) with the corresponding signal (δH 5.37) of H-1 of glucose-1 and the H-3 signals (δH 5.63, 5.26, and 5.46)

8 glucose-1 1 2 3 4 5 6 glucose-2 1 2 3 4 5 6 glucose-3 1 2 3 4 5 6 glucose-4

1

α-anomer

β-anomer

2

93.6 71.6 73.5 70.5 72.9 63.0

90.89 72.64 71.56 71.01 66.89

96.28 73.98 73.98 70.94 71.86

93.6 71.6 73.5 70.6 72.9 63.0

93.7 71.4 73.6 70.7 72.9 63.1 95.5 72.1 75.6 70.7 72.7 63.2

63.5 93.6

70.5 72.78 62.94

93.7 71.4 73.5 70.7 72.9 63.0

93.49 71.68 73.34 70.58 72.93 63.13

93.7 71.4 73.4 70.7 72.9 63.1

71.2 73.34

71.25 73.39

95.5 72.1 75.6 70.7 72.7 63.2

of glucoses-1−3 (Table 1) through the carbonyl carbons (δC 164.55, 166.85, 166.5, and 167.1), respectively. The axial chirality of the HHDP groups was determined as (aS) because of the large positive Cotton effect at 237 nm, [θ]237 +3.2 × 105, in the electronic circular dichroism (ECD) spectrum (Figure S30, Supporting Information) of 1.20 On the basis of the aforementioned spectroscopic data, the structure of nilotinin T2 (1) (Figure 1) was proposed as an ellagitannin trimer where its sugar cores are interconnected by two DHDG moieties (O-2 to O-1) in the fashion commonly reported among the tannins of the same plant family (Figure 2). Structure of Nilotinin Q1. Nilotinin Q1 (2) was isolated as an off-white, amorphous powder. Its long retention time (tR = 24.6 min) revealed by the NP-HPLC analysis corresponded to that of an ellagitannin tetramer.16 The molecular formula, C157H110O100, was established by HRESIMS (Experimental Section). The 1H NMR spectrum of 2 exhibited three oneproton singlets (δH 7.07, 7.06, and 7.043) and three pairs of meta-coupled doublets [δH (7.36, 6.52), (7.22 and 6.44), and (7.18 and 6.50); each 1H, d, J = 1.8 Hz] characteristic of the DHDG moieties.13a−c,15,17 The spectrum also displayed five two-proton singlets (δH 7.037, 7.00, 6.95, 6.89, and 6.85) assignable to five galloyl units. Eight one-proton singlets comprising four singlets with relatively downfield shifts (δH 6.63, 6.61, 6.605, and 6.598) and the other with relatively upfield shifts (δH 6.52, 6.482, 6.479, and 6.46) are diagnostic for four of the HHDP units. Additionally, the proton signals (Table 1) in the aliphatic region of the spectrum were assigned to four C



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Table 3. 13C NMR Assignments of the Aromatic Residues in 1, 2, and 8 (151 MHz, Acetone-d6−D2O, 9:1, v/v) position DHDG C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-1′ C-2′ C-3′ C-4′ C-5′ C-6′ C-7′ HHDP C-1 C-1′ C-2, C-2′ C-3 C-3′ C-4, C-4′ C-5 C-5′ C-6, C-6′ C-7 C-7′ galloyl C-1 C-2/C-6 C-3/C-5 C-4 C-7 a

1

8

2

119.8, 120.0 107.2, 107.4 147.7, 147.8 139.88, 140.2 145.9, 146 112.9. 113.1 164.6, 165.1 113.25, 113.48 136.62, 136.9 139.95, 140.3, 140.7 (2C)

(119.0, 119.34),a (119, 37, 119.45)a (107.4, 107.5),a (107.91, 107.93)a 147.69, 147.81, 147.86 140.15, 140.7 (2C in total) 145.95, 146.03, 146.06, 146.18 (2C in total) 112.63, 112.74 (164.57, 164.58),a (164.80, 164.85)a (113.33, 113.35),a (113.56, 113.74)a (136.74, 136.76),a (136.8, 137.26)a 140.2, 140.3, 140.4, 140.5, 140.7 (4C in total)

143.3, 143.4 110.1, 110.3 164.53, 164.56 115.6 (2C), 115.7 115.8 (2C), 115.9 125.4, 125.6, 125.7, 126.1, 126.12, 126.2 107.88, 107.99, 108.1 107.77, 107.82, 107.88 145.07, 145.1 (4C), 145.2 136.25, 136.29 (2C) 136.43, 136.44, 136.6 144.2, 144.27, 144.3 (2C each) 168.33, 168.35, 168.41 167.7, 167.8, 167.9 119.4, 119.94, 119.96, 120.8 110.12, 110.16, 110.2, 110.4 (2C each) 145.56 (2C), 145.72 (4C), 145.8 (2C) 139.0, 139.2, 139.4, 139.7 164.55, 166.5, 166.85, 167.1

143.1, 143.23, 143.29 (2C in total) (109.9, 110.05),a (110.05, 110.14)a 164.05, 164.4 (2C in total) 115.6 (3C) 115.8, 115.84, 115.9 125.39, 125.48, 125.62, 126.02, 126.12, 126..15, 126.2 (6C in total) 107.93, 107.98, 108.1 107.75, 107.76, 107.81 145.10, 145.13 (6C in total) 136.22, 136.26 (2C) 136.47, 136.53, 136.56 144.3 (6C in total) 168.32, 168.35, 168.44, 168.46, 168.5 (3C in total) 167.71, 167.76, 167.85 (3C in total) 119.79, 119.9, 120, 120.1, 120.2 (4C in total) 110.05, 110.14 (4C each)

119.35, 120.01, 120.05 107.3, 107.4, 107.8 147.67, 147.71, 147.8 140, 140.18, 140.22, 145.9, 146, 146.04 112.9 (2C), 113.1 164.5, 164.8, 165.1 113.25, 113.44, 113.51 136.7, 136.9, 137.1 140.29 (2C), 140.35, 140.66, 140.75, 140.78

143.23, 143.3, 143.4 110.05, 110.09, 110.3 164, 164.6 (2C) 115.6 (2C), 115.67, 115.74 115.8 (2C), 115.86, 115.92 125.4, 125.6 (2C), 125.7, 126.1 (2C), 126.16, 126.22 107.9, 108, 108.1, 108.2 107.85, 107.88, 107.9 (2C) 145.07 (2C), 145.1, 145.2 (4C), 145.15 136.26 (3C), 136.30 136.45, 136.46, 136.5, 136.6 144.2 (2C), 144.25 (4C), 144.35 (2C) 168.34, 168.36, 168.43, 168.48 167.6, 167.7, 167.8, 167.9 119.4, 119.7, 119.92, 119.98, 120.8 110.14, 110.17, 110.4 (2C each), 110.2 (4C in total) 145.63, 145.66, 145.69, 145.72, 145.83 (8C in total) 145.54 (2C), 145.59 (2C), 145.7 (4C), 145.8 (2C) 139.20, 139.24, 139.42 (4C in total) 139.0, 139.16, 139.3, 139.7, 139.9 165.89, 165.92, 166.65, 166.68, 167.07, 167.26 (4C in total) 164.5, 166.5, 166.6, 166.8, 167.1

Chemical shifts between parentheses equal 1C in total.

4

C1 glucopyranose cores based on the 1H−1H COSY correlations. Among the glucose anomeric protons, those of the glucoses-2−4 are readily recognized [δH 5.87, 5.80, and 5.83 (each 1H, d, J = 7.8 Hz)] (see the 1H NMR spectrum of 2, Figure S8, Supporting Information). However, that of glucose-1 (δH 5.47) was associated with H-3 of glucose-3 (δH 5.48) and H-3 of glucose-4 (δH 5.46), and it was recognized by an HSQC correlation with the glucose anomeric carbon at δC 93.6. The 1 H−1H COSY data of the glucose residues (Table 1) indicated that proton signals of three glucose cores (glucoses-1−3) resonate in the lower field and are thus fully acylated, whereas the shielding of the H-2 resonance of glucose-4 (δH 3.99) indicated the presence of a free hydroxy group at C-2 of glucose-4. The acyloxy units at the anomeric positions of the glucose cores have the β-configuration due to the large coupling constant (J = 7.8 or 8.4 Hz) of the glucoses’ anomeric protons. The 13C NMR spectrum (Figures S11−S13, Supporting Information) also displayed aliphatic (Table 2), aromatic, and carbonyl carbons (Table 3) consistent with the structural components recognized from the 1H NMR data. In summary, the 1D NMR and MS data indicated the structure of 2 as an ellagitannin tetramer composed of four glucose cores, four HHDP units, five galloyl units, and three DHDG moieties. The exact locations of these acyl functions on the glucose cores were defined on the basis of spectroscopic findings aided by comparison with the closely related nilotinin T2 (1) as follows.

An HHDP unit was located at O-4/O-6 of each glucose core because the large chemical shift differences of the C-6 diastereotopic protons of glucose-1 (δH 5.28, 3.83), glucose-2 (δH 5.23, 3.84), glucose-3 (δH 5.27, 3.92), and glucose-4 (δH 5.22 and 3.79).19 The HHDP locations were further supported by the HMBC correlations among the HHDP H-3′ proton signals (δH 6.52, 6.482, 6.479, and 6.46) and the glucose cores H-4 (δH 5.12, 5.04, 5.13, and 5.00) via the common carbonyl carbons (δC 167.6, 167.7, 167.8, and 167.9) and also the correlations of the HHDP H-3 proton signals (δH 6.63, 6.61, 6.605, and 6.60) and the glucose cores’ H-6 signals [δH (5.28, 3.83), (5.23, 3.84), (δH 5.27, 3.92), and (δH 5.22 and 3.79)] through the common carbonyl carbons (δC 168.34, 168.36, 168.43, and 168.48). Similar to nilotinin T2 (1), the HMBC spectrum of 2 also evidenced bridging of the DHDG moieties between its monomeric constituent units via the same mode as in 1. The DHDG one-proton singlets (δH 7.07 and 7.06) showed HMBC correlations with the overlapped H-2 (δH 5.49, 2H) of both glucose-2 and -3 through two equivalent ester carbonyl carbons [δC 166.6 (2C)] and correlation of the DHDG singlet (δH 7.042) with H-2 of glucose-1 (δH 5.52) through an ester carbonyl carbon (δC 164.0). On the other hand, the HMBC spectrum also exhibited correlations of metacoupled doublets of a DHDG moiety (δH 7.35, 6.52) with H-1 (δH 5.85) of glucose-3 through the ester carbonyl carbon at δC 165.1 and correlations of the DHDG doublets (δH 7.22, 6.44) D



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Figure 1. Structures of the new ellagitannins trimer (1) and tetramer (2). Arrows (→) indicate important HMBC correlations. The dotted arrows (···>) indicate the nondetected HMBC correlations.

5.62, and 5.26) of H-3 of glucose-4, glucose-1, and glucose-2 through the respective carbonyl carbons (δC 167.9, 166.8, and 166.5) substantiated attachments of these galloyl units to the corresponding positions on the glucopyranose rings. Although the HMBC correlations of the proton signals (δH 7.00 and 6.85) of the other galloyl groups with the respective carbonyl carbons (δC 164.5 and 166.5) were detected, the suspected correlations with the nearly overlapped signals of H-1 of glucose-1 (δH 5.47) and H-3 of glucose-3 (δH 5.48) were not detected, probably due to their broadening by the restricted rotation about the C−C and O−C bonds in the vicinity of the protons. The axial chirality of the HHDP units was defined as aS based on a large positive Cotton effect at 237 nm, [θ]237 + 6.3 × 105, in the ECD spectrum (Figure S30, Supporting Information) of 2. On the basis of these findings, nilotinin Q1

with H-1 of glucose-2 (δH 5.87) through the ester carbonyl carbon at δC 164.5. It is noteworthy that the signal at δC 164.8 was assigned to C-7 of the DHDG moiety based on the HMBC correlations with the two DHDG meta-coupled doublets (δH 7.18 and 6.50). Although the correlation cross-peak of H-1 of glucose-3 (δH 5.80) with the same carbon peak (δC 164.8) was not detected, which may be due to line broadening (Figure S9, Supporting Information) of the proton signal (δH 5.80), attachment of the corresponding galloyl portion of the DHDG moiety to the anomeric center of glucose-3 was supported by the close similarities of the chemical shifts in 2 to the corresponding ones in nilotinin T2 (1) (Tables 1−3). The five galloyl units should thus necessarily be located at C-3 of each of the four glucose cores in addition to C-1 of glucose-1. The HMBC correlations of the galloyl proton signals (δH 7.037, 6.95, and 6.89) with the respective proton signals (δH 5.46, E



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Figure 2. Structures of the known ellagitannins 3−6.

total)], and two DHDG moieties [δH 7.24, 7.23, 7.19, 7.18, 6.61, 6.60, 6.48, and 6.47 (each d, J = 1.8 Hz, 4H in total), δH 7.09, 7.06, and 7.05 (each s, 2H in total)]. The aliphatic region of the spectrum displayed three pairs of doublets {δH [5.33 (J = 3.6 Hz) and 4.52 (J = 7.8 Hz)], [5.55 (J = 7.8 Hz) and 5.61 (J = 7.8 Hz)], and [6.10 (J = 8.4 Hz) and 6.11 (J = 8.4 Hz)]} assignable to three sets of the glucose anomeric protons based on the HSQC correlations with respective anomeric carbons [δC 96.28 and 90.89 (1C in total), 93.6 (1C), and 93.49 (1C)]. The chemical shifts of the glucose anomeric proton signals and their coupling constants (Table 1) indicated that the anomeric center of glucose-1 is unacylated, while the anomeric centers of glucose-2 and -3 are linked to β-oriented acyloxy units. Assignments of the other protons of the glucose cores (H-2− H-6) were achieved based on the correlations in the 1H−1H COSY spectrum. The large coupling constants of the glucose protons (Table 1) evidenced the presence of the sugar cores in the 4C1 conformation. The presence of the aromatic acyl moieties as well as the glucose cores was also evident from the 13 C NMR data (Tables 2 and 3) (see the 13C NMR spectrum of

was proposed as the ellagitannin tetramer presented by the structure 2 (Figure 1). Structure of Nilotinin T3. Nilotinin T3 (8) was isolated as an off-white, amorphous powder. Its trimeric structure having the molecular formula C116H82O74 was indicated by the HRESIMS and spectroscopic data shown below. The long retention time (tR = 14.96 min) revealed by NP-HPLC analysis of 8 indicated its nature as an ellagitannin trimer isomeric to 1. It was suggested to possess a free anomeric center at one of its sugar cores because of the elution of 8 with two different retention times (tR 4.58 and 6.86 min) when analyzed by RPHPLC, despite the elution of 8 as a single peak upon NPHPLC analysis. Duplication of the signals in the 1D NMR spectra of 8 also indicated the presence of 8 as an equilibrium mixture of α- and β-anomers. Although it showed a complicated 1H NMR spectrum (Figures S15−S17, Supporting Information), its aromatic proton signals were attributed to the presence of four galloyl units [δH 6.984, 6.982, 6.976, 6.971, 6.946, 6.944, 6.911, and 6.907 (each s, 8H in total)], three HHDP units [δH 6.66, 6.65, 6.61, 6.48, and 6.469 (each s, 6H in F



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Figure 3. Structures of the new ellagitannins trimer (8) and the known ellagtannins 7 and 9−11. The arrows (→) indicate important HMBC correlations.

glucose-3 based on the HMBC correlations of the galloyl proton signals [δH (6.984, 6.982), (6.976, 6.971), (6.946, 6.944), and (6.911, 6.907), each pair of signals equaling 2H in total] with the corresponding signals of H-2 of glucose-3 (δH 5.55), H-3 of glucose-1 [δH 5.79 and 5.49 (1H in total)], H-3 of glucose-2 (δH 5.51), and H-3 of glucose-3 [δH 5.76 and 5.75 (1H in total)] via the carbonyl carbons [δC (165.89, 165.92), (167.07, 167.26), and (166.65, 166.68)], respectively. Bridging of the DHDG moieties as the interglucosdic linkages among the monomeric units of 8 was indicated by the HMBC correlations of the DHDG singlets [δH 7.09 and (7.06, 7.05)] with glucose-1 [H-2α (δH 5.04) and H-2β (δH 5.14)] and H-2 of glucose-2 [δH 5.44, 5.46 (1H in total)] through the carbonyl carbons [δC 164.05, 164.4 (2C in total)] and the HMBC correlations of the DHDG doublets [δH 7.24, 7.23, 6.61, 6.60 (2H in total), 7.19, 7.18, 6.48, 6.47 (2H in total)] with H-1 of

8, Figures S18−S20, Supporting Information). The HHDP units were located at O-4/O-6 of the glucose cores because of the large chemical shift differences between the C-6 diastereotopic protons of glucose-1 [δH (5.18, 3.74) and (5.20, 3.78)], glucose-2 (δH 5.22, 3.81), and glucose-3 [δH (5.31, 3.82) and (5.31, 3.80)]19 and the HMBC correlations among these protons and the HHDP protons with the relatively downfield shifts (δH 6.66, 6.65, and 6.61) via the carbonyl carbons [δC 168.32, 168.35, 168.44, 168.46, 168.5 (3C in total)], as well as the HMBC correlations of the H-4 signals of glucose-1 [δH 5.06 and 5.04 (1H in total)], glucose-2 (δH 5.08), and glucose-3 [δH 5.19 and 5.18 (1H in total)], and the HHDP protons with the relatively upfield shift [δH 6.48, and 6.469 (each s, 3H in total)] via the carbonyl carbons [δC 167.71, 167.76, and 167.85 (3C in total)]. The galloyl units were positioned at C-3 of each of the glucose cores and C-2 of G



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Figure 4. Suggested biosynthetic mechanism toward the new ellagitannins 1 and 2. Step1: Biosynthesis of nilotinin D1 from the deacylated monomer 13 and tellimagrandin II. Step 2: Biosynthesis of nilotinin T2 (1) from the deacylated precursor nilotinin D1 and a molecule of tellimagrandin II. Step 3: Biosynthesis of nilotinin Q1 (2) from the deacylated precursor nilotinin T2 (1) and a molecule of tellimagrandin II.

carcinogens,21,22 inhibition of tumor promotion,23,24 and hostmediated antitumor activities14 have been reported. Among various hydrolyzable tannins screened, those with large Mr, such as oenothein A and woodfordin D (trimer), and woodfordin F (tetramer), specifically inhibited the growth of tumors (Sarcoma-180 and MM2) by a host-mediated mechanism in mice.14 In the preceding contributions,13c,e the effects of numerous ellagitannin monomers and dimers and a macrocyclic-type trimer from T. nilotica on OSCC (HSC-2, HSC-3, and HSC-4) and HL-60 cell lines in comparison with those on the human oral normal cells (HGF, HPC, and HPLF) were reported. Since the cytotoxic effects of large ellagitannin molecules from Tamarix plants were not investigated, the cytotoxic effects of the trimers 3, 4, 6, and 8 were examined in this investigation. Additionally, the effects of simple ellagitannin monomers, tellimagrandin I (11), gemin D (12), and 1,3-di-Ogalloyl-4,6-O-(aS)-hexahydroxydiphenoyl-β-D-glucose (13), a DHDG-type dimer, nilotinin D3 (9), and for the first time the isoDHDG-type dimer tamarixinin C (10) are also shown in comparison with those of the trimeric ellagitannins in the present study. The results listed in Table 4 provided the following evidence: Generally all the examined tannins except for tellimagrandin I (11) and 1,3-di-O-galloyl-4,6-O-(aS)-hexahydroxydiphenoyl-βD-glucose (13) showed noticeable cytotoxic effects on HL-60 cells. Furthermore, except for the macrocyclic-type ellagitannin trimer nilotinin T1 (3), all the examined tannins showed cytotoxic effects on OSCC cell lines. Cytotoxicity per unit molecular weight tended to decrease with polymerization, i.e., monomer > dimer > trimer. The new ellagitannin trimer 8, which occurs as a mixture of α- and β-anomers, is the most

glucose-3 [δH 6.11, 6.10 (1H in total)] and H-1 of glucose-2 [δH 5.55 and 5.61 (1H in total)] through the respective carbonyl carbons [δC 164.80, 164.85, 164.57, and 164.58 (2C in total)]. A positive Cotton effect at 236 nm, [θ]236 +5.3 × 105, in the ECD spectrum (Figure S30, Supporting Information) of 8 is indicative of the aS configuration of the HHDP unit. In view of the aforementioned data, the structure 8 (Figure 3) was assigned to nilotinin T3. Biosynthesis of the Ellagitannins in Tamarix Species. The trimer nilotinin T2 (1) is assumed to be biosynthesized by C− O oxidative coupling involving a carbon of the galloyl unit at glucose O-2 of tellimagrandin II and a meta hydroxy group on the galloyl unit at O-1 of the glucose core of 1,3-di-O-galloyl4,6-O-(aS)-hexahydroxydiphenoyl-β-D-glucose (13), leading to the formation of nilotinin D1 (Figure 4, step 1), a dimeric ellagitannin that we also previously isolated from the same Tamarix species.13a Repeated coupling of another molecule of tellimagrandin II with nilotinin D1 by the same oxidative pattern would lead to nilotinin T2 (1, Figure 4, step 2). Similarly, nilotinin Q1 (2) is produced by further coupling of a molecule of tellimagrandin II with the trimer 1 by the same mode (Figure 4, step 3). The deacylated anomeric center of glucose-1 of nilotinin T3 (8) strongly suggested the biogenesis of 8 via analogous C−O oxidative couplings between a galloyl unit at O-2 of the glucose core of 11 and a galloyl unit at O-1 of the coexisting tellimagrandin II, leading to the formation of nilotinin D3 (9, Figure 3). Repeating the same reaction between the intermediate (9) and another molecule of tellimagrandin II would give nilotinin T3 (8, Figure 3). Cytotoxicity of Ellagitannins. Several kinds of antitumor activities of tannins including inhibition of mutagenicity of H


0.23 0.31 0.13 0.89 0.45 0.76 6.4 3.2 2.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.31 0.89 0.55 1.2 1.7 2.1 16.6 0.25 1.6

HL-60 30.5 9.39 16.4 6.48 19.5 29.7 46.7 30.5 39.9 1.1 2.1 2.4 0.15 0.45 1.0 2.8 1.4 7.1 ± ± ± ± ± ± ± ± ± 2.0 0.61 0.85 7.0 0.70 1.9 7.2 0.42 3.5 ± ± ± ± ± ± ± ± ±

human tumor cell lines

HSC-4 42.9 38.2 34.3 33.9 37.9 27.7 45.7 34.4 38.0 HSC-3 34.2 37.4 25.5 21.2 32.6 29.4 19.5 23.3 28.0 HSC-2 22.3 18.7 18.8 23.2 17.7 16.3 14.6 5.82 14.1 compound 3 4 6 8 9 10 11 12 13

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effective cytotoxic compound with tumor specificity (TS/HL60 = 13.5 and TS/HSC = 3.3). This is comparable with the popular anticancer drug 5-fluorouracil (5-FU) (TS > 3.3), albeit slightly lower than those for melphalan (TS = 7.1), doxorubicin (TS = 9.8), and peplomycin (TS = 15.9). The DHDG-type trimer hirtellin T1 (4, Figure 2) and the hellinoyl-type trimer (6, Figure 2) also showed good cytotoxicity with increased specificity {TS/HSC = 3 and TS/HL-60 = 10.8 (for the trimer 4) and TS/HSC = 3.5 and TS/HL-60 = 5.5 (for the trimer 6)} toward the HL-60 cells. Likewise, the dimeric tannin with a free anomeric center, nilotinin D3 (9), also showed cytotoxic effects comparable to its structural analogue 8. The isoDHDG-type ellagitannin dimer tamarixinin C (10) exhibited comparable cytotoxicity against all of the tumor cell lines examined. In conclusion, liver diseases, especially liver cancer, are among the major causes of mortality and morbidity in Egypt; among the cancers in male patients, 33.6% is liver cancer.25 Ellagitannins, including those isolated from Tamarix plants, were reported to exhibit strong antitumor activity upon a single intraperitoneal injection into mice 4 days before intraperitoneal inoculation of Sarcoma-180 cells.14 The suppression of the chemically mediated tumor promotion response in rats and hepatoprotective, antihepatotoxic, and/or antioxidant effects often shown by a number of Tamarix plants, T. gallica, T. nilotica, T. ramosissima, T. hispida, Tamarix boveana, and T. aphylla, have also been demonstrated.3a,4−11 These findings, along with the previous13c,e and current results of the noticeable tumor-specific cytotoxicity of ellagitannins from T. nilotica and T. tetrandra, agree with the possibility of developing antitumor ellagitannins with significant tumor specificity from tamaricaceous plants. However, it is required to perform more specific in vivo studies to elucidate the mechanism of the antitumor actions as well as the possible hepatoprotective and/or antihepatotoxic role of the tamaricaceous ellagitannins with characteristic structural features. In spite of the sporadic distribution of Tamarix plants in arid environments, accumulation of the same type of ellagitannins in cultured shoots of T. tetrandra warrants a sustainable supply of this unique class of ellagitannins.

a Each value represents the mean of at least three independent experiments. bTS/HSC, tumor specificity toward HSC cells = {[CC50(HGF) + CC50(HPC) + CC50(HPLF)]/[CC50(HSC-2) + (HSC-3)] + (HSC-4)]}. cTS/HL-60, tumor specificity toward HL-6 cells = {[CC50(HGF) + CC50(HPC) + CC50(HPLF)]/[CC50(HL-60)]} × 1/3}.

2.1 1.5 1.5 2.6 0.6 0.0 6.1 ± ± ± ± ± ± ± TS/HSC 2.9 >3 3.5 3.3 >3.2 >3.9 >3.3 >4.68 >3.74

b

CC50 (μM)a

Table 4. Cytotoxicity of the Ellagitannins 3, 4, 6, 8, and 9−13 against Human Normal and Tumor Cells

HGF 96.6 96.0 87.7 88.0 92.0 95.3 >100 >100 >100

± ± ± ± ± ±

2.5 4.0 5.8 1.0 1.0 2.1

HPC 90.7 87.3 85.0 86.0 86.6 89.0 64.0 >100 >100

human normal oral cells

HPLF >100 >100 98.6 ± 3.1 88.3 ± 3.5 >100 >100 >100 >100 >100

c

TS/HL-60 3.1 >10.8 5.5 13.5 >4.8 >3.2 >1.9 >3.3 >2.5


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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO DIP-1000 digital polarimeter. UV spectra were recorded using a JASCO V-530 spectrophotometer. The ECD spectra were measured using a JASCO J-720W spectrophotometer. ESIMS data were acquired on an API-4000 instrument (AB Sciex, Framingham, MA, USA). The solvent used was MeOH−H2O (1:1, v/ v) + 0.1% NH4OAc. The HRESIMS data were recorded using a Micromass AutoSpec OA-Tof spectrometer. The solvent used was MeOH−H2O (1:1, v/v) + 0.1% NH4OAc, and the flow rate was set at 20 μL/min. The 1D (1H and 13C) and 2D (1H−1H COSY, HSQC, and HMBC) NMR spectra were recorded on a Varian INOVA AS 600 instrument (600 MHz for 1H and 151 MHz for 13C; Agilent, Santa Clara, CA, USA). Chemical shifts are given in δ (ppm) values relative to that of the solvent signals [acetone-d6 (δH 2.04; δC 29.8) and DMSO- d6 (δH 2.50; δC 39.5)] on the TMS scale. NP-HPLC was conducted on a YMC-Pack SIL A-003 (YMC, Japan) column (4.6 i.d. × 250 mm) developed using n-hexane−MeOH−THF−formic acid (55:33:11:1, v/v/v/v) containing oxalic acid (450 mg/L) at a flow rate of 1.5 mL/min at room temperature. Detection was effected with UV absorption at 280 nm. RP-HPLC was performed on a YMC-Pack ODS-A A-303 (YMC, Japan) column (4.6 i.d. × 250 mm) developed using 0.01 M H3PO4−0.01 M KH2PO4−MeOH (2:2:1, v/v/v) at a flow rate of 1 mL/min in an oven set at 40 °C. Detection was performed on UV absorption at 280 nm. Preparative RP-HPLC was I



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performed at 40 °C on a YMC-Pack ODS-A A-324 column (10 i.d. × 300 mm) using 0.01 M H3PO4−0.01 M KH2PO4−MeOH [either 41.5:41.5:17 (solvent I), 42:42:16 (solvent II), 43:43:14 (solvent III), 37.5:37.5:25 (solvent IV), or 2:2:1 (solvent V)], at a flow rate of 2 mL/min with UV detection at 280 nm. Diaion HP-20 and MCI-gel CHP-20P (Mitsubishi Chemical, Japan), Toyopearl HW-40C (TOSOH, Japan), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Sweden) were used for column chromatography. The human normal oral cells HGF, HPLF, and HPC were obtained from the first premolar tooth extracted from the lower jaw of a 12year-old girl,26 and the OSCC cell lines (HSC-2, HSC-3, HSC-4), purchased from the Riken Cell Bank, Tsukuba, Japan, were cultured at 37 °C in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin G, and 100 μg/mL streptomycin sulfate under a humidified 5% CO2 atmosphere. Cells were harvested by treatment with 0.25% trypsin−0.025% EDTA-2Na in PBS (−) and either subcultured or used for experiments. Normal human oral cells (HGF, HPC, and HPLF) were used at 8−15 population doubling levels. The HL-60 cells were cultured at 37 °C in RPMI1640 supplemented with 10% heat-inactivated FBS. Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco BRL, Grand Island, NY, USA. FBS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were from Wako Pure Chem. Ind., Osaka, Japan. Fluorouracil (5-FU) was from Kyowa, Tokyo, Japan. Culture plastic dishes and plates (96-well) were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Plant Materials. Leaves of T. nilotica were collected at Al-Wadi AlAssiuty, 20 km northeast of Assiut city, Egypt, in October 2006, and identified by Prof. Mo’men M. Zare, Department of Botany, Faculty of Science, Assiut University. A voucher specimen (No. 1024) is deposited in the same department. Multiple shoot cultures of T. tetrandra were induced on basal Linsmaier-Skoog agar medium supplemented with 30 g L−1 sucrose, 2.13 mg L−1 indole acetic acid, and 2.25 mg L−1 benzyl adenine. The obtained cultures were maintained and propagated as described in the previous report.13d Fresh tissues from the dark-grown cultures were routinely harvested after 30 days from the inoculum time. Collected fresh tissues (∼1 kg) were used for the hydrolyzable tannins isolation procedures. Extraction and Isolation. Powdered leaves of T. nilotica (1 kg) were homogenized in H2O−acetone [3:7, v/v (22 L)] at room temperature. The homogenate was filtered and concentrated to 1.5 L below 40 °C under reduced pressure. The extract was subjected to a Diaion HP-20 (5 i.d. × 43 cm) column and eluted with H2O, MeOH− H2O (2:8, 4:6, 6:4, and 10:0, v/v), and acetone−H2O (7:3, 10:0, v/v), successively. A part (∼10 g) of the MeOH−H2O (4:6, v/v) eluate (52 g) was subjected to a Toyopearl HW-40C (2.2 i.d. × 43 cm) column with EtOH−H2O (5:5, 7:3, v/v), EtOH/H2O (7:3, v/v)−acetone/ H2O (7:3, v/v) (9:1, 8:2, 7:3, 6:4, and 5:5, v/v) and acetone−H2O (7:3, v/v), collecting 1000-drop fractions. The 70% aqueous EtOH− 70% acetone(aq) (8:2, v/v) eluate (1.433 g), containing mainly ellagitannin trimers as revealed from their long retention times (tR > 13 min) upon NP-HPLC analyses, was subjected to column chromatography on MCI-gel CHP-20P (1.1 i.d. × 40 cm) with H2O, then with H2O−MeOH (9:1) (8.5:1.5, 8:2, 7.5:2.5, 7:3, 6:4, 1:1, and 0:10, v/v), collecting 600-drop fractions. Similar fractions were collected to give six MCI fractions (MCI A−F). The H2O−MeOH (75:25, v/v) eluate (MCI-C, 268 mg) was rechromatographed over MCI-gel CHP-20P (1.1 i.d. × 37 cm) with H2O−MeOH (7:3, v/v) in the isocratic elution mode. A preparative RP-HPLC separation with solvent I of part (65 mg) of the eluate afforded hirtellins T2 [5 (7.6 mg)] and T3 [6 (19.6 mg)] and nilotinin T2 [1 (2.1 mg)]. Similarly, a preparative RP-HPLC purification with solvent II of a subsequent eluate (57 mg) yielded hirtellin T3 [6 (14.3 mg)] and nilotinin T2 [1 (9 mg)], along with nilotinins T1 [3 (9 mg)]. The MCI-D fraction (496 mg), eluted with H2O−MeOH (7:3, v/v), was further chromatographed over a Sephadex LH-20 column (1.1 i.d. × 37 cm) eluted with EtOH/H2O (7:3, v/v)−acetone/H2O (7:3, v/v) (9:1, v/v), isocratically. The preparative RP-HPLC separation with solvent II of the middle part of the eluate (87 mg) afforded hirtellin T1 [4

(18.7 mg)], in addition to hirtellin T3 [6 (5.7 mg)], nilotinins T1 [3 (9.3 mg)] and T2 [1 (6 mg)], and tamarixinin B [7 (10.2 mg)]. The eluate with a mixture of EtOH/H2O (7:3, v/v) and acetone/H2O (7:3, v/v; 7:3, v/v; 1.117 g) showed tR > 17 min upon the NP-HPLC analyses, suggesting ellagitannin tetramers. The fraction was subjected to column chromatography on Sephadex LH-20 (2.2 i.d. × 27 cm) with the following mixtures: EtOH/H2O (7:3, v/v)−acetone/H2O (7:3, v/v) (9:1, 8:2, 7:3, 5:5, and 0:10, v/v). The eluate with (EtOH/ H2O; 7:3, v/v)−(acetone/H2O; 7:3, v/v) (9:1, v/v) and part of the (8:2, v/v) elute (343 mg) were combined and rechromatographed on MCI-gel CHP-20P (1.1 i.d. × 37 cm) with H2O−MeOH (9:1, 8.5:1.5, 8:2, 75:25, 7:3, 6:4, 1:1, and 0:10, v/v). The preparative RP-HPLC purification with solvent III of the H2O−MeOH (7:3, v/v) eluate (181 mg) afforded nilotinin Q1 [2 (15.7 mg)]. Fresh tissues (∼1 kg) from the dark-grown cultures were collected at the age of 30 days from the inoculum time. The tissues were homogenized in 12 L of H2O−acetone (3:7, v/v). The homogenate was filtered, and the filtrate was concentrated in vacuo to ca. 500 mL below 40 °C. The concentrated syrup was applied to a Diaion HP-20 (5 i.d. × 50 cm) column eluted with H2O, MeOH−H2O (5:5, v/v), MeOH, and acetone, successively. The H2O−MeOH (5:5, v/v) fraction (12.6 g) was subjected to a Toyopearl HW-40C (2.2 i.d. × 60 cm) column with EtOH−H2O (5:5, 7:3, v/v), (EtOH/H2O; 7:3, v/ v)−(acetone/H2O; 7:3, v/v) (9:1, 8:2, 7:3, and 5:5, v/v), and acetone−H2O (7:3, v/v), collecting 700 Toyopearl fractions [(Tfr), 1000 drops each]. The eluate with H2O−EtOH (5:5, v/v) was applied to a Sephadex LH-20 (2.2 i.d. × 21 cm) column with EtOH−H2O (5:5 then 7:3, v/v) and acetone−H2O (7:3, v/v). A part (70 mg) of the H2O−EtOH (5:5, v/v) eluate (700 mg) was further purified by RP-HPLC with solvent IV and yielded tellimagrandin I [11 (42 mg)]. The subsequent fraction (229 mg) of the H2O−EtOH (5:5, v/v) eluate was subjected to an MCI gel CHP-20P (1.1 i.d. × 37 cm) column with H2O−MeOH (9:1, 8.5:1.5, 8:2, 7.5:2.5, 7:3, and 0:10, v/ v). The H2O−MeOH (7.5:2.5, v/v) eluate afforded nilotinin D3 [9 (7.1 mg)] after preparative HPLC purification with solvent IV. The (EtOH−H2O; 7:3, v/v)−(acetone/H2O; 7:3, v/v) (8:2, v/v) eluate was subjected to a Sephadex LH-20 (2.2 i.d. × 20 cm) column with EtOH−H2O (7:3, v/v), (EtOH−H2O; 7:3, v/v)−(acetone/H2O; 7:3, v/v) (9:1, v/v), and acetone−H2O (7:3, v/v), successively. The eluate was collected into 245 Sephadex LH-20 fractions (1000 drops/ fraction). The Sephadex LH-20 fractions [S76−95 (118 mg) and S96−130 (217 mg)], eluted with EtOH−H2O (7:3, v/v), were subjected separately to partial purifications on MCI gel CHP-20P (1.1 i.d. × 37 cm) columns with H2O−MeOH (7.5:2.5, 7:3. and 0:10, v/v). The eluate (142 mg) with H2O−MeOH (7:3, v/v) was rechromatographed on the same column with the same elution mode. The eluate (7.5 mg) with H2O−MeOH (7.5:2.5, v/v) and the early eluate (7.8 mg) with H2O−MeOH (7:3, v/v) were subjected to preparative HPLC purification with solvent II and yielded nilotinin T2 [1 (1.9 mg)] and hirtellin T3 [6 (2.2 mg)], whereas the preparative HPLC purification with solvent IV of the MeOH eluate (52.5 mg) yielded tamarixinin C [10 (8 mg)]. The Sephadex LH-20 fractions S145−164 (237 mg) and S165−220 (198 mg) were also subjected separately to column chromatography on MCI gel CHP-20P (1.1 i.d. × 37 cm) with H2O−MeOH (7.5:2.5, 7:3, and 0:10, v/v). The eluates with H2O− MeOH (7.5:2.5, v/v) from both columns afforded pure samples (67 and 41 mg) of nilotinin T3 (8), respectively. The eluate with H2O− MeOH (7:3, v/v) from the first column afforded hirtellin T1 [4 (30 mg)], and the early eluate (40 mg) with H2O−MeOH (7:3, v/v) from the second column afforded another pure sample of hirtellin T1 [4 (19 mg)] upon preparative HPLC purification with solvent V. Nilotinin T2 (1): off-white, amorphous powder; [α]19D +49 (c 1, MeOH); UV (MeOH) λmax (log ε) 219 (5.50), 271 (5.14); ECD (MeOH) [θ] (nm) +3.2 × 105 (237), −5.4 × 104 (265), +4.2 × 104 (286); 1H NMR (acetone-d6−D2O, 9:1) δH 7.35 and 7.19 [each 1H, d, J = 1.8 Hz, (DHDG H-6) × 2], 7.07 and 7.06 [each 1H, s, (DHDG H6′) × 2], 7.03, 7.00, 6.95, and 6.86 [each 2H, s, (galloyl H-2/H-6) × 4], 6.62, 6.602, and 6.598 [each 1H, s, (HHDP H-3) × 3], 6.51, 6.49, and 6.46 [each 1H, s, (HHDP H-3′) × 3], 6.52 and 6.42 [each 1H, d, J = 1.8 Hz, (DHDG H-2) × 2], glucose protons (Table 1); 13C NMR J



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(Tables 2 and 3); HRESIMS m/z 1352.11992 [M + 2Na]2+ (calcd for 1 /2C116H82O742Na, 1352.12190). Nilotinin Q1 (2): off-white, amorphous powder; [α]21D +65 (c 1, MeOH); UV (MeOH) λmax (log ε) 219 (5.66), 274.5 (5.30); ECD (MeOH) [θ] (nm) +6.3 × 105 (237), −1.1 × 105 (262), +1.1 × 105 (286); 1H NMR (acetone-d6−D2O, 9:1) δH 7.36, 7.22, and 7.18 [each 1H, d, J = 1.8 Hz, (DHDG H-6) × 3], 7.07, 7.06, and 7.043 [each 1H, s, (DHDG H-6′) × 3], 7.037, 7.00, 6.95, 6.89, and 6.85 [each 2H, s, (galloyl H-2/H-6) × 5], 6.63, 6.61, 6.605, and 6.598 [each 1H, s, (HHDP H-3) × 4], 6.52, 6.482, 6.479, and 6.46 [each 1H, s, (HHDP H-3′) × 4], 6.52, 6.50, and 6.44 [each 1H, d, J = 1.8 Hz, (DHDG H-2) × 3], glucose protons (Table 1); 13C NMR (Tables 2 and 3); HRES IM S m/z 1820.17123 [M + 2Na] 2 + (calcd for 1 /2C157H110O1002Na, 1820.16535). Nilotinin T3 (8): off-white, amorphous powder; [α]21D +70 (c 1, MeOH); UV (MeOH) λmax (log ε) 219 (5.66), 275.5 (5.30); ECD (MeOH) [θ] (nm) +5.3 × 105 (237), −1.3 × 105 (262), +1.3 × 105 (286); 1H NMR (α- and β-anomers) (acetone-d6−D2O, 9:1) δH 7.24, 7.23, 7.19, and 7.18 [each d, J = 1.8 Hz, 2H in total, (DHDG H-6) × 2], 7.09, 7.06, and 7.05 [each s, 2H in total, (DHDG H-6′) × 2], 6.984, 6.982, 6.976, 6.971, 6.946, 6.944, 6.911, and 6.907 [each s, 8H in total, (galloyl H-2/H-6) × 4], 6.66, 6.65, and 6.610 [each s, 3H in total, (HHDP H-3) × 3], 6.48, and 6.469 [each s, 3H in total (HHDP H-3′) × 3], 6.61, 6.60, 6.48, and 6.47 [each d, J = 1.8 Hz, 3H in total (DHDG H-2) × 2]; glucose protons (Table 1); 13C NMR (Tables 2 and 3); HRESIMS m/z 2681.25305 [M + Na]+ (calcd for C116H82O74Na, 2681.25458). Assay for Cytotoxic Activities. The cells (other than HL-60) were inoculated at 5 × 103 cells/well in 96-microwell plates. After 48 h, the medium was replaced with 0.1 mL of fresh medium containing different test compound concentrations with three replicate wells. Each test compound was dissolved in DMSO at a concentration of 20 mM. The first well contained 100 μM of the test compound and was sequentially diluted 2-fold. The cells were incubated for an additional 48 h, and the relative viable cell number was determined by the MTT method.27 The HL-60 cells were inoculated at 3.0 × 104 cells/0.1 mL in 96-microwell plates, and different concentrations of test compounds were added. After a 48 h incubation, the viable cell number was determined with a hemocytometer under a light microscope after trypan blue staining. The 50% cytotoxic concentration (CC50) was determined from the dose−response curve. The antitumor potential was evaluated by the tumor-specificity index (TS). Tumor-specificity toward the OSCC cell lines (TS/OSCC) was calculated by dividing the mean CC50 against normal oral cells by that against OSCC cell lines. The tumor specificity toward the HL-60 cell line (TS/HL-60) was calculated by dividing the mean CC50 against normal oral cells by that against the HL-60 cell line. We have previously confirmed that the TS value determined by this method reflects the antitumor potential of test samples, although these normal oral cells and OSCC cell lines are classified as mesenchymal or epithelial cells.28

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ACKNOWLEDGMENTS M.A.A.O. thanks the Intractable Infectious Diseases Research Project Okayama (IIDPO) for financial support. This study was supported in part by a Grant-in-Aid for Scientific Research of JSPS (No. 25450170). The NMR instrument used in this study is the property of the SC-NMR Laboratory of Okayama University.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01065. 1 H and 13C NMR, ECD, and HR-ESIMS spectra of the new ellagitannins 1, 2, and 8 and the related known ellagitannin hirtellin T1 (4) (PDF)

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