Ellagitannin Oligomers from Eucalyptus camaldulensis Leaves and Their Role in the Detoxification of Aluminum

Abstract

Eucalyptus camaldulensis of the Myrtaceae family shows high resistance to aluminum (Al) ions and contains various compounds such as steroids, terpenoids, saponins, flavonoids, glycosides, alkaloids, and tannins. Although the ellagitannin oenothein B (12) isolated from E. camaldulensis exhibits remarkable properties for Al detoxification, likely contributing to its Al resistance, other ellagitannin oligomers present in E. camaldulensis have not been investigated in detail. In this study, novel dimeric and trimeric ellagitannin oligomers eucarpanin D₂ (1) and eucamalin A (2), together with known gallotannins (7, 8, and 10), monomeric ellagitannins (4–6, and 11), and dimeric ellagitannins (3, 9, and 12–14), were isolated from E. camaldulensis leaves. The structures of these novel compounds were elucidated based on their chemical and physicochemical properties, including the orientations of tergalloyl groups in compounds 1 and 2. Similar to compound 12, previously isolated from the roots of E. camaldulensis, the ellagitannins demonstrated good Al detoxification properties. Hence, these tannins may play a critical role in the high Al resistance of E. camaldulensis in acidic soils. This paper reports for the first time the isolation of ellagitannin oligomers from the leaves of E. camaldulensis.

1. Introduction

Eucalyptus camaldulensis Dehnh. is an evergreen tree that belongs to the Myrtaceae family. Native to Australia, it is widespread in tropical and subtropical regions and commonly grows on riverbanks. This plant is tolerant to extreme drought and high temperatures, grows rapidly, and exhibits a good coppicing ability [1,2]. Therefore, it shows significant variations among different regions. E. camaldulensis exhibits high resistance to Al ions and can grow in acidic soils, which account for 30% of the Earth land area [3,4]. We conducted an ingredient search to elucidate the mechanism of this ability of E. camaldulensis.

E. camaldulensis contains various compounds such as steroids, terpenoids, saponins, flavonoids, glycosides, alkaloids, and tannins [5,6]. Among them, we previously identified the Al-binding ligand oenothein B (12), a dimeric ellagitannin, in the roots of E. camaldulensis. It contains large amounts of compound 12, and the bioassays using Arabidopsis (Arabidopsis thaliana) showed that the inhibition of root elongation by Al was alleviated by compound 12 [7]. That is, it is suggested that compound 12 contributes to the Al detoxification in E. camaldulensis. Initially, compound 12 was isolated from the leaves of Oenothera erythrosepala (Onagraceae), which is widely distributed among species belonging to the Myrtaceae, Onagraceae, and Lythraceae families [8]. These species have oligomeric analogs, such as eucarpanin D₁ (15) (dimer), oenothein T₁ (trimer), eucarpanin T₁ (16), and T₂ (trimer) extracted from the leaves of Eucalyptus cypellocarpa and woodfordin I (dimer), E (trimer), and F (tetramer) isolated from the flowers of Woodfordia fruticosa [8,9,10]. However, the ellagitannin oligomers present in E. camaldulensis have not been investigated in detail to date. Therefore, we focused on high molecular weight tannins to identify those having higher ability to reduce the toxic effects of Al than compound 12, as well as the leaves of E. camaldulensis, which are rich in such tannins. In this study, we isolated and characterized monomeric and oligomeric ellagitannin, along with gallotannins, from the leaves of E. camaldulensis and examined their Al detoxification properties.

2. Results and Discussion

2.1. Isolation and Structural Elucidation of Novel and Known Compounds

A concentrated solution of a 70% aqueous acetone homogenate from the frozen leaves of E. camaldulensis was sequentially extracted with Et₂O, EtOAc, and water-saturated n-BuOH. The EtOAc extract yielded three known ellagitannins: pedunculagin (4) [11], tellimagrandin I (5) [12], and tellimagrandin II (6) [12], two known gallotannins: 1,2,6-tri-O-galloyl-β-d-glucose (7) [13] and 1,2,3,6-tetra-O-galloyl-β-d-glucose (8) [14], and dimeric ellagitannin: eucalbanin C (9) [15]. In addition, two novel dimeric and trimeric ellagitannins: eucarpanin D₂ (1) and eucamalin A (2), were isolated from this extract (Figure 1). The n-BuOH extract gave gallotannin: 1,6-di-O-galloyl-β-d-glucose (10) [16], three monomeric ellagitannins: compounds 4 and 5, and strictinin (11) [11,17], two dimeric ellagitannins: compound 9 and oenothein B (12) [8,18], and two highly oxidized ellagitannins: eurobustin C (3) [19] and eugeniflorin D₂ (14) [8,18,19] (Figure 1). The water-soluble extract obtained dimeric ellagitannin: eugeniflorin D₁ (13) [8,19] (Figure 1). The known compounds were identified by comparing their spectroscopic properties to data obtained from previous isolations (Figure S1).

We further characterized the novel dimeric and trimeric ellagitannins identified in this work and named them eucarpanin D₂ (1) and eucamalin A (2), respectively. These ellagitannins contained tergalloyl groups serving as acyl units. Eurobustin C (3) was first isolated from Eucalyptus robusta, and its structure was identified by Yoshida et al. in their book, however, a detailed chemical structure determination was not reported [19]. Therefore, we performed a detailed structural characterization of compound 3. The chemical structures of these ellagitannins are described below.

Eucarpanin D₂ (1) was obtained as a pale-brown amorphous powder. The molecular formula of compound 1 (C₇₅H₅₄O₄₈) was determined by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) from its singly and doubly charged molecular ions at m/z 1721.1649 [M − H]⁻ and 860.0840 [M − 2H]²⁻, indicating that compound 1 was an ellagitannin dimer. Its ¹H-nuclear magnetic resonance (NMR) spectrum is similar to that of eucalbanin C (9) and indicated the presence of four sets of 2H singlets (δ 7.11, 7.11, 7.02, 7.02, 7.01, 7.01, 7.00, and 6.99), two sets of 1H singlets (δ 6.65, 6.64, 6.49, and 6.48), and three sets of 1H singlets (δ 6.87, 6.84, 6.60, 6.59, 6.53, and 6.51), which were ascribed to four galloyl, one hexahydroxy diphenoyl (HHDP), and one tergalloyl groups, respectively. This compound also exists as an equilibrium mixture of two tautomers and is characterized by the presence of two ⁴C₁-conformation glucopyranose cores. The absolute configuration of glucopyranose in compound 1 was determined from the D-series using the method described by Tanaka et al. [20]. The H-1 signal of the β-anomer of compound 1 shifted to a lower field (δ 6.18, d, J = 8.4 Hz) than that of compound 9 (δ 4.98). The corresponding ¹³C-NMR spectrum contains a signal characteristic of the tergalloyl group in the hydroxyl carbon region; the tergalloyl-4’ carbon was present in a lower field (δ 149.0) than that of the valoneoyl group, similar to that of compound 9 (Figure S2). The enzymatic hydrolysis of compound 1 with tannase yielded compound 9. Tellimagrandin I (5), tellimagrandin II (6), and gemin D (17) [21] were produced via partial hydrolysis. This isomerization via a Smiletype rearrangement was successfully applied to establish a chemical correlation between compound 1 and eucarpanin D₁ (15). The tergalloyl group in the crowded substitution mode is isomerized to the valoneoyl group, which has a less sterically hindered structure because of a Smiles-type rearrangement. An aqueous solution containing a small amount of 0.02 M phosphate buffer (pH 7.4) was left standing at room temperature for 6 h to produce an isomerized product of compound 15. The methylation of compound 1 with dimethyl sulfate and potassium carbonate in acetone followed by methanolysis yielded methyl tri-O-methylgallate (18), dimethyl hexamethoxydiphenate (19), and trimethyl octa-O-methyltergallate (20) (Figure 2). The positive cotton effect in the short wavelength region of the circular dichroism (CD) spectrum indicated that the absolute configuration of HHDP and tergalloyl group was S-series, respectively [22]. Based on these data, eucarpanin D₂ is represented by the structure of compound 1.

Eucamalin A (2) was obtained as a pale-brown amorphous powder. The molecular formula of this material, which was determined by HR-ESI-MS from the singly, doubly, and triply charged molecular ions at m/z 2353.2400 [M − H]⁻, 1176.1117 [M − 2H]²⁻, and 783.7383 [M − 3H]³⁻, respectively, was C₁₀₂H₇₄O₆₆, indicating that compound 2 was a trimeric ellagitannin. The ¹H-NMR spectrum pattern of compound 2 was strongly resembled those of compound 9 and eucarpanin T₁ (16) [23], suggesting that compound 2 was linked through its tergalloyl groups. Although the ¹H-NMR spectrum of compound 2 exhibited intricate signals caused by the presence of anomeric mixtures at the glucose cores, the presence of galloyl, HHDP, tergalloyl groups, and glucose moieties was confirmed by the signals at δ 7.06–6.96 (galloyl-H), δ 6.87–6.83 (tergalloyl-H), δ 6.64–6.62 (HHDP-H), δ 6.60–6.56 (tergalloyl-H), δ 6.52–6.46 (tergalloyl and HHDP-H), and δ 3.74–5.90 (glucose-H) (Figure S3). Furthermore, the signal pattern of glucose carbon atoms in the ¹³C-NMR spectrum of compound 2 was similar to those of compounds 9 and 16. The absolute configuration of glucopyranose in compound 2 was identified from the D-series [20]. This isomerization via a Smiles-type rearrangement was successfully applied to establish a chemical correlation between compounds 2 and 16. An aqueous solution of compound 2 containing a small amount of the 0.02 M phosphate buffer (pH 7.4) was left to stand at room temperature for 6 h and was nearly identical to those of compound 16. The phenolic acyl units of compound 2 were confirmed by the methylation of compound 2 and subsequent methanolysis to produce compounds 18–20. The configurations of the chiral biphenyl moieties of the HHDP and tergalloyl units in compound 2 represent the S-series because of the strong positive cotton effects in the CD spectrum at 228 and 220 nm (shoulder), respectively [22]. The partial hydrolysis of compound 2 generated compounds 5 and 17 (Figure 3). Based on these data, we determined the structure of compound 2.

Eurobustin C (3) was obtained as a pale-brown amorphous powder. The molecular formula of this material (C₆₇H₄₈O₄₃) was determined by HR-ESI-MS from its singly and doubly charged molecular ions with m/z 1539.1491 [M − H]⁻ and 769.0657 [M − 2H]²⁻, indicating that compound 3 was an ellagitannin dimer. Some of the aromatic and sugar proton signals in the obtained ¹H-NMR spectrum was broadened probably because of the poor flexibility of the macro-ring arising from restricted rotation. The ¹H-NMR spectrum of compound 3 exhibited two 2H singlets at δ 7.15 and 7.08 attributable to the protons of one galloyl group. Three 1H singlets at δ 7.18, 6.85, and 6.56, and two 1H singlets at δ 6.39 and 6.26 indicated the presence of an HHDP group and a new acyl unit named a eurobustinoyl group. This unit is metabolized by the dehydrovaloneoyl group of eugeniflorin D₂ (14). Therefore, the ¹H-NMR spectrum of compound 3 is similar to that of compound 14. ¹H-¹H correlation spectroscopy (COSY) experiment revealed the presence of two ⁴C₁ conformation glucopyranose cores. The doublet at δ 6.94 (J = 3.6 Hz) and δ 4.84 (J = 8.8 Hz) of anomeric protons resonated at a higher field than those expected for the C-1 signals of acylated α- and β-anomers, demonstrating that both anomeric centers were unacylated. A methine proton signal at δ 4.45 and methylene proton signals at δ 2.14 and 1.89 were coupled to each other. The ¹³C-NMR spectrum of compound 3 showed the presence of an α, β-unsaturated ketone system (δ 136.9, 160.6, and 197.2). Heteronuclear multiple bond connectivity (HMBC) experiment revealed the existence of correlations between methyl protons and carbonyl carbon (δ 197.2), which were separated by two bonds. The ¹³C-NMR spectrum was similar to those of brevifolincarboxylic acid [19]. Therefore, this compound contains a five-membered ring according to the HMBC experiment of compound 3. The connectivity between a methine protons (δ 4.45) and one of the methylene protons (δ 2.14), and H-2 of glucose II (δ 4.82, t, J = 8.8 Hz) was confirmed by a long-range correlation through the same ester carbonyl carbon signal (δ 170.5). Similarly, H-3’ of the valoneoyl group (δ 6.85) correlated with H-4 of glucose II (δ 4.82, t, J = 8.8 Hz) through the signal at δ 166.9. The allocations of galloyl groups at O-3 of glucose I and O-3 of glucose II were also established by the cross peaks of H-3 of glucose I (δ 5.42, t, J = 8.8 Hz) and H-3 of glucose II (δ 5.92, t, J = 6.8 Hz), respectively, formed with the ester carbonyl carbon at δ 168.2 and 166.2, which correlated with the galloyl proton signals (Figure S4). These data suggested the formation of structure 3, which was supported by the following chemical reaction. The partial hydrolysis of compound 3 in hot water containing 1% trifluoroacetic acid (TFA) produced gallic acid (21), valoneic acid dilactone (22), oenothein C (23) [24], isocoriariin F (24) [25,26], and cornusiin B (25) [26,27]. The valoneoyl group of compound 3 was of the isorugosin type because this reaction yielded compounds 23–25. The methylation of compound 3 with dimethyl sulfate and potassium carbonate in acetone followed by methanolysis resulted in compounds 18 and 19, trimethyl octa-O-methylvalonate (26), and trimethyl hexa-O-methyleurobustinate (27). Compound 19 was produced by the cleavage of the ether bond of the valoneoyl group (Figure 4). The S-configuration was identified for both the valoneoyl and eurobustinoyl group in compound 3 by the positive cotton effects in the CD spectrum [22]. Finally, the structure of compound 3 was established based on these data.

2.2. Detoxification of Al

The extracts in the liquid–liquid distributions and ellagitannins isolated from leaves of E. camaldulensis were added to the seedlings of Arabidopsis thaliana, and their Al detoxification properties were evaluated after 24 h. The EtOAc, n-BuOH, and water-soluble extracts in the liquid–liquid distributions demonstrated Al detoxification abilities (Figure 5). Therefore, we focused on isolating tannins from these extracts. Among the ellagitannins isolated from these extracts, oenothein B (12), eucarpanin D₂ (1), eucalbanin C (9), and eugeniflorin D₂ (14) exhibited the same degree of root elongation, whereas eucamalin A (2) and eugeniflorin D₁ (13) at 8 μM pyrogallol-equivalent differed significantly from compound 12 in the Tukey-Kramer test (Figure 6). We also evaluated the effect of the addition of 16 μM pyrogallol-equivalent ellagitannin alone, to the seedlings of A. thaliana. Among these ellagitannins: compounds 9, 12, and 13 did not inhibit root elongation; however, compounds 1, 2, and 14 slightly inhibited the root elongation process (Figure 7). These results revealed that ellagitannin did not promote root elongation and that compounds 1, 9, and 14 possessed the same Al detoxification ability as that of compound 12, whereas the detoxification abilities of compounds 2 and 13 were stronger than that of compound 12. Ellagitannins have structure of pyrogallol, and metal ions form chelate there [28,29]. Compound 12 has eight pyrogallols, which are presumed to contribute to Al detoxification by binding to Al ions [7]. Similarly, ellagitannins isolated from the leaves of E. camaldulensis (compounds 1, 2, 9, 13 and 14) have some or greater number of pyrogallols in their molecules as compound 12. Further isolation of the compounds is necessary to elucidate the detailed mechanisms of Al detoxification potential, including differences in the number of pyrogallol and the steric structure of the compounds.

3. Materials and Methods

3.1. Instrumentation

Optical rotations were recorded using a Jasco DIP-1000 polarimeter (Jasco, Tokyo, Japan). The ultraviolet (UV) and CD spectra were obtained using a Jasco V-530 spectrophotometer (Jasco, Tokyo, Japan) and a Jasco J-710 spectropolarimeter (Jasco, Tokyo, Japan), respectively. The ¹H-NMR (600 MHz) and ¹³C-NMR (151 MHz) spectra including ¹H-¹H COSY, heteronuclear single quantum correlation (HSQC), and HMBC were recorded on a Varian NMR instrument (Varian, Palo Alto, CA, USA), and the chemical shifts are listed in part-per-million (ppm) values relative to acetone-d₆ (2.04 ppm for ¹H and 29.8 ppm for ¹³C). The ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) spectra including ¹H-¹H COSY, HSQC, and HMBC were recorded on a JNM-ECS400 instrument (JEOL, Akishima, Tokyo, Japan), and the chemical shifts are provided in part-per-million (ppm) values relative to acetone-d₆ (2.04 ppm for ¹H and 29.8 ppm for ¹³C). The high-resolution mass spectra were obtained using a Bruker MicrOTOF II instrument (Bruker, Billerica, MA, USA) and a Xevo G3 QTof (Waters, Milford, MA, USA) with an ESI source in negative-ion mode. Reversed-phase HPLC was conducted with a Wakosil II 5C18HG (250 × 4.6 mm i.d., Wako, Osaka, Japan) column using a mobile phase composed of 0.1% aqueous TFA (solvent A) and CH₃CN (solvent B) (82:18) and isocratic program at a temperature of 35 °C, UV detection wavelength of 250 nm, and flow rate of 0.8 mL/min (System A). Reversed-phase HPLC was performed on an Inertsustain C18 column (150 × 4.6 mm i.d., 5 µm, GL Sciences, Tokyo, Japan) using a mobile phase composed of H₂O:CH₃CN:HCOOH (90:5:5) (solvent A) and H₂O:CH₃CN:HCOOH (50:45:5) (solvent B) at 40 °C, and the gradient was programmed as follows: 0–30 min (solvent B: 0–100%) (system B). Reversed-phase HPLC was performed on a YMC-pack ODS-A column (250 × 4.6 mm i.d., 5 µm, YMC Co., Ltd., Kyoto, Japan) using a mobile phase consisting of 0.01 M H₃PO₄:0.01 M KH₂PO₄:EtOH:EtOAc = 47.5:47.5:3:2 at a column temperature of 40 °C, flow rate of 1.0 mL/min, and linear gradient. Detection was performed at 280 nm (system C). Normal-phase HPLC was conducted on a CHIRALCEL OD-H column (250 × 4.6 mm i.d., 5 µm, DAICEL Co., Ltd., Osaka, Japan) using a mobile phase composed of n-hexane: EtOH = 20:1 at 24 ± 1 °C. The flow rate was 2.0 mL/min, and the UV detection wavelength was 280 nm (system D). Purification by column chromatography were conducted using Diaion-HP-20 (Mitsubishi Kasei Co., Tokyo, Japan), Toyopearl HW-40 (coarse grade) (Tosoh Co., Tokyo, Japan), Sephadex LH-20 (GE Healthcare, Chicago, IL, USA), and MCI GEL CHP-20P (75–150 µm) (Mitsubishi Chemical Co., Tokyo, Japan) resins.

3.2. Plant Material

A clone of Eucalyptus camaldulensis Dehnh. (seed lot: 19708; Australian Tree Seed Center, CSIRO) was propagated by cutting as described previously. The obtained plantlets were transferred to vermiculite in plastic pots (18.4 × 11.3 cm i.d.) and cultured in a growth chamber (16 h light/8 h dark; 28/25 °C; photosynthetic photon flux density: 200 µmol m⁻² s⁻¹). The plantlets were watered twice a day with a nutrient solution [7]. After two months of cultivation, leaves were harvested from the plantlets with heights of approximately 1 m, frozen in liquid nitrogen, and stored at −80 °C until use.

3.3. Extraction and Isolation

The frozen leaves of E. camaldulensis (2.4 kg) were homogenized with 70% aqueous acetone (4.0 L × 3), filtered, and evaporated in vacuo. Extracts were obtained sequentially with Et₂O (3.0 L × 3), EtOAc (3.0 L × 3), and water-saturated n-BuOH (3.0 L × 3) to yield Et₂O (1.7 g), EtOAc (15.1 g), n-BuOH (40.7 g), and a water-soluble fraction (117 g). A portion of the EtOAc extract (7.0 g) was subjected to column chromatography over Toyopearl HW-40 (coarse grade) (40 × 2.2 cm i.d.) using 30%, 40%, 50%, 60%, and 70% aqueous MeOH; MeOH; MeOH:H₂O:acetone (7:2:1), MeOH:H₂O:acetone (7:1:2), and 70% aqueous acetone as eluents. The 50%MeOH fraction resulted in tellimagrandin I (5) (924 mg), which was purified over MCI GEL CHP-20P (30 × 1.1 cm i.d.) with aqueous MeOH and Mega Bond Elut C18 cartridge column with aqueous MeOH to yield pedunculagin (4) (148 mg) and 1,2,6-tri-O-galloyl-β-d-glucose (7) (143 mg). 1,2,3,6-Tetra-O-galloyl-β-d-glucose (8) (104 mg) and tellimagrandin II (6) (30 mg) was obtained from the 60% and 100% MeOH fractions, respectively. The MeOH:H₂O:acetone (7:1:2) fraction yielded eucalbanin C (9) (682 mg) and a novel ellagitannin: eucarpanin D₂ (1) (175 mg). The 70% aqueous acetone fraction resulted in eucamalin A (2) (37 mg). A portion of the n-BuOH extract (6.3 g) was chromatographed over Diaion HP-20 (70 × 1.5 cm i.d.) using H₂O; 10%, 30%, and 50% aqueous MeOH; MeOH; and 70% aqueous acetone as eluents. The 30% MeOH fraction (1.5 g) was subjected to column chromatography over Toyopearl HW-40 (coarse grade) (40 × 2.2 cm i.d.) using 30%, 40%, 50%, 60%, and 70% aqueous MeOH; MeOH; MeOH:H₂O:acetone (7:2:1), MeOH:H₂O:acetone (7:1:2), and 70% aqueous acetone as eluents to give 1,6-di-O-galloyl-β-d-glucose (10) (20 mg), strictinin (11) (28 mg), compounds 4 (44 mg) and 5 (53 mg), and oenothein B (12) (360 mg). The water-soluble extract (120 g) was chromatographed over Diaion HP-20 (60 × 5 cm i.d.) using H₂O; 10%, 30%, and 50% aqueous MeOH; MeOH; and 70% aqueous acetone as eluents. The 50% MeOH fraction (19 g) was further chromatographed over Toyopearl HW-40 (coarse grade) (40 × 2.2 cm i.d.) using 30%, 40%, 50%, 60%, and 70% aqueous MeOH; MeOH; MeOH:H₂O:acetone (7:2:1); MeOH:H₂O:acetone (7:1:2); and 70% aqueous acetone as eluents. The 60%MeOH fraction purified over MCI GEL CHP-20P (30 × 1.1 cm i.d.) with aqueous MeOH yielded Eugeniflorin D₁ (13) (39 mg). For further ellagitannin studies, the leaves of E. camaldulensis were extracted again. The frozen leaves of E. camaldulensis (1.6 kg) were homogenized in 70% aqueous acetone (4.0 L × 3), filtered, and evaporated in vacuo. Extracts were obtained sequentially with Et₂O (2.0 L × 3), EtOAc (2.0 L × 3), and water-saturated n-BuOH (2.0 L × 3) to produce Et₂O (2.3 g), EtOAc (12.3 g), n-BuOH (31.8 g), and a water-soluble fraction (92.4 g). The EtOAc extract (10.0 g) was subjected to column chromatography over Toyopearl HW-40 (coarse grade) (40 × 2.2 cm i.d.) using 30%, 40%, 50%, 60%, and 70% aqueous MeOH; MeOH; MeOH:H₂O:acetone (7:2:1); MeOH:H₂O:acetone (7:1:2); and 70% aqueous acetone as eluents. The 50% MeOH fraction produced compound 5 (758 mg); the MeOH:H₂O:acetone (7:1:2) fraction resulted in compounds 1 (340 mg) and 9 (677 mg); and the 70% aqueous acetone fraction produced compound 2 (20 mg). A portion of the n-BuOH extract (31.8 g) was separated by column chromatography over Diaion HP-20 (20 × 8 cm i.d.) using H₂O with increasing amounts of MeOH (H₂O, 10%, 20%, 40% and 100% MeOH) in a stepwise mode followed by 70% aqueous acetone. The 50% MeOH fraction (1.0 g) was chromatographed over Toyopearl HW-40 (coarse grade) (30 × 2.2 cm i.d.) with 30%, 40%, 50%, 60%, and 70% aqueous MeOH; MeOH:H₂O:acetone (7:2:1); MeOH:H₂O:acetone (7:1:2); and 70% aqueous acetone. The 50% aqueous MeOH fraction produced compound 12 (1.0 g) and was purified by MCI GEL CHP-20P (30 × 1.1 cm i.d.) and preparative reversed-phase HPLC to yield eurobustin C (3) (14 mg) and eugeniflorin D₂ (14) (35 mg).

The physicochemical data obtained for compounds 1–3, 9, 13, and 14 are provided below.

Eucarpanin D₂ (1): pale brown amorphous powder; [a${]}_{\mathrm{D}}^{23}$ + 24.5° (c 0.002, MeOH); UV (MeOH) λ_max (log ε) 218 (5.17), 275 (4.83) nm; CD (MeOH) [θ] (nm) + 1.6 × 10⁵ (225), +1.7 × 10⁵ (237), −8.7 × 10⁵ (262), +7.9 × 10⁵ (285), −1.4 × 10⁵ (322); ¹H-NMR [600 MHz, acetone-d₆-D₂O (9:1)] δ 7.11, 7.02, 7.02, 7.01, 6.10, 6.99 (2H, s, galloyl-H), 6.65, 6.64, 6.49, 6.48 (1H, s, HHDP-H), 6.87, 6.84, 6.60, 6.59, 6.53, 6.51 (1H, s, tergalloyl-H), 6.18 (1H, d, J = 8.4 Hz, Glc (I) H-1), 5.87 (1H, t, J = 9.6 Hz, Glc (II) H-3α), 5.85 (1H, t, J = 10.6 Hz, Glc (I) H-3), 5.67 (1H, t, J = 9.6 Hz, Glc (II) H-3β), 5.63 (1H, dd, J = 1.2, 8.4 Hz, Glc (I) H-2), 5.60 (1H, d, J = 5.4 Hz, Glc (II) H-1α), 5.33–5.23 (1H, m, Glc (I) H-4, H-6, Glc (II) H-2β, 6αβ), 5.16–5.11 (1H, m, Glc (II) H-2α, 4αβ), 5.00 (1H, d, J = 7.8 Hz, Glc (II) H-1β), 4.66 (1H, m, Glc (II) H-5α), 4.57 (1H, m, Glc (I) H-5), 4.26 (1H, m, Glc (II) H-5β), 3.93 (1H, brd, J = 13.2 Hz, Glc (I) H-6), 3.89–3.87 (2H, d, J = 12.6 Hz, Glc (II) H-6αβ); ¹³C-NMR [151 MHz, acetone-d₆-D₂O] δ 96.3 (Glc (II) C-1β), 93.5 (Glc (I) C-1), 90.8 (Glc (II) C-1α), 74.8 (Glc (II) C-2β), 73.5 (Glc (II) C-2α, 3β), 73.1 (Glc (II) C-3α), 72.7 (Glc (II) C-5α), 71.9 (Glc (II) C-5β), 71.8 (Glc (I) C-2), 71.3 (Glc (I) C-3), 71.0 (Glc (I) C-4, Glc (II) 3C-4β), 71.0 (Glc (II) C-4α), 67.0 (Glc (I) C-5), 63.4 (Glc (I) H-6, Glc (II) C-6αβ), 168.5, 168.0, 168.0, 167.8, 167.6, 167.2, 166.6, 166.0, 165.1 (ester carbonyl-C); HR-ESI-MS: m/z 1721.1649 [M − H]⁻, 860.0840 [M − 2H]²⁻ (calcd for C₇₅H₅₄O₄₈ − H, 1721.1712).

Absolute configuration of glucopyranose. A solution of compound 1 (5.0 mg) in 5% H₂SO₄ was heated in boiling water for 6 h. The reaction mixture was purified using Sep-Pack Plus tC18 cartridges with H₂O and MeOH. The aqueous layer was neutralized with Amberlite IRA-400 (OH⁻ form) and the resin was removed by filtration. After the removal of the solvent in vacuo, the residue was heated with l-cysteine methyl ester hydrochloride (0.5 mg) in pyridine (0.5 mL) to 60 °C for 1 h. Subsequently, O-tolyl isothiocyanate (0.5 mg) in pyridine (0.1 mL) added to the reaction mixture and further heated to 60 °C for 1 h. After the reaction, the solution was analyzed via reversed-phase HPLC (system A) (t_R: 9.8 min).

Isomerization of compound 1 to eucarpanin D₁ (15): A solution of compound 1 (1.0 mg) in the 0.02 M phosphate buffer (pH 7.4) (1.0 mL) was left standing at room temperature for 6 h. The isomerized product was identical to compound 15 according to the results of reversed-phase HPLC (system B) (t_R: 10.13 min, 10.39 min).

Enzymatic hydrolysis of compound 1: A solution of compound 1 (1.0 mg) in H₂O (1.0 mL) was incubated with tannase (Aspergillus ficuum) at 37 °C for 2 h. The product was identified as compound 9 using reversed-phase HPLC (system B) (t_R: 7.02 min, 8.11 min).

Partial hydrolysis of compound 1: A solution of compound 1 (20 mg) in water was heated to 98 °C for 5 h. The reaction mixture was purified using Sephadex LH-20 (30 × 1.0 cm i.d.) and MCI GEL CHP-20P (30 × 1.0 cm i.d.) and subjected to reversed-phase HPLC (system B) to reveal the presence of compounds 5 (1.6 mg), 6 (3.5 mg), and 17 (1.5 mg) in the hydrolysate.

Methylation of compound 1 followed by methanolysis: A mixture of compound 1 (20 mg), potassium carbonate (200 mg), unhydrate, and dimethyl sulfate (100 µL) in super dehydrate acetone (2.0 mL) was stirred and refluxed for 6 h. After removing the inorganic material via centrifugation, the supernatant was evaporated. The reaction mixture was directly methanolyzed in 1% sodium methoxide (0.5 mL) and superdehydrated MeOH (0.1 mL) and left standing overnight at room temperature. After adding acetic acid (two drops), the solution was evaporated under nitrogen gas. The solution was treated with excess diazomethane (0.8 mL) and Et₂O (0.4 mL) for 3 h and then evaporated in vacuo. The residue was subjected to preparative TLC (TLC Silica gel 60 PF₂₅₄, Merck KGaA) with toluene:acetone (4:1) to compounds 18 (6.0 mg), 19 (1.5 mg), and 20 (1.7 mg). All three materials were characterized by HR-ESI-MS, normal-phase HPLC (system D), and CD spectroscopy, which revealed that these compounds were identical to the authentic samples. Compound 19: CD (MeOH) [θ] (nm) + 5.3 × 10⁴ (229), −4.0 × 10⁴ (252), +6.9 × 10³ (313); HR-ESI-MS: m/z 473.1419 [M + Na]⁺ (calcd for C₂₂H₂₆O₁₀ + Na, 473.1418); normal-phase HPLC (system D): t_R 6.1 min. Compound 20: CD (MeOH) [θ] (nm) + 4.6 × 10⁴ (221), −3.6 × 10⁴ (254), +7.5 × 10³ (317); HR-ESI-MS: m/z 683.1937 [M + Na]⁺ (calcd for C₃₂H₃₆O₁₅ + Na, 683.1946); normal-phase HPLC (system D): t_R 6.5 min.

Eucamalin A (2): pale brown amorphous powder; [a${]}_{\mathrm{D}}^{25}$ + 49.2° (c 0.002, MeOH); UV (MeOH) λ_max (log ε) 218 (5.33) nm, 272 (4.50) nm; CD (MeOH) [θ] (nm) + 2.9 × 10⁵ (222), +2.7 × 10⁵ (237), −1.7 × 10⁵ (262), +1.4 × 10⁵ (286), −2.5 × 10⁴ (323); ¹H-NMR [400 MHz, acetone-d₆-D₂O (9:1)] δ 7.06–6.96 (2H, s, galloyl-H), 6.87–6.83 (1H, s, tergalloyl-H), 6.65–6.62 (1H, s, HHDP-H), 6.60–6.56 (1H, s, Tergalloyl-H), 6.52–6.47 (1H, s, tergalloyl and HHDP-H), 5.90–5.82 (m, Glc H-3αβ), 5.67 (t, J = 9.6 Hz, Glc H-3β), 5.63–5.54 (m, Glc H-1α), 5.56 (d, J = 3.6 Hz, Glc H-1α), 5.28–5.22 (m, Glc H-2α, 6αβ), 5.16–5.09 (m, Glc H-1β, 2α, 4), 5.00 (d, J = 7.2 Hz, Glc H-1β), 4.70–4.64 (m, Glc H-5α), 4.30–4.24 (m, Glc H-1β, H-5β), 3.91–3.79 (Glc H-6αβ); ¹³C-NMR [100 MHz, acetone-d₆-D₂O (9:1)]: δ 96.6, 96.4 (Glc C-1β), 91.1, 90.8 (Glc C-1α), 75.1 (Glc C-2β), 74.1 (Glc C-2β), 73.7 (Glc C-3, 4), 73.5 (Glc C-2α), 73.0 (Glc C-2α), 71.9, 71.7 (Glc C-5β), 71.2 (Glc C-3αβ), 71.0 (Glc C-4), 67.0, 66.91 (Glc C-5α), 63.9, 63.4 (Glc C-6αβ), 168.3, 168.1, 167.8, 167.8, 169.0, 166.9, 166.6, 166.3, 165.9 (ester carbonyl-C); HR-ESI-MS: m/z 2353.2400 [M − H]⁻, 1176.1117 [M − 2H]²⁻, 783.7383 [M − 3H]³⁻.

Absolute configuration of glucopyranose. A solution of compound 2 (2.0 mg) in 5% H₂SO₄ was heated in boiling water for 1 h. The reaction mixture was purified using Sep-Pack Plus tC18 cartridges with H₂O and MeOH. The aqueous layer was neutralized with Amberlite IRA-400 (OH⁻ form) and the resin was removed by filtration. After removal of the solvent in vacuo, the residue was heated with l-cysteine methyl ester hydrochloride (0.5 mg) in pyridine (0.5 mL) to 60 °C for 1 h. Subsequently, O-tolyl isothiocyanate (0.5 mg) in pyridine (0.1 mL) added to the reaction mixture and further heated to 60 °C for 1 h. After the reaction, the solution was analyzed via reversed-phase HPLC (system A) (t_R: 9.8 min).

Isomerization of compound 2 to eucarpanin T₁ (16): A solution of compound 2 (4.0 mg) in the 0.02 M phosphate buffer (pH 7.4) (2.0 mL) was stored at room temperature for 9 h. The isomerized product was identical to compound 16 according to ¹H-NMR.

Partial hydrolysis of compound 2: A solution of compound 2 (10 mg) in water was heated to 98 °C for 2 h. The reaction mixture was purified by MCI GEL CHP-20P (20 × 1.0 cm i.d.) and subjected to reversed-phase HPLC (system B) to reveal the presence of compounds 5 (0.5 mg) and 17 (0.7 mg) in the hydrolysate.

Methanolysis of compound 2: A mixture of compound 2 (5.0 mg), potassium carbonate (100 mg), unhydrate, and dimethyl sulfate (100 µL) in super dehydrated acetone (1.0 mL) was stirred and refluxed for 6 h. After removing the inorganic material via centrifugation, the supernatant was evaporated. The reaction mixture was directly methanolyzed in 1% sodium methoxide (0.5 mL) and superdehydrated MeOH (0.1 mL) and left standing overnight at room temperature. After adding acetic acid (two drops), the solution was evaporated under nitrogen gas. The solution was treated with an excess of diazomethane (0.4 mL) and Et₂O (0.2 mL) for 3 h and then evaporated in vacuo. The residue was subjected to preparative TLC (TLC Silica gel 60 PF₂₅₄, Merck KGaA) with toluene: acetone (4:1) to yield compounds 18 (1.6 mg), 19 (1.3 mg), and 20 (0.9 mg). All three compounds were characterized by HR-ESI-MS and CD spectroscopy, which revealed that they were identical to the authentic samples. Compound 18: HR-ESI-MS: m/z 227.0968 [M + H]⁺ (calcd for C₁₁H₁₅O₅ + H: 227.0919). Compound 19: CD (MeOH) [θ] (nm) + 5.3 × 10⁴ (228), −4.1 × 10⁴ (252), +7.1 × 10³ (313); HR-ESI-MS: m/z 450.1517 [M]⁺ (calcd for C₂₂H₂₆O₁₀, 450.1526); normal-phase HPLC (system D): t_R 6.1 min. Compound 20: CD (MeOH) [θ] (nm) + 5.0 × 10⁴ (220), −4.0 × 10⁴ (254), +8.6 × 10³ (314); HR-ESI-MS: m/z 661.2113 [M + H]⁺ (calcd for C₃₂H₃₅O₁₅ + H, 661.2132); normal-phase HPLC (system D): t_R 6.4 min.

Eurobustin C (3): pale brown amorphous powder; [α${]}_{\mathrm{D}}^{23}$ + 13° (c 1, MeOH); UV (MeOH) λ_max (log ε) 219 (4.94), 685 (4.69) nm; CD (MeOH) [θ] (nm) − 2.4×10⁴ (206), +1.9 × 10⁵ (218), +8.4 × 10⁴ (228), +2.0 × 10⁵ (240), −1.3 × 10⁵ (263), +5.7 × 10⁴ (284), −1.7 × 10⁴ (308); ¹H-NMR [400 MHz, acetone-d₆-D₂O (9:1)] δ 7.15, 7.08 (2H, s, galloyl-H), 7.20, 6.85, 6.56 (1H, s, valoneoyl-H), 6.39, 6.26 (1H, s, eurobustinoyl-H), 5.94 (1H, d, J = 3.6 Hz, Glc (I) H-1α), 5.93 (1H, t, J = 4.0 Hz, Glc (I) H-2), 5.92 (1H, t, J = 9.6 Hz, Glc (I) H-3α), 5.42 (1H, t, J = 8.8 Hz, Glc (II) H-3β), 5.21 (1H, dd, J = 6,13 Hz, Glc (I) H-6α), 5.08 (1H, dd, J = 6.4, 13.2 Hz, Glc (II) H-6β), 4.99 (1H, t, J = 10.0 Hz, Glc (I) H-4α), 4.90 (1H d, J = 8.8 Hz, Glc (II) H-1β), 4.82 (1H, t, J = 8.8 Hz, Glc (II) H-2β, 4β), 4.80 (1H, dd, J = 6.4, 10.0 Hz, Glc (I) H-5α), 4.45 (1H, dd, J = 2.0, 8.0 Hz, methin-H), 3.65 (2H, d, J = 13.2 Hz, Glc (II) H-6β), 3.57 (2H, d, J = 12.8 Hz, Glc (I) H-6α), 2.14 (1H, dd, J = 1.6, 18.8 Hz, methylene-H), 1.89 (1H, dd, J = 8.0, 19.2 Hz, methylene-H); ¹³C-NMR [100 MHz, acetone-d₆-D₂O] δ 96.2 (Glc (II) C-1), 91.9 (Glc (I) C-1), 74.7 (Glc (I) C-2), 73.9 (Glc (II) C-2), 73.3 (Glc (II) C-3), 72.1 (Glc (II) C-4), 70.6 (Glc (I) C-4), 70.4 (Glc (II) C-5), 69.8 (Glc (I) C-3),68.2 (Glc (I) C-5), 63.2, 62.9 (Glc (I) (II) C-6), 170.5, 168.2, 167.9, 167.6, 166.9, 166.2, 165.0 (ester carbonyl-C), 197.2 (ketone-C); HR-ESI-MS: m/z 1539.1423 [M − H]⁻, 769.0657 [M − 2H]²⁻ (calcd for C₆₇H₄₈O₄₃ − H, 1539.1491).

Partial hydrolysis of compound 3: A solution of compound 3 (0.7 mg) in hot water containing 1% TFA (0.7 mL) was heated in a water bath for 5 h. The reaction mixture was subjected to reversed-phase HPLC (System C) to reveal the presence of compounds 21–25 in the hydrolysate.

Methylation of compound 3 followed by methanolysis: diazomethane-EtOH (1 mL) was added to an EtOH solution (0.5 mL) of compound 3 (10 mg), and the obtained mixture was stored for 3 h at room temperature. After removing the solvent using an evaporator, a MeOH solution (1.0 mL) of the residue was added to 1% sodium methoxide (20 drops) and left standing overnight at room temperature. After the addition of AcOH, the solution was evaporated, and the residue was partitioned between EtOAc and H₂O. An EtOAc layer was added, the residue was further treated with diazomethane (10 drops)–Et₂O for 2 h, and the solvent was evaporated. The residue was subjected to preparative thin layer chromatography (TLC) (TLC silica gel 60 PF₂₅₄, Merck KGaA) with toluene:acetone (4:1) to yield compounds 18, 19, 26, and 27. All four materials were characterized via CD spectroscopy, which revealed that these compounds were identical to the authentic samples. Compound 26: CD (MeOH) [θ] (nm) − 4.4 × 10⁴ (202), +1.5 × 10⁵ (220), −7.3 × 10⁴ (251), +2.0 × 10³ (278), −2.0 × 10³ (294), +1.7 × 10⁴ (315). Compound 27: CD (MeOH) [θ] (nm) − 5.7 × 10⁴ (204), +1.1 × 10⁵ (228), −8.2 × 10⁴ (254), +1.0 × 10⁴ (278), −1.1 × 10⁴ (297), +5.1 × 10³ (316).

Eucalbanin C (9): pale brown amorphous powder; ¹H-NMR [600 MHz, acetone-d₆-D₂O (9:1)] δ 7.06, 7.05, 7.05, 7.04, 7.02, 7.02, 7.01, 6.99, 6.99, 6.96 (2H in total each, s, galloyl-H), 6.65, 6.64, 6.64, 6.63, 6.49, 6.48 (1H, s, HHDP-H), 6.87, 6.86, 6.84, 6.83, 6.59, 6.58, 6.58, 6.52, 6.50, 6.50 (1H, s, tergalloyl-H), 5.86 (2H, t, Glc (I) H-3α, Glc (I) H-3β), 5.66 (1H, t, Glc (I) H-3β), 5.60 (2H, m, Glc (II) H-1α, H-3α), 5.54 (1H, d, J = 3.6 Hz, Glc (I) H-1α), 5.28–5.20 (1H, m, Glc (I) H-2β, Glc (II) H-2β, Glc (I) (II) H-6αβ), 5.17–5.07 (1H, m, Glc (I) H-2α, Glc (II) H-2α, Glc (I) (II) H-4αβ), 4.37 (1H, m, J = 7.8 Hz, Glc (II) H-1β), 4.98 (1H, d, J = 7.8 Hz, Glc (I) H-1β), 4.66 (2H, m, Glc (I) (II) H-5α), 4.26 (2H, m, Glc (I) (II) H-5β), 3.83 (2H, m, Glc (I) (II) H-6’αβ); ¹³C-NMR [151 MHz, acetone-d₆-D₂O] δ 96.5 (Glc (II) C-1β), 96.3 (Glc (I) C-1β), 91.1 (Glc (I) C-1α), 90.7 (Glc (II) C-1α), 74.8 (Glc (I) (II) C-2β), 74.0 (Glc (I) (II) C-2α), 73.7, 73.6 (Glc C-3β), 71.9, 71.7 (Glc (I) (II) C-5β), 71.4 (Glc (I) (II) C-3α), 73.0, 71.2, 71.1 (Glc (I) (II) C-4β), 71.0 (Glc (I) (II) C-4α), 66.9, 66.8 (Glc (I) (II) C-5α), 63.9, 63.5 (Glc (I) (II) C-6αβ); HR-ESI-MS: m/z 1569.1569 [M − H]⁻, 785.0816 [M − 2H]²⁻ (calcd for C₆₈H₅₀O₄₄ − H, 1569.1602).

Eugeniflorin D₁ (13): pale brown amorphous powder; ¹H-NMR [600 MHz, acetone-d₆-D₂O (9:1)] δ 6.94, 6.92, 6.91 (2H in total each, s, galloyl-H), 6.97, 6.82, 6.65, 6.61, 6.52, 6.33 (1H, s, valoneoyl-H), 6.40 (1H, d, J = 8.4 Hz, Glc (I) H-1), 5.77 (1H, t, J = 9.6 Hz, Glc (I) H-3), 5.40 (1H, dd, J = 7.2 Hz, Glc (II) H-6), 5.36 (1H, t, J = 9.0 Hz, Glc (II) H-3), 5.22–5.15 (3H, m, Glc (I) H-2, Glc (II) H-4, 6), 5.03 (1H, t, J = 9.0 Hz, Glc (II) H-2), 4.61–4.58 (1H, m, Glc (I) H-5), 4.42 (1H, d, J = 7.8 Hz, Glc (II) H-1), 4.38 (1H, dd, J = 6.0, 10.2 Hz, Glc (II) H-5), 4.00 (1H, dd, J = 2.4, 13.2 Hz, Glc (I) H-6), 3.91 (1H, d, J = 13.2 Hz, Glc (II) H-6’), 3.88–3.84 (1H, m, Glc (I) H-6’); ¹³C-NMR [151 MHz, acetone-d₆-D₂O] 95.8 (Glc (II) C-1), 93.0 (Glc (I) C-1), 75.6 (Glc (I) C-2), 74.5 (Glc (II) C-2), 73.9 (Glc (II) C-4), 72.1 (Glc (II) C-3), 72.0 (Glc (I) C-5), 71.8 (Glc (II) C-5), 71.7 (Glc (I) C-3), 71.3 (Glc (I) C-4), 66.0 (Glc (II) C-6), 63.1 (Glc (I) C-6), 170.4, 168.9, 168.8, 168.1, 166.9, 166.9, 165.9, 164.8 (ester carbonyl-C); HR-ESI-MS: m/z 1719.1532 [M − H]⁻, 859.0759 [M − 2H]²⁻ (calcd for C₇₅H₅₂O₂₈ − H, 1719.1555).

Eugeniflorin D₂ (14): pale brown amorphous powder; ¹H-NMR [400 MHz, acetone-d₆-D₂O (9:1)] δ 7.30, 7.16 (2H in total each, s, galloyl-H), 7.17, 6.77 (1H, s, dehydrovaloneoyl-H), 7.10 (1H, d, J = 2.0 Hz, dehydrovaloneoyl-H), 6.57, 6.36, 5.87 (1H, s, valoneoyl-H), 5.98 (1H, d, J = 3.2 Hz, Glc (I) H-1α), 5.90 (1H, t, J = 10.0 Hz, Glc (I) H-3α), 5.69 (1H, dd, J = 3.2, 10.0 Hz, Glc (I) H-2α), 5.55 (1H, t, J = 10.0 Hz, Glc (II) H-3β), 5.24 (1H, t, J = 8.0 Hz, Glc (II) H-2β), 5.19 (1H, d, J = 8.0 Hz, Glc (II) H-1β), 5.09 (1H, dd, J = 6.4, 13.6 Hz, Glc (II) H-6β), 4.91 (1H, t, J = 10.0 Hz, Glc (II) H-4αβ), 4.70 (1H, dd, J = 5.6, 12.8 Hz, Glc (I) H-6α), 4.49 (1H, dd, J = 5.6, 10.0 Hz, Glc (I) H-5α), 4.05 (1H, dd, J = 6.4, 10.0, Glc (II) H-5β), 3.74–3.70 (2H, m, Glc (I) H-6α, Glc (II) H-6β); ¹³C-NMR [100 MHz, acetone-d₆-D₂O] δ 96.2 (Glc (II) C-1β), 91.7 (Glc (I) C-1α), 75.4 (Glc (I) C-2α), 74.9 (Glc (II) C-2β), 72.5 (Glc (I) C-4α), 72.4 (Glc (II) C-3β), 71.3 (Glc (II) C-5β), 71.0 (Glc (I) C-4α), 70.3 (Glc (I) C-3α), 68.5 (Glc (I) C-5α), 64.4 (Glc (I) C-6α), 63.5 (Glc (II) C-6β), 168.4, 168.3, 167.4, 166.2, 164.5, 163.9 (ester carbonyl-C), 194.1 (ketone-C); HR-ESI-MS: m/z 1583.1389 [M − H]⁻, 791.0640 [M − 2H]²⁻ (calcd for C₆₈H₄₈O₄₅ − H, 1583.148).

3.4. Bioassay for Detoxification Studies

The Al detoxification properties of the fractionated extracts and isolated ellagitannins were evaluated by a bioassay using the Al-sensitive model plant Arabidopsis thaliana, as described previously [7]. Briefly, pretreated A. thaliana seeds were germinated on culture equipment comprising glass slides and nylon mesh inside a growth chamber. Seedlings were grown hydroponically in a 2% strength-modified Molecular Genetics Research Laboratory (MGRL) medium (without phosphate ions and with 200 μM CaCl₂ instead of calcium salts). After 4 d of culture, the seedlings were treated with a modified MGRL medium (pH 5.0) containing 0 or 12 μM AlCl₃ and 0, 8, or 16 μM ellagitannin for 24 h [30]. To evaluate the detoxification properties of the extracts, 3 mg of each dried extract was added to 500 mL of the medium instead of the ellagitannin. Images of the root were taken with a scanner before and after treatment and expressed relative to controls (0 µM Al, 0 µM ellagitannin). The elongation of each primary root was measured using the WinRHIZO Pro image analysis software (https://regent.qc.ca/assets/winrhizo_software.html, Régent Instruments, Quebec, QC, Canada).

4. Conclusions

In summary, we isolated two undescribed ellagitannin oligomers, i.e., eucarpanin D₂ (1, dimer) and eucamalin A (2, trimer), along with 14 known compounds from the leaves of E. camaldulensis, including gallotannins (7, 8 and 10), monomeric ellagitannins (4–6 and 11), and dimeric ellagitannins (3, 9, 12 and 14). Compounds 1–3 were identified via HR-ESI-MS, NMR, chemical reactions, and physicochemical data. The detailed chemical structure of 3 was elucidated for the first time. Furthermore, the ellagitannins obtained from E. camaldulensis, especially compound 2 and eugeniflorin D₁ (13), demonstrated high abilities to alleviate Al toxicity. These results suggest that ellagitannins other than oenothein B (12) possess good Al detoxification properties. Therefore, further studies should focus on ellagitannins that are present not only in the leaves, but also in the roots of E. camaldulensis. Although further studies are required to achieve a better understanding of the Al detoxification mechanism of ellagitannins, these results suggest that these tannins promote the growth of E. camaldulensis in acidic soils.