Two new phenolic compounds from the leaves of Alnus sibirica Fisch. ex Turcz.

Abstract

Two new phenolic compounds, 4-O-glucopyranosyl-5-O-caffeoylshikimic acid (1) and 2,3-digalloyl oregonin (2), were isolated along with eight known phenolic compounds (3–10) from an 80% acetone extract of Alnus sibirica leaves. The chemical structures of these compounds were elucidated using 1D/2D nuclear magnetic resonance and high resolution-MS. The anti-oxidative activities of these compounds were determined by assaying their 1,1-diphenyl-2-picrylhydrazyl radical and nitroblue tetrazolium superoxide anion scavenging activity. All of the isolated phenolic compounds (1–10) exhibited potent anti-oxidative activities. In particular, 2 and 4, which are diarylheptanoids, and 10 which is ellagitannin exhibited excellent anti-oxidative activities with almost the same potency as that of the positive controls L-ascorbic acid and allopurinol.

1. Introduction

Members of the Alnus species have been used in a number of traditional medicines such as cathartics, emetics, galactogogues, febrifuges, hemostatics, parasiticides, vermifuges, skin tonics and astringents (Guo et al. 2001). Alnus sibirica Fisch. ex Turcz. (AS) is geographically distributed in Korea, Japan, Northeast China and Russia, and the bark of this plant has been used as an antipyretic, expectorant, antiasthmatic and a health tea for alcoholism (Lee 1966). Previous studies on the chemical constituents of the Alnus species have led to the isolation of various tannins, flavonoids, diarylheptanoids and triterpenoids (Suga et al. 1972; Terazawa et al. 1984; Aoki et al. 1990; Lee et al. 1992, 1999; Jeong et al. 2000; Choi et al. 2012). These studies have shown that the Alnus species is a good source of diarylheptanoids, and that plants of this genus exhibit anti-oxidative, anti-inflammatory, anti-atopic, anti-bacterial and anti-adipogenic activities (Joo et al. 2009; Lee et al. 2010, 2013; Choi et al. 2012). This paper describes the isolation and structure elucidation of two new phenolic compounds along with eight known phenolic compounds. In addition, the anti-oxidative activities of these compounds were determining by assessing their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and nitroblue tetrazolium (NBT) superoxide anion scavenging activity.

2. Results and discussion

The 80% acetone extract of AS leaves was dissolved in water and filtered using Celite. The resulting filtrate was concentrated and applied to column chromatography using Ambelite XAD-2, Sephadex LH-20, MCI-gel, CHP 20P and ODS-B gel with a reversed phase medium pressure liquid chromatography (MPLC) system, which afforded 10 compounds including two new phenolic compounds (1 and 2) (Figure 1). The known compounds (3–10) were identified as alnuside A (3, Kuroyanagi et al. 2005), alnuside C (4, Kuroyanagi et al. 2005), quercetin (5, Dutta et al. 2007), quercitrin (6, Lee et al. 2003), hyperoside (7, Lee et al. 2003), quercetin-3-O-β-d-glucuronide-methylester (8, Pacifico et al. 2013), kaempferol-3-O-α-l-(4″E-p-coumaroyl)-rhamnoside (9, Yang et al. 2010) and isocorilagin (10, Liu et al. 2008), respectively, by comparing their spectroscopic (MS and nuclear magnetic resonance (NMR)) data with literature values.

Figure 1 . Structures of compounds 1–10.

Compound 1 was isolated as a pale yellow amorphous powder. High-resolution (HR)-negative FAB-MS (m/z 497.1295 [M − H]⁻ , calcd for C₂₂H₂₅O₁₃, 497.1299) indicated its molecular formula was C₂₂H₂₆O₁₃. In thin-layer chromatography (TLC), 1 was detected using a UV lamp at 254 nm as a dark brown spot by spraying with FeCl₃ solution and a yellow spot by spraying with H₂SO₄ solution and heating.

The ¹H NMR spectrum of 1 revealed three aromatic protons [δ_H 7.18 (d, J = 2.4 Hz, H-2′), 7.03 (dd, J = 2.4, 7.8 Hz, H-6′), 6.84 (d, J = 7.8 Hz, H-5′)] in an ABX spin system and two doublets at δ_H 7.70 (d, J = 16.2 Hz, H-7′), 6.33 (d, J = 16.2 Hz, H-8′) of a trans-double bond. The ¹³C NMR spectrum of 1 exhibited one carboxyl group (δ_C 167.3) and two hydroxyl bearing aromatic carbons (δ_C 148.2 and 146.2). These findings suggested the presence of a caffeoyl moiety.

The ¹H NMR spectrum of 1 showed the presence of a proton singlet at δ_H 6.72 (1H, m, H-6), two coupled germinal protons at δ_H 2.79 (1H, dd, J = 5.4, 18.6 Hz, H-2a) and 2.30 (1H, dd, J = 5.4, 18.6 Hz, H-2b) and three oxymethine groups appearing at δ_H 5.83 (1H, brs, H-5), 4.24 (1H, dd, J = 5.1, 12.6 Hz, H-3) and 4.10 (1H, dd, J = 5.1, 12.6 Hz, H-4). ¹³C NMR spectrum of 1 revealed the presence of a shikimic acid moiety [δ_C 167.7 (C-7), 133.4 (C-1), 132.5 (C-2), 78.6 (C-4), 68.2 (C-5), 66.3 (C-3), 31.2 (C-6)].

The ¹H and ¹³C NMR spectra of 1 also revealed a glucopyranosyl moiety, one methylene [δ_H 3.85 (m, H-6″a), 3.65 (m, H-6″b)], and five additional methine groups [δ_H 4.50 (d, J = 7.8 Hz, H-1″), 3.45 (m, H-3″), 3.34 (m, H-4″, H-5″), 3.23 (m, H-2″)] and at δ_C 103.6 (C-1″), 76.8 (C-5″), 76.4 (C-3″), 73.6 (C-2″), 70.2 (C-4″), 61.5 (C-6″). In addition, the large coupling constant (J = 7.8 Hz) of H-1″ at δ_H 4.50 in the ¹H NMR spectrum of 1 suggested that the glucopyranoside was in a β-configuration.

The connectivities of caffeoyl and shikimic acid and glucose were elucidated by heteronuclear multiple bond coherence (HMBC) correlation signals. The HMBC spectrum of 1 showed a correlation between the H-1″ of glucose and C-4 of shikimic acid, and also showed the correlation between H-5 of shikimic acid and carbonyl C-9′ of caffeoyl moiety. Based on these results, compound 1 was elucidated as 4-O-β-d-glucopyranosyl-5-O-caffeoylshikimic acid. Compound 1 is the first reported shikimic acid conjugated with glucose and caffeic acid.

Compound 2 was isolated as a dark yellow amorphous powder. HR-negative FAB-MS (m/z 781.1978 [M − H]⁻ , calcd for C₃₈H₃₇O₁₈, 781.1980) indicated its molecular formula was C₃₈H₃₈O₁₈. In TLC, 2 was detected as a dark blue spot by spraying with FeCl₃ solution, and a brown spot by spraying with H₂SO₄ solution and heating.

The ¹H and ¹³C NMR spectra of 2 showed two galloyl groups in the aromatic region [δ_H 7.04 (2H, s, galloyl-2, 6) and 7.06 (2H, s, galloyl-2′, 6′)] and [(δ_C 165.7 (C-7″″), 165.0 (C-7″″′), 145.0 (C-3″″, 3″″′, 5″″, 5″″′), 138.1 (C-4″″), 138.0 (C-4″″′), 120.4 (C-1″″), 120.3 (C-1″″′), 109.2 (C-6″″, 6″″′), 109.1 (C-2″″, 2″″′)] and one diarylheptanoid glycoside moiety compose of five methylenes [δ_H 1.70–1.75 (2H in total, m, H-6), 2.33–2.68 (8H, m, H-1, 2, 4, 6, 7] and a methine [δ_H 4.15 (1H, m, H-5) and 1.70–1.75 (2H in total, m, H-6)] and carbonyl carbon C-3 (δ_C 207.7), a secondary carbonyl carbon C-5 (δ_C 75.2), and five carbons [δ_C 28.6 (C-1), 44.8 (C-2), 47.6 (C-4), 37.3 (C-6) and 30.5 (C-7)] as heptanes moiety and two sets of caffeoyl groups [δ_H 6.67 (1H, d, J = 7.8 Hz, H-5′), 6.69 (1H, d, J = 7.8 Hz, H-5″, 6.59 (1H, d, J = 2.4 Hz, H-2′), 6.67 (1H, d, J = 2.4 Hz, H-2″), 6.39 (1H, dd, J = 2.4, 7.8 Hz, H-6′) and 6.48 (1H, dd, J = 2.4, 7.8 Hz, H-6″; δ_C 115.0 (C-2′), 115.1 (C-2″), 115.3 (C-5′, C-5″) and 119.4 (C-6′, C-6″), 144.6 (C-3′), 144.7 (C-3″), 143.0 (C-4″) and 142.9 (C-4′)] and a xylopyranosyl moiety [δ_H 5.32 (1H, m, H-3″′), 5.07 (1H dd, J = 7.8, 9.6 Hz, H-2″′), 4.81 (1H, d, J = 7.8 Hz, H-1″′), 4.04 (1H, m, H-5″′a), 3.95 (1H, m, H-4″′), and 3.48 (1H, m, H-5′b), and δ_C 101.2 (C-1″′), 75.7 (C-3″′), 71.7 (C-2″′), 68.4 (C-4″′) and 65.7 (C-5″′)] were observed.

These results indicated that 2 consisted of a diarylheptanoid glycoside (oregonin) and two galloyl groups. The locations of the galloyl units were determined as the C-3″′ and C-2″′ of the xylopyranosyl moiety by a downfield shift of C-3″′ and C-2″′ at δ_C 75.7 (C-3″′) and 71.7 (C-2″′) together with an upfield shift of C-4″′ to δ_C 68.4 (C-4″′) compared with oregonin (Lee et al. 1992).

The connectivity of the diarylheptanoid with xylose and galloyl groups was further confirmed by HMBC correlations. Especially, the HMBC spectrum of 2 showed a correlation between the H-1″′ of xylose and C-5 of the diarylheptanoid moiety. The HMBC spectrum of 2 also revealed correlations between H-2″′ and H-3″′ of xylose and each C-7″″, 7″″′ of the galloly moiety. Based on these results, the structure of compound 2 was elucidated as 2,3-digalloyl oregonin. Interestingly, a diarylheptanoid-conjugated ellagitannin from the leaves of Alnus hirsuta var. microphylla was previously reported (Lee et al. 1992); however, compound 2 is the first report of a diarylheptanoid-conjugated gallotannin.

Most of the isolated phenolic compounds from the leaves of AS exhibited potent scavenging activities of DPPH radical and NBT superoxide anion (Table 1). The new compound (2) which is diarylheptanoid with galloyl moiety and isocorilagin (10) which is ellagitannin showed more strong anti-oxidative activity than L-ascorbic acid and almost same potency compared with allopurinol (Table 1).

Table 1 . DPPH radical and superoxide anion scavenging activities of compounds 1–10.

Compounds	DPPH radical scavenging activity (μM)	Superoxide anion scavenging activity (μM)
1	10.28 ± 0.13c	41.38 ± 0.52l
2	6.66 ± 0.08a	8.57 ± 0.36d
3	33.85 ± 0.16n	23.87 ± 0.28j
4	11.43 ± 0.06d	9.04 ± 0.09e
5	21.82 ± 0.34k	61.37 ± 1.47p
6	17.74 ± 0.08i	18.45 ± 0.74i
7	18.56 ± 0.43j	42.96 ± 0.23m
8	29.31 ± 0.85l	71.06 ± 0.62q
9	41.24 ± 0.99o	>100r
10	6.92 ± 0.25a	8.26 ± 0.420c
Vit-C	20.50 ± 0.71k	–
Allopurinol	–	7.51 ± 0.53b

Values represent the mean ± SD of three determinations. In the IC₅₀ column, values that are not significantly different from one another are designated with the same letter, whereas values that are significantly different from one another (p < 0.05) are designated with different letters.

In particular, the anti-oxidative activity of 2, which contains a diarylheptanoid with two galloyl moieties, is more potent than that of 3 and 4 whose structures are similar to 2 (Table 1). This result indicates the importance of polyhydroxy groups in the structure for the anti-oxidative effect.

3. Experimental

3.1. General experimental procedures

Column chromatographic isolations were performed using Ambelite XAD-2 (20–50 μm, Fluka AG, Buchs, Switzerland), Sephadex LH-20 (10–25 μm, GE Healthcare Bio-Science AB, Uppsala, Sweden), MCI-gel CHP 20P (75–150 μm, Mitsubishi Chemical, Tokyo, Japan), Toyopearl HW-40F (30–60 μm, Tosoh Corp., Tokyo, Japan) and ODS-B gel (40–60 μm, Daiso, Osaka, Japan). ODS-B gel was also used as a stationary phase for the middle pressure liquid chromatography (MPLC) system. TLC was carried out using a pre-coated silica gel 60 F₂₅₄ plate (Merck, Darmastadt, Germany) with chloroform, methanol, and water (70:30:4, 80:20:2, volume ratio). Spots were detected under UV radiation (254 nm) and spraying with FeCl₃ and 10% H₂SO₄ or anisaldehyde–H₂SO₄ followed by heating. The chemical structures were elucidated by several instrumental analyses. 1D NMR such as ¹H (300 or 600 MHz) and ¹³C (75 or 150 MHz) NMR, 2D NMR such as proton–proton correlation spectroscopy (¹H–¹H COSY), heteronuclear single quantum coherence, and HMBC experiments were recorded with Gemini 2000 and VNS instruments (Varian, Palo Alto, CA, USA) at the centre for research facilities of Chung-Ang University. HR fast atom bombardment mass spectra were recorded with JMS-600W and JMS-700 instruments (JEOL, Tokyo, Japan) at the National Center for Inter-University Research facilities at Seoul National University.

3.2. Plant material

AS leaves were collected from Mt. Guksabong in Dongjak-gu, Seoul, Korea in August 2010 and its identity was confirmed by Prof. M.W. Lee (Pharmacognosy Lab, Laboratory of Pharmacognosy and Natural Product Derived Medicine, College of Pharmacy, Chung-Ang University) and C.I. Lee (Kwang-Leung Korean National Arboretum in Pocheon, Korea). A voucher specimen (MR2010-08) has been deposited at the herbarium of the College of Pharmacy, Chung-Ang University.

3.3. Extraction and isolation

AS leaves (15 kg) were extracted with 80% acetone at room temperature. The resulting extract was concentrated by removing the acetone under vacuum, which afforded 1062 g of material. After acetone evaporation, water liquid was filtered through Celite 545 (Duksan Pure Chemicals Co. Ltd, Korea). The resulting filtrate (764 g) was applied to Ambelite XAD-2 (20–50 μm, 10 kg, 70 × 50 cm) and eluted using a graded H₂O, 50–100% MeOH solvent system yielding six fractions (AS-1 to AS-6). Repeated column chromatography of fraction AS-4 (225 g) using a Sephadex LH-20 column (25–100 μm, 2000 g, 10 × 120 cm, 0–100% MeOH in H₂O) yielded seven subfractions (AS-4-1 to AS-4-7). Fraction AS-4-3 (94 g) was applied to a Sephadex LH-20 column (25–100 μm, 2000 g, 10 × 120 cm, 0–100% MeOH in H₂O), which yielded eight subfractions (AS-4-3-1 to AS-4-3-8). Repeated column chromatography of fraction AS-4-3-2 using an ODS gel column (50 μm, 150 g, 3 × 50 cm, 0–100% MeOH in H₂O) and Toyopearl HW-40F (40 μm, 120 g, 3 × 40 cm) yielded [4-O-glucopyranosyl-5-O-caffeoylshikimic acid] (1, 370 mg). Fraction AS-4-3-4 (9.79 g) was applied to an MCI gel column (50 μm, 400 g, 3 × 50 cm, 0–100% MeOH in H₂O) and an ODS gel column (50 μm, 250 g, 3 × 50 cm, 10–100% MeOH in H₂O) to yield alnuside A (3, 80 mg). Subfraction AS-4-3-7 (3.78 g) was applied to an ODS gel column (50 μm, 250 g, 3 × 50 cm, 20–100% MeOH in H₂O), which yielded quercitrin (6, 300 mg) and hyperoside (7, 22 mg). Subfraction AS-4-4 was applied to an MCI gel column (50 μm, 400 g, 3 × 50 cm, 0–100% MeOH in H₂O), from which 13 subfractions (AS-4-4-1 to AS-4-4-13) were obtained. Subfraction AS-4-4-3 was sequentially applied to an ODS gel column (50 μm, 250 g, 3 × 50 cm, 0–100% MeOH in H₂O), of which subfraction AS-4-4-3-11 was applied to a Toyopearl HW-40F column (40 μm, 120 g, 3 × 40 cm, 0–100% MeOH in H₂O) to yield isocorilagin (10, 55 mg). Likewise, the subfraction AS-4-4-5 was applied to an ODS gel column (50 μm, 250 g, 3 × 50 cm, 0–100% MeOH in H₂O), which yielded quercetin-3-O-β-d-glucuronide-methylester (8, 62 mg). In addition, 2,3-digalloyl oregonin (2, 66 mg) was obtained from subfraction AS-4-4-8 by column chromatography with an ODS gel column (50 μm, 250 g, 3 × 50 cm, 0–100% MeOH in H₂O). Repeated column chromatography of fraction AS-5 (40.85 g) using a Sephadex LH-20 column (25–100 μm, 2000 g, 10 × 120 cm, 50–100% MeOH in H₂O), yielded seven subfractions (AS-5-1 to AS-5-7). The subfraction AS-5-2 (1.37 g) was applied to an MCI gel column (50 μm, 400 g, 3 × 50 cm, 40–100% MeOH in H₂O) and an ODS gel column (50 μm, 150 g, 3 × 50 cm, 20–100% MeOH in H₂O), which yielded alnuside C (4, 45 mg). Fraction AS-5-5 (0.97 g) was continuously applied to an ODS gel column (50 μm, 150 g, 3 × 50 cm, 50–100% MeOH in H₂O), which resulted in kaempferol-3-O-α-l-(4″E-p-coumaroyl)-rhamnoside (9, 800 mg). Lastly, quercetin (5, 125 mg) was obtained by recrystallisation of AS-5-6 (0.49 g).

3.3.1. 4-O-Glucopyranosyl-5-O-caffeoylshikimic acid (1)

Pale yellow amorphous powder. [α]_D: − 149.7° (c = 0.01, MeOH). IR (KBr) cm^–1: 3391, 1697, 1601, 1517. HR-negative FAB-MS m/z: 497.1295 [M − H]^– (calcd C₂₂H₂₅O₁₃, 497.1299). ¹H NMR (600 MHz, acetone-d ₆ + D₂O): δ 7.70 (1H, d, J = 16.2 Hz, H-7′), 7.18 (1H, d, J = 2.4 Hz, H-2′), 7.03 (1H, dd, J = 2.4, 7.8 Hz, H-6′), 6.84 (1H d, J = 7.8 Hz, H-5′), 6.72 (1H, m, H-6), 6.33 (1H, d, J = 16.2 Hz, H-8′), 5.83 (1H, brs, H-5), 4.50 (1H, d, J = 7.8 Hz, H-1″), 4.24 (1H, m, H-3), 4.10 (1H, m, H-4), 3.85 (1H, m, H-6″a), 3.65 (1H, m, H-6″b), 3.45 (1H, t, J = 8.7 Hz, H-3″), 3.34 (2H, m, H-4″, H-5″), 3.23 (1H, m, H-2″), 2.79 (1H, m, H-2a), 2.30 (1H, m, H-2b). ¹³C NMR (150 MHz, acetone-d ₆ + D₂O): δ 167.3 (C-7), 166.9 (C-9′), 148.2 (C-4′), 146.2 (C-3′), 145.4 (C-7′), 133.0 (C-1), 132.1 (C-2), 126.4 (C-1′), 121.9 (C-6′), 115.5 (C-5′), 114.3 (C-2′), 114.0 (C-8′), 103.6 (C-1″), 78.1 (C-4), 76.8 (C-5″), 76.4 (C-3″), 73.6 (C-2″), 70.2 (C-4″), 67.8 (C-5), 65.9 (C-3), 61.5 (C-6″), 30.7 (C-6).

3.3.2. 2,3-Digalloyl oregonin (2)

Dark yellow amorphous powder. IR (KBr) cm^− 1: 3346, 1708, 1611, 1526. HR-negative FAB-MS m/z: 781.1978 [M − H]⁻  (calcd C₃₈H₃₇O₁₈, 781.1980) ¹H NMR (600 MHz, acetone-d ₆ + D₂O): δ 7.06 (2H, s, H-2″″′, 6″″′), 7.04 (2H, s, H-2″″, 6″″), 6.69 (1H, d, J = 7.8 Hz, H-5″, 6.67 (1H, d, J = 2.4 Hz, H-2′), 6.67 (1H, d, J = 7.8 Hz, H-5′), 6.59 (1H, d, J = 2.4 Hz, H-2″), 6.48 (1H, dd, J = 2.4, 7.8 Hz, H-6″), 6.39 (1H, dd, J = 2.4, 7.8 Hz, H-6′), 5.32 (1H, m, H-3″′), 5.07 (1H, dd, J = 7.8, 9.6 Hz, H-2″′), 4.81 (1H, d, J = 7.8 Hz, H-1″′), 4.15 (1H, m, H-5), 4.04 (1H, m, H-5″′a), 3.95 (1H, m, H-4″′), 3.48 (1H, m, H-5″′b), 2.33–2.68 (8H in total, m, H-1, 2, 4, 7), 1.70–1.75 (2H in total, m, H-6). ¹³C NMR (150 MHz, acetone-d ₆ + D₂O): δ 207.7 (C-3), 165.7 (C-7″″), 165.0 (C-7″″′), 145.0 (C-3″″, 3″″′, 5″″, 5″″′), 144.7 (C-3″), 144.6 (C-3′), 143.0 (C-4″), 142.9 (C-4′), 138.1 (C-4″″), 138.0 (C-4″″′), 133.7 (C-1″), 132.8 (C-1′), 120.4 (C-1″″), 120.3 (C-1″″′), 119.4 (C-6′, 6″), 115.3 (C-5′, 5″), 115.1 (C-2″), 115.0 (C-2′), 109.2(C-6″″, 6″″′), 109.1 (C-2″″, 2″″′), 101.2 (C-1″′), 75.7 (C-3″′), 75.2 (C-5), 71.7 (C-2″′), 68.4 (C-4″′), 65.7 (C-5″′), 47.6(C-4), 44.8 (C-2), 37.3 (C-6), 30.5 (C-7), 28.6 (C-1).

3.4. Measurement of DPPH radical scavenging activity

Each sample was dissolved in absolute EtOH and added to a DPPH solution (0.1 mM, in absolute EtOH). After mixing gently for 30 min, optical densities were measured at 518 nm using a microplate reader (TECAN, Salzburg, Austria). L-ascorbic acid was used as a positive control.

3.5. Measurement of NBT/superoxide anion scavenging activity

Each sample was dissolved in 50 mM phosphate buffer (pH 7.5) containing 0.05 mM EDTA, 0.2 mM hypoxanthine, and 0.1 mM NBT. Next, xanthine oxidase (1.2 U/μL) was added to the mixture. After mixing gently for 30 min, optical densities were measured at 612 nm using a microplate reader (TECAN). Allopurinol was used as a positive control.

3.6. Statistical analysis

All data were expressed as the mean ± SD. Values were analysed by Student–Newman–Keuls test, and values of p < 0.05 were considered to be significantly different.

4. Conclusion

The activity-guided isolation of A. sibirica yielded 10 phenolic compounds (1–10) including two new compounds, 4-O-β-d-glucopyranosyl-5-O-caffeoylshikimic acid (1) and 2,3-digalloyl oregonin (2) which are the first reported shikimic acid conjugated with glucose and caffeic acid (1) and diarylheptanoid conjugated with gallotannin (2).

The phenolic compounds (1–10) showed potent antioxidative activities against DPPH and NBT radicals. Especially, 2 and 4 which are diarylheptaoid and 10 which is ellagitannin showed excellent anti-oxidative activities. The results suggest that the leaves of A. sibirica and the phenolic compounds isolated from these leaves are promising source of natural products that can be developed as anti-oxidant agents.

Supplementary material

The underlying research materials for this article can be accessed at http://dx.doi.org/10.1080/14786419.2015.1053087

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding Statement

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology [grant number 2010-0022929].