Phytochemical Constituents of the Leaves of Hosta longipes

Abstract

Phytochemical investigation of the 80% MeOH extract from the leaves of Hosta longipes resulted in the isolation of sixteen compounds (1 - 16). The structures of the compounds were elucidated by spectroscopic methods to be methyl 10,10-dimethoxydecanoate (1), methyl 10-hydroxy-8E,12Z-octadecadienoate (2), methyl coriolate (3), trans-phytol (4), phytene-1,2-diol (5), phyton (6), (3S,5R,6S,7E,9R)-7-megastigmene-3,6,9-triol (7), (3S,5R,6S,9R)-3,6,9-trihydroxymegastigman-7-ene (8), shikimic acid (9), p-coumaramide (10), trans-N-p-coumaroyltyramine (11), cis-N-coumaroyltyramine (12), tryptophan (13), thymidine (14), adenosine (15), and deoxyadenosine (16). Compound 1 was synthesized, but not yet isolated from natural source, and compounds 2-16 were isolated for the first time from this plant source.

Introduction

Hosta longipes (Fr. et Sav.) Matsumura (Liliaceae), widely distributed throughout Korea, China, and Japan, is an edible vegetable in Korea. It has long been used as a traditional Korean medicine for treating cough, sputum, laryngopharyngitis, burns, swelling, snake bites and inflammation.1,2 Previous phytochemical investigations of this plant led to the isolation of steroidal saponins.3,4 In the course of our continuing search for biologically active components from Korean medicinal plants, we investigated the constituents of the leaves of H. longipes and reported the isolation of steroidal saponins and flavonoids and their anti-inflammatory effects.5,6 In our continuing study on this source, we further isolated sixteen compounds (1 - 16). Their structures were elucidated by physicochemical and spectroscopic methods. Compound 1 was isolated for the first time from nature and compounds 2 - 16 were isolated for the first time from this plant source.

 

Experimental

General experimental procedures – Optical rotations were measured on a Jasco P-1020 polarimeter in MeOH. IR spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer. HRFABMS were obtained on a JEOL JMS700 mass spectrometer. NMR spectra were recorded on a Varian UNITY INOVA 500 NMR spectrometer operating at 500 MHz (1H) and 125 MHz (13C) with chemical shifts given in ppm (δ). Preparative HPLC was conducted using a Gilson 306 pump with Shodex refractive index detector and Apollo Silica 5 μ column (250 × 22 mm i.d.). Silica gel 60 (Merck, 70 - 230 mesh and 230 - 400 mesh) was used for column chromatography. The packing material for molecular sieve column chromatography was Sephadex LH-20 (Pharmacia Co.). TLC was performed using Merck precoated silica gel F254 plates. Spots were detected on TLC under UV light or by heating after spraying with 10% H2SO4 in C2H5OH (v/v).

Plant materials – Leaves of H. longipes were collected in Taebaek City, Korea, in June 2010. The plant was identified by one of the authors (K. R. Lee). A voucher specimen (SKKU-NPL 1103) of the plant has been deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Extraction and isolation – Leaves of H. longipes (2.5 kg) were extracted with 80% MeOH at room temperature and filtered. The filtrate was evaporated under reduced pressure to give a MeOH extract (190 g), which was suspended in water (800 mL) and solvent-partitioned to give n-hexane (3 g), CHCl3 (14 g), EtOAc (3 g), and n-BuOH (24 g) layers. The n-hexane (3 g) layer was separated over a silica gel column (n-hexane: EtOAc = 7 : 1 – 1 : 1) to yield nine fractions (H1 – H9). Fraction H2 (370 mg) was chromatographed on an RP-C18 silica gel column (90% MeOH) to give three subfractions (H21 – H23). Subfraction H21 (100 mg) was purified over a silica gel semi-prep. HPLC (hexane : CHCl3 : EtOAc = 9 : 2 : 1) to afford compounds 2 (10 mg, Rt = 13.1 min) and 3 (3 mg, Rt = 16.0 min). Subfraction H22 (20 mg) was purified by an RP-C18 semi-prep. HPLC (95% MeCN) to afford compound 4 (8 mg, Rt = 18.7 min). Fraction H7 (150 mg) was purified with an RP-C18 silica Lobar A®-column (80% MeOH) and silica gel semi-prep. HPLC (hexane : EtOAc = 3 : 1) to afford compound 5 (6 mg, Rt = 11.0 min). The CHCl3 (14 g) layer was separated over a silica gel column (n-hexane : EtOAc = 7 : 1 – 1 : 1) to yield seven fractions (C1 – C7). Fraction C1 (1.0 g) was separated over an RP-C18 silica gel column (90% MeOH) and purified by a silica gel semiprep. HPLC (hexane : EtOAc = 2 : 1) to afford compound 1 (11 mg, Rt = 13.4 min). Fraction C2 (200 mg) was purified with an RP-C18 silica Lobar A®-column (90% MeOH) and silica gel semi-prep. HPLC (hexane : EtOAc = 7 : 1) to afford compound 6 (4 mg, Rt = 17.0 min). The EtOAc (3 g) layer was chromatographed over a Sephadex LH-20 column (90% MeOH) to yield nine fractions (E1 – E9). Fraction E2 (800 mg) separated over a silica gel column (CHCl3 :MeOH = 12 : 1) and further purified with RP-C18 semi-prep. HPLC (50% MeOH) to afford compounds 7 (2 mg, Rt = 11.8 min) and 8 (3 mg, Rt = 12.9 min). Fraction E3 (700 mg) was separated over an RP-C18 silica gel column (90% MeOH) to give six subfractions (E31 – E36). Subfraction E31 (200 mg) was purified with an RP-C18 silica Lobar A®-column (80% MeOH) and silica gel semi-prep. HPLC (CHCl3 :MeOH= 2 : 1) to afford compounds 9 (7 mg, Rt = 16.7 min), 14 (6 mg, Rt = 18.7 min), 15 (3 mg, Rt = 20.3 min), and 16 (3mg Rt = 25.3min). Subfraction E33 (80mg) was purified by an RP-C18 semi-prep. HPLC (30% MeCN) to afford compounds 11 (3 mg, Rt = 11.8 min) and 12 (3 mg, Rt = 10.4 min). Compound 10 (3 mg) was obtained by purification of subfraction E4 (210 mg) using a silica gel semiprep. HPLC (CHCl3 : MeOH :H2O = 9 : 2 : 0.2). Fraction E9 (100 mg) was purified with a silica gel semi-prep. HPLC (CHCl3:MeOH:H2O= 2 : 1 : 0.2) to yield compound 13 (7 mg, Rt = 15.0 min).

Methyl 10,10-dimethoxydecanoate (1) − Colorless gum. IR (KBr) νmax cm−1: 2951 (C-H), 1723 (C = O), 1284, 1032; 1H and 13C NMR: see Table 1; HRFABMS m/z 269.1733 [M+ Na]+; (calcd for C13H26O4 Na, 269.1729).

Table 1.aThe assignments were based on HMQC and HMBC experiments.

Methyl 10-hydroxy-8E,12Z-octadecadienoate (2) − Colorless gum. : −3.0 (c 0.25, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 5.67 (1H, dt, J = 15.5, 6.5 Hz, H- 12), 5.54 (1H, m, H-9), 5.48 (1H, m, H-13), 5.37 (1H, m, H-8), 4.08 (1H, m, H-10), 3.67 (3H, s, OCH3), 2.30 (2H, t, J = 7.5 Hz, H-2), 2.25 (2H, m, H-11), 2.04 (4H, m, H-7 and 14), 1.62 (2H, m, H-17), 1.31 - 1.40 (12H, m, H-3 to H-6, H-15, H-16), 0.90 (3H, t, J = 7.0 Hz, H-18); 13C NMR (CDCl3, 125 MHz): δ 174.5 (C-1), 133.4 (C-8), 132.5 (C-9), 132.4 (C-13), 125.0 (C-12), 72.7 (C-10), 51.6 (OCH3), 35.7 (C-11), 34.3 (C-7), 32.1 (C-2), 31.5 (C-16), 29.8 (C-4), 29.3 (C-5, C-6, C-15), 27.6 (C-14), 25.2 (C-3), 22.4 (C-17), 14.1 (C-18). FABMS m/z: 311.3 [M + H]+.

Methyl coriolate (3) − Colorless gum. : +10.2 (c 0.30, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 6.49 (1H, dd, J = 15.0, 10.5 Hz, H-11), 5.97 (1H, t, J = 10.5 Hz, H-10), 5.66 (1H, dd, J = 15.0, 7.0 Hz, H-12), 5.45 (1H, dt, J = 10.5, 8.0 Hz, H-9), 4.16 (1H, q, J = 7.0, H-13), 3.67 (3H, s, H-OCH3) 2.30 (2H, t, J = 7.5 Hz, H-2), 2.18 (2H, m, H-8), 1.26 - 1.62 (18H, m, H-3 to H-7, H-14 to H-17), 0.89 (3H, t, J = 7.5 Hz, H-18). FABMS m/z: 311.3 [M+H]+.

trans-Phytol (4) − Coloress oil. 1H NMR (500 MHz, CD3OD): δ 5.35 (1H, t, J = 7.0 Hz, H-2), 4.07 (2H, d, J = 6.5 Hz, H-1), 2.00 (2H, t, J = 6.5 Hz, H-4), 1.65 (3H, s, H-20), 1.52 - 1.05 (19H, m), 0.87 (9H, d, J = 6.5 Hz, H-16, 18, 19), 0.88 (3H, d, J = 6.5 Hz, H-17); 13C NMR (125 MHz, CD3OD): δ 138.4 (C-3), 123.6 (C-2), 58.2 (C- 1), 39.8 (C-4), 39.4 (C-14), 37.4 (C-8), 37.3 (C-10), 37.2 (C-12), 36.6 (C-6), 32.8 (C-7), 32.7 (C-11), 27.9 (C-15), 25.1 (C-5), 24.7 (C-13), 24.3 (C-9), 22.0 (C-17), 21.9 (C- 16), 19.1 (C-19), 19.0 (C-18), 15.0 (C-20). FABMS m/z: 319.3 [M + Na]+

Phytene-1,2-diol (5) − Coloress gum. 1H NMR (500 MHz, CD3OD): δ 5.07 (1H, s, H-20a), 4.89 (1H, s, H- 20b), 4.07 (1H, dd, J = 7.0, 3.5 Hz, H-2), 3.58 (1H, dd, J = 6.5, 4.0 Hz, H-1a), 3.44 (1H, dd, J = 6.5, 2.5 Hz, H-1b), 2.09 - 1.97 (2H, m, H-4), 1.57 - 1.06 (19H, m), 0.88 (9H, d, J = 7.0 Hz, H-16, 18, 20), 0.86 (3H, d, J = 7.0 Hz, H-19); 13C NMR (125 MHz, CD3OD): δ 149.6 (C-3), 109.6 (C-17), 75.4 (C-2), 65.3 (C-1), 39.3 (C-14), 37.3 (C-8), 37.2 (C-10, 12), 36.8 (C-6), 32.7 (C-4), 32.5 (C-7, 11), 27.9 (C-15), 25.5 (C-5), 24.6 (C-13), 24.3 (C-9), 21.9 (C-20), 21.8 (C-16), 19.0 (C-19), 18.9 (C-18). EIMS m/z: 312.3 [M]+.

Fig. 1.The structures of 1 - 16 isolated from H. longipes.

Phyton (6) − Colorless gum. : +2.1 (c 0.15, CHCl3); 1H NMR (500 MHz, CDCl3): δ 2.40 (2H, t, J = 7.5 Hz, H-3), 2.12 (3H, s, H-1), 1.03 - 1.58 (17H, m, H-4 to H-14), 0.85 (12H, d, J = 7.0 Hz, H-15 to H-18); 13C NMR (125 MHz, CDCl3): 209.5 (C-2), 44.4 (C-3), 39.6 (C-13), 37.6, 37.5, 37.5 and 36.7 (C-5, C-7, C-9, C-11) 33.0 and 32.9 (C-6, C-10), 30.0 (C-1), 28.2 (C-4), 25.0 (C-14), 24.6 (C-12), 22.9 (C-8), 22.8 and 21.7 (C-15, C- 18), 20.0 and 19.8 (C-16, C-17). FABMS m/z: 269.3 [M + H]+.

(3S,5R,6S,7E,9R)-7-Megastigmene-3,6,9-triol (7) − Amorphous powder. : −11.9 (c 0.15, CH3OH); 1H NMR (500 MHz, CD3OD): δ 5.72 (1H, dd, J = 16.0, 6.0 Hz, H-8), 5.55 (1H, dd, J = 16.0, 1.0 Hz, H-7), 4.29 (1H, m, H-9), 3.80 (1H, m, H-3), 1.93 (1H, m, H-5), 1.67 (1H, m, H-4a), 1.66 (1H, m, H-2a), 1.40 (1H, m, H-2b), 1.39 (1H, m, H-4b), 1.24 (3H, d, J = 6.0 Hz, H-10), 0.98 (3H, s, H-11),0.89 (3H, s, H-12), 0.81 (3H, d, J = 6.0 Hz, H-13); 13C NMR (125 MHz, CD3OD): δ 135.7 (C-8), 134.0 (C-7), 78.3 (C-6), 69.4 (C-9), 67.6 (C-3), 46.1 (C-2), 40.6 (C-1), 40.1 (C-4), 35.6 (C-5), 25.9 (C-11), 25.3 (C-12), 24.3 (C-10), 16.6 (C-13). FABMS m/z: 229.2 [M + H]+.

Fig. 2.Key HMBC (→) and 1H-1H COSY () correlations of 1.

(3S,5R,6S,9R)-3,6,9-Trihydroxymegastigman-7-ene (8) − Amorphous powder. : −15.9 (c 0.20, CH3OH). 1H-NMR (500 MHz, CD3OD): 5.73 (1H, dd, J = 16.0, 6.0 Hz, H-8), 5.55 (1H, dd, J = 16.0, 1.0 Hz, H-7), 4.29 (1H, m, H-9), 3.80 (1H, m, H-3), 1.94 (1H, m, H-5), 1.68 (1H, m, H-4a), 1.66 (1H, t, J = 12.0 Hz, H-2a), 1.40 (1H, ddd, J = 12.0, 4.0, 2.0 Hz, H-2b), 1.39 (1H, q, J = 12.0 Hz, H- 4b), 1.24 (3H, d, J = 6.0 Hz, H-10), 0.96 (3H, s, H-11), 0.86 (3H, s, H-12), 0.84 (3H, d, J = 7.0 Hz, H-13); 13C NMR (125 MHz, CD3OD): δ 135.6 (C-8), 133.9 (C-7), 78.1 (C-6), 69.3 (C-9), 67.5 (C-3), 40.5 (C-1), 40.0 (C-4), 35.5 (C-5), 25.9 (C-12), 25.2 (C-11), 24.2 (C-10), 16.5 (C-13). FABMS m/z: 229.2 [M + H]+.

Shikimic acid (9) − Amorphous powder. : −12.1 (c 0.20, CH3OH). 1H-NMR (500 MHz, CD3OD): δ 6.48 (1H, m, H-6), 4.28 (1H, t, J = 4.0 Hz, H-5), 3.90 (1H, m, H-4), 3.53 (1H, m, H-3), 2.81 (1H, dd, J = 18.0, 5.5 Hz, H-2a), 2.16 (1H, dd, J = 18.0, 4.0 Hz, H-2b); 13C-NMR (125 MHz, CD3OD): δ 174.2 (C-7), 136.7 (C-1), 130.4 (C-6), 73.2 (C-5), 67.4 (C-4), 66.8 (C-3), 33.4 (C-2). FABMS m/z: 175.1 [M + H]+.

p-Coumaramide (10) − Amorphous powder. 1H-NMR (500 MHz, CD3OD): δ 7.35 (2H, d, J = 8.0 Hz, H-2, H-6), 7.32 (1H, d, J = 16.0 Hz, H-7), 6.75 (2H, d, J = 8.0 Hz, H-3, H-5), 6.33 (1H, d, J = 16.0 Hz, H-8). FABMS m/z: 164.1 [M + H]+.

trans-N-p-Coumaroyltyramine (11) −Amorphous powder. 1H-NMR (500 MHz, CD3OD): δ 7.33 (1H, d, J = 15.5 Hz, H-7), 7.02 (1H, d, J = 1.5 Hz, H-2), 7.00 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.95 (1H, dd, J = 8.5, 1.5 Hz, H-6), 6.69 (1H, d, J = 8.5 Hz, H-5), 6.62 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.30 (1H, d, J = 15.5 Hz, H-8), 3.38 (2H, t, J = 7.5 Hz, H-8), 2.66 (2H, t, J = 7.5 Hz, H-7); 13C-NMR (125 MHz, CD3OD): δ 167.2 (C-9), 155.6 (C-4'), 147.5 (C-4), 145.5 (C-3), 141.0 (C-7), 131.0 (C-1), 130.7 (C-2', C-6'), 127.1 (C-1), 121.0 (C-6), 117.3 (C-8), 116.3 (C-5), 116.0 (C-3', C-5'), 114.1 (C-2), 42.0 (C-8'), 34.0 (C-7'). FABMS m/z: 284.2 [M + H]+.

cis-N-Coumaroyltyramine (12) − Amorphous powder. 1H-NMR (500 MHz, CD3OD): δ 7.26 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.91 (2H, d, J = 8.5 Hz, H-2, H-6), 6.65 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.62 (2H, d, J = 8.5 Hz, H-3, H-5), 6.51 (1H, d, J = 12.5 Hz, H-8), 5.69 (1H, d, J = 12.5 Hz, H-7), 3.29 (2H, t, J = 7.5 Hz, H-8'), 2.59 (2H, t, J = 7.5 Hz, H-7'); 13C-NMR (125 MHz, CD3OD): δ 170.4 (C-9), 159.4 (C-4), 156.9 (C-4'), 138.1 (C-7), 132.3 (C-2', C-6'), 131.2 (C-1'), 130.7 (C-2, C-6), 127.9 (C-1), 121.4 (C-8), 116.2 (C-3, C-5), 116.0 (C-3', C-5'), 42.3 (C-8'), 35.5 (C-7'). FABMS m/z: 284.2 [M + H]+.

Tryptophan (13) − Amorphous powder. 1H-NMR (500 MHz, DMSO-d6): δ 10.95 (1H, s, H-NH), 7.55 (1H, d, J = 7.5 Hz, H-4), 7.34 (1H, d, J = 7.5 Hz, H-7), 7.22 (1H, s, H-2), 7.05 (1H, t, J = 7.5 Hz, H-5), 6.96 (1H, t, J = 7.5 Hz, H-6), 3.53 (1H, m, H-12), 3.33 (1H, m, H-10a), 3.03 (1H, m, H-10b); 13C-NMR (125 MHz, DMSO-d6): δ 171.0 (C-12), 137.1 (C-8), 127.9 (C-9), 124.8 (C-2), 121.6 (C-4), 119.1 (C-5), 118.9 (C-6), 112.0 (C-7), 110.3 (C-3), 55.5 (C-11), 27.9 (C-10).

Thymidine (14) − Amorphous powder. 1H-NMR (500 MHz, CD3OD): δ 7.81 (1H, s, H-6), 6.28 (1H, t, J = 7.0 Hz, H-1'), 4.40 (1H, m, H-3'), 3.90 (1H, dd, J = 6.5, 3.5 Hz, H-4'), 3.79 (1H, dd, J = 12.0, 3.5 Hz, H-5'a), 3.72 (1H, dd, J = 12.0, 3.5 Hz, H-5'b), 2.20 (2H, m, H-2'), 1.88 (3H, s, H-7).

Adenosine (15) − Amorphous powder. 1H-NMR (500 MHz, CD3OD): δ 8.30 (1H, s, H-2), 8.18 (1H, s, H-8), 5.96 (1H, d, J = 6.0 Hz, H-1'), 4.74 (1H, t, J = 6.0 Hz, H-2'), 4.33 (1H, dd, J = 6.0, 2.0 Hz, H-3'), 4.17 (1H, m, H- 4'), 3.88 (1H, dd, J = 12.5, 2.5 Hz, H-5'a), 3.74 (1H, dd, J = 12.5, 3.0 Hz, H-5'b)

Deoxyadenosine (16) − Amorphous powder. 1H-NMR (500 MHz, CD3OD): δ 8.32 (1H, s, H-2), 8.18 (1H, s, H-8), 6.43 (1H, d, J = 7.0 Hz, H-1'), 4.58 (1H, m, H-3'), 4.07 (1H, m, H-4'), 3.84 (1H, dd, J = 12.5, 2.5 Hz, H-5'a), 3.74 (1H, dd, J = 12.5, 3.0 Hz, H-5'b), 2.80 (1H, m, H-2'a), 2.41 (1H, m, H-2'b)

 

Result and Discussion

Column chromatographic separation of the 80% MeOH extract from the leaves of H. longipes led to the isolation of known compounds 2 - 16, which were identified as methyl 10-hydroxy-8E,12Z-octadecadienoate (2),7 methyl coriolate (3),8 trans-phytol (4),9 phytene-1,2-diol (5),10 phyton (6),11 (3S,5R,6S,7E,9R)-7-megastigmene-3,6,9-triol (7),12 (3S,5R,6S,9R)-3,6,9-trihydroxymegastigman-7-ene (8),13 shikimic acid (9),14 p-coumaramide (10),15 trans-N-p-coumaroyltyramine (11),16 cis-N-coumaroyltyramine (12),16 tryptophan (13),17 thymidine (14),17 adenosine (15),17 and deoxyadenosine (16)17 by comparing the spectroscopic data. All compounds were isolated for the first time from this plant source. The following describes the structure elucidation of compound 1, which was synthesized18 but was not yet isolated from natural source. 269.1733 [M+ Na]+; (calcd for C13H26O4 Na, 269.1729).

Compound 1 was obtained as a colorless oil and had a molecular formula of C13H26O4, as determined from the ion peak [M+ Na]+ at m/z 269.1733 [M + Na]+; (calcd for C13H26O4 Na, 269.1729) in positive ion HRFABMS. The IR spectrum indicated that 1 possessed C-H bond (2951 cm−1) and carbonyl (1723 cm−1) groups. The 1H NMR spectrum showed an oxygenated methine [δH 4.34 (1H, t, J = 5.5 Hz, H-10)], three methoxy groups [δH 3.66 (3H, s, 1-OCH3), 3.30 (6H, s, 10-OCH3)], a methylene adjacent to carbonyl group [δH 2.29 (2H, t, J = 7.5 Hz, H-2)], and seven methylenes [δH 1.61 (2H, m, H-9), 1.59 (2H, m, H-3), 1.30-1.34 (10H, m, H-4 to H-8)]. The 13C NMR spectrum contained 13 signals, including a carboxylic carbon [δC 174.5 (C-1)], an acetal carbon [δC 104.8 (C-10)], three methoxy carbons [δC 52.8 (×2) (10-OCH3), 51.6 (1-OCH3)], and eight methylene carbons [δC 34.3 (C-2), 32.7 (C-9), 29.5 (C-7), 29.4 (C-4), 29.2 (C-6), 29.1 (C-5), 25.1 (C-8), and 24.7 (C-3)]. This spectroscopic data were very similar to those of methyl 8,8- dimethoxyoctanoate19 except that the presence of additional two methylene groups [δH 1.30-1.34; δC 29.2, 29.5]. The HMBC cross-peaks of 1-OCH3/C-1 and 10 OCH3/C-10 confirmed the location of three methoxy groups. Analyses of 1H-1H COSY, HMQC and HMBC spectra corroborated the gross structure of 1, which was elucidated as methyl 10,10-dimethoxydecanoate. Compound 1 was previously reported as a synthetic18 without NMR assignment. We isolated compound 1 from natural source and performed full NMR assignment of 1 for the first time. But we suggest that compound 1 could be an artifact because MeOH was used as solvent during purification.