The persimmon Diospyros kaki Thunb. is a fruit that is indigenous to Japan, and China, Korea, and Japan produce most of the fruits used for commercial consumption. Persimmons (Kaki) are also used traditionally for medicinal purposes, because of their preventive action against infection, diabetes, and arteriosclerosis, and because of their diuretic effect.1) The persimmon contains many compounds such as different sugars, starch, organic acids, ascorbic acid, tannins, flavonoids, carotenoids, triterpenoids, and fatty acids.2–7) With regard to its pharmacological activities, many studies have reported pharmacological effects including antioxidant activity and tyrosinase inhibitory activity.3,8–10) The peels of fresh astringent persimmons are usually removed and dried, which yields sweet dried persimmon peels. The peel was found to contain more polyphenols11) and carotenoids12) than the fruit body. The persimmon flesh contained large amounts of carotenoids, which are responsible for the color of the fruits.4,5) Unlike vitamin C and vitamin E, β-carotene and other carotenoids can scavenge singlet oxygen.13) Also, carotenoids are known to exist as free compounds or as fatty acid esters in plants. Because the peels of persimmons are usually discarded, we believe that it might be possible to recover the peels for industrial utilization or for use as a food supplement.
Breithaupt and Bamedi14) suggested, on the basis of HPLC results, the occurrence of carotenoid esters as β-cryptoxanthin ester and zeaxanthin ester in persimmons. Weller and Breithaupt15) also suggested, on the basis of LC/MS results, the presence of zeaxanthin esters as diesters of dipalmitate and of palmitate and stearate in persimmons. However, they did not isolate these compounds as pure single entities. Differences in the chemical structure of each carotenoid and its ester would lead to different pharmacological effects and physicochemical characteristics. For example, synthetic lutein myristate ester was more stable than free lutein after heat or UV treatment.16) Sugawara et al.17) found that Caco-2 cells easily took up more hydrophobic carotenoids than less hydrophobic carotenoids. One can easily envision that different carotenoids with or without ester moieties would have different polar characteristics and that differences in structure would lead to different biochemical activities or in vivo functions. Therefore, more detailed structural characterizations of each carotenoid and its esters, obtained by means of physical and chemical techniques such as high-resolution (HR) mass spectrometry (MS) and 1H- and 13C-NMR, are required. For this purpose, we isolated the functional components, the main carotenoids and their esters, and, after their purification via silica gel column chromatography, characterized their structures by using HR-FAB-MS, 1H-, and 13C-NMR.
SeparationDried and powdered persimmon peels (504.7 g) were extracted by refluxing with chloroform. After the chloroform was removed, the resulting extract (12.4 g) was used. Part of the extract (3.0 g) was separated via silica gel column chromatography (L=28 cm, ϕ=3 cm) and five components were obtained: compound 1 (50.2 mg), compound 2 (3.4 mg), compound 3 (96.2 mg), compound 4 (88.4 mg), and compound 5 (152.0 mg).
Structural Characterization of Compounds 1 to 5Compounds 1 and 2 were identified as β-carotene and lycopene, respectively, on the basis of Rf values obtained by TLC (solv. n-hexane–chloroform=3 : 1, Rf=0.80, 0.64) and 1H- and 13C-NMR.18,19)
Compound 3 was obtained as a red amorphous powder. The UV-VIS spectrum (n-hexane) demonstrated absorptions at λ=447.5 nm (ε=78400). The IR spectrum had absorptions at νmax (KBr) 1735 cm−1 (ester carbonyl group). Positive HR-FAB-MS had molecular ion peaks at m/z 763.6395 [M+H]+ (Calcd for C54H83O2: 763.6393) and m/z 789.6546 [M+H]+ (Calcd for C56H85O2: 789.6550) and in a ratio of ca. 6 : 1 intensity, respectively. The 1H-NMR spectrum (CDCl3) showed signals for a fatty acid moiety at δ 0.86 (terminal methyl group) and 1.24 (strong, long methylene chain), and a set of signals of the carotenoid moiety at δ 1.01, 1.06, 1.10, 1.71, and 1.97 (each s, methyl groups), 5.10 (m, oxygen-bearing methine proton), 5.35, 6.10, and 6.61 (each m, olefinic protons), and 6.34 and 6.35 (each 1H, d, J=14.9 Hz, olefinic protons). On the other hand, the 13C-NMR spectrum of compound 3 exhibited carbon signals for methyl, methylene, methine, quaternary carbons at δ 12.9–39.9 and 44.2, one oxygenated carbon at δ 68.2, many olefinic carbons at δ 124.4–138.8, and one ester carbonyl carbon at δ 173.7. From these data, compound 3 was estimated to be composed of a fatty acid and a carotenoid. Therefore, compound 3 was treated with 3% NaOH in MeOH at room temperature for 1 h. After neutralization with 1 m HCl–MeOH, the reaction mixture was concentrated to give a fraction that was subjected to silica gel chromatography with n-hexane–ethyl acetate=4 : 1 to obtain a carotenoid, compound 6, a major fatty acid derivative (compound 7), and minor fatty acid derivative (compound 7′).
Compound 6 was obtained as a red amorphous powder, and the UV-VIS spectrum (n-hexane) showed absorptions at λ=448.0 nm (ε=113800). Its HR-FAB-MS exhibited a quasi-molecular ion peak at m/z 553.4407 [M(C40H56O)+H]+. Measurements with 1H- and 13C-NMR for 1H–1H correlation spectroscopy (COSY), heteronuclear multiple-quantum coherence (HMQC), and heteronuclear multiple-bond connectivity (HMBC) allowed its structural assignments. The 1H-NMR spectrum of compound 6 was assigned as follows: δ 1.02 (s, H3-16′, 17′), 1.05 (s, H3-16, 17), 1.44 (m, H2-2′, H-2), 1.60 (m, H2-3′), 1.70 (s, H3-18′), 1.72 (s, H3-18), 1.77 (overlapped, Hb-2), 1.97 (s, H3-19, 19′, 20, 20′), 1.97–2.00 (overlapped, H2-4′, Ha-4), 2.32 (m, Hb-4), 3.98 (m, H-3), 6.09–6.16 (m, H-7, 7′, 8, 8′, 10, 10′), 6.25 (m, H-14, 14′), 6.33 (d, J=14.9 Hz, H-12′), 6.35 (d, J=14.9 Hz, H-12), and 6.61 (m, H-11, 11′, 15, 15′), which are identical to data for β-cryptoxanthin.20) The 13C-NMR spectrum confirmed that compound 6 is identical to β-cryptoxanthin composed of the following 40 signals at δ: 12.9 (C-19, 19′, 20, 20′), 19.4 (C-3′), 21.7 (C-18), 21.9 (C-18′), 29.1 (C-16), 29.5 (C-16′), 29.7 (C-17′), 30.4 (C-17), 33.2 (C-4′), 34.4 (C-1′), 37.2 (C-1), 39.7 (C-2′), 42.6 (C-4), 48.5 (C-2), 65.2 (C-3), 125.0 (C-11), 125.2 (C-11′), 125.6 (C-7), 126.2 (C-5), 126.8 (C-7′), 129.5 (C-5′), 130.0 (C-15′), 130.2 (C-15), 130.9 (C-10′), 131.4 (C-10), 132.5 (C-14′), 132.7 (C-14), 135.7 (C-9), 136.2 (C-9′), 136.2 (C-13′), 136.5 (C-13), 136.7 (C-12′), 137.3 (C-12) 137.7 (C-8′), 137.9 (C-6), 138.0 (C-6′), 138.6 (C-8). Moreover, compound 6 displayed the CD spectrum (n-hexane) at 222 nm (−2.70 mdeg), 248 (+2.53), 286 (−4.01), and 342 (+0.67). Taking into consideration of its UV and circular dichroism (CD) spectra, the structure of compound 6 was identified as all-E-(3R)-β-cryptoxanthin.
For compound 7, on the other hand, its HR-FAB-MS of major fatty acid moiety indicated at m/z 243.2323 [C15H30O2+H]+. The 1H-NMR spectrum (CDCl3) showed a simple pattern: a terminal methyl group at δ 0.87 (t, J=6.9 Hz), long methylene groups at δ 1.26, one methylene group adjacent to a carboxylic acid carbonyl function at δ 2.29 (t, J=7.5 Hz), and one methoxy group at δ 3.66 (s). The 13C-NMR spectrum showed many methyl and methylene carbon signals at δ 14.1, 22.8, 25.1, 29.3, 29.4, 29.5, 29.6, 29.8×4, 32.0, and 34.2; one methoxy carbon at δ 51.3; and one carboxylate carbonyl carbon at δ 174.2, which were identified as signals of myristic acid methyl ester.
For compound 7′, the HR-FAB-MS of minor fatty acid moiety indicated at m/z 269.2482 [C17H32O2+H]+. The 1H-NMR spectrum (CDCl3) showed a simple pattern: a terminal methyl group at δ 0.86 (t, J=7.2 Hz), long methylene groups at δ 1.28, one methylene group adjacent to a carboxylic acid carbonyl function at δ 2.28 (t, J=7.2 Hz), one methoxy group at δ 3.65 (s), and olefinic protons at δ 5.33 (m). The 13C-NMR spectrum showed many methyl and methylene carbon signals at δ 14.5, 23.0, 25.3, 27.5, 27.6, 29.3, 29.4, 29.5×2, 30.0, 30.1, 32.1, and 34.4; one methoxy carbon at δ 51.7; olefinic carbons at δ 129.8 and 130.1; and one carboxylate carbonyl carbon at δ 174.3, which were identified as signals of palmitoleic acid methyl ester.
Therefore, the structure of compound 3 was characterized as the ester of β-cryptoxanthin and the fatty acid constituted with myristic acid and palmitoleic acid (ca. 6 : 1) as shown in Fig. 1.
Fig. 1. Structures of Compound 3 and Compound 4
Although the existence of this ester between carotenoid and fatty acid was suggested by HPLC-MS data that were reported earlier,14) the actual characterization of this compound 3 are, as far we know, presented here for the first time.
Compound 4 was obtained as a redish-orange amorphous powder. The IR spectrum showed absorptions at νmax (KBr) 1732 cm−1 (ester carbonyl group), and UV-VIS spectrum (n-hexane) had absorptions at λ=445.0 nm (ε=42300). Positive HR-FAB-MS (m/z) showed molecular ion peaks at m/z 989.8326 [M+H]+ (Calcd for C68H109O4: 989.8326) and at m/z 1015.8486 [M+H]+ (Calcd for C70H111O4: 1015.8482), in a ratio of ca. 7 : 1 intensity, respectively. The 1H-NMR spectrum (CDCl3) showed signals of methyl groups at δ 0.85, 1.05, 1.09, 1.70, and 1.94; an oxygen-bearing methine proton at δ 5.04; and olefinic protons at δ 5.33, 6.08, 6.10, 6.12, 6.14, 6.24, 6.33, 6.34 (d, J=14.9 Hz), and 6.62. The 13C-NMR spectrum (CDCl3), however, showed methyl, methylene, methine, and quaternary carbon signals at δ 12.9, 14.2, 21.6, 22.8, 25.1, 32.0, 36.7, 38.8, and 44.2; an oxygen-bearing methine carbon at δ 68.2; olefinic carbons at δ 124.3, 125.4, 125.8, 130.1, 131.5, 132.7, 135.7, 136.6, 137.7, 137.9, and 138.7; and one carboxylic carbonyl carbon at δ 173.7. Compound 4 was also treated with 3% NaOH in MeOH at room temperature for 1 h. After neutralization, the reaction mixture was subjected to silica gel chromatography with n-hexane–ethyl acetate=2 : 1 to obtain a carotenoid, compound 8, and a fatty acid derivatives, compounds 7 and 7′.
Compound 8 was obtained as a red amorphous powder. The UV-VIS had absorptions at λ=445.0 nm (ε=115200). The HR-FAB-MS showed a quasi-molecular ion peak at m/z: 569.4356 [M(C40H56O2)+H]+. The 1H-NMR spectrum (CDCl3) of compound 8 was assigned as follows: δ 1.06 (s, H3-16, 16′, 17, 17′), 1.46 (t, H-2, 2′, J=12.0 Hz), 1.72 (s, H3-18, 18′), 1.75 (m, H-2, 2′), 1.97 (s, H3-19, 19′, 20, 20′), 2.05 (m, H-4, 4′), 2.37 (br dd, H-4, 4′, J=5.0, 16.9 Hz), 3.98 (m, H-3, 3′), 6.11 (m, H-7, 7′, 8, 8′), 6.34 (d, J=15.0 Hz, H-12, 12′), and 6.61 (m, H-11, 11′, 15, 15′). The 13C-NMR spectrum (CDCl3) showed signals at δ 12.9 (C-19, 19′), 12.9 (C-20, 20′), 21.7 (C-18, 18′), 28.8 (C-17, 17′), 30.4 (C-16, 16′), 37.2 (C-1, 1′), 42.6 (C-4, 4′), 48.5 (C-2, 2′), 65.2 (C-3, 3′), 125.0 (C-11, 11′), 125.7 (C-7, 7′), 126.3 (C-5, 5′), 130.2 (C-15, 15′), 131.7 (C-10, 10′), 132.7 (C-14, 14′), 135.8 (C-9, 9′), 136.6 (C-13, 13′), 137.7 (C-12, 12′), 137.8 (C-6, 6′), and 138.6 (C-8, 8′). These data confirm identification of compound 8 as zeaxanthin.21)
The CD spectrum (n-hexane) showed the peaks at 222 nm (−1.22 mdeg), 247 (+0.97), 284 (-1.56), and 341 (+0.34). Based on the above UV and CD spectra, the structure of compound 8 was identified with all-E-(3R,3′R)-zeaxanthin.
Therefore, the structure of compound 4 was characterized as the diester of zeaxanthin and the fatty acid, myristic acid, accompanied by a small of palmitoleic acid (ca. 1/7) as shown in Fig. 1.
Compound 5 was identical to oleanolic acid.22)
Therefore, this study reports the first separation and structural characterization of solvent-soluble components of persimmon peels. Biological studies of these compounds will be reported in the near future.
Experimental
General Experimental ProceduresThe IR spectra were measured with a JASCO FT/IT-4200 spectrometer, the UV-VIS spectra were recorded on a Shimadzu UV-2500PC detector, and CD was measured with a JASCO (Model J-720). The HR-FAB-MS was carried out on JEOL JMS-DX 300 and JMS-DX 303 HF spectrometers in an m-nitrobenzyl alcohol matrix in the positive ion mode. A matrix-assisted laser desorption-ionization time-of-flight mass spectrometer (Bruker AutoflexIITOF/TOF) was also used with a dithranol matrix in the positive ion mode. The 1H- and 13C-NMR spectra were measured in chloroform-d3 (for compounds 1–8, except for compound 5), and pyridine-d5 for compound 5, with a JEOL α-500 spectrometer. Chemical shifts were obtained on a δ (ppm) scale with tetramethylsilane as the internal standard. In this Experimental, the 1H- and 13C-NMR spectra data, except those for compound 5, are omitted because they appear earlier in the text. Column chromatography was performed with silica gel (Kieselgel 60, 230–400 mesh, Merck). TLC was performed on thin-layer silica gel plates (Kieselgel 60 F254, Merck AG). The TLC spots were visible or were visualized by using UV light (254/366 nm), exposure to I2 vapor, and spraying with 10% H2SO4, and spraying with 5% phosphorus molybdenum with EtOH followed by heating.
Extraction and SeparationDried and powdered persimmon peels (504.7 g) were extracted by refluxing with chloroform. After removal of chloroform, an extract (12.4 g) was obtained. Part of the extract (3 g) was applied to silica gel column chromatography [solvent: n-hexane–chloroform=5 : 1→1 : 1 (gradient)→CHCl3–MeOH=20 : 1], and five compounds were obtained: compound 1 (50.2 mg), compound 2 (3.4 mg), compound 3 (96.2 mg), compound 4 (88.4 mg), and compound 5 (152.0 mg).
Compounds 1 and 2TLC and 1H- and 13C-NMR identified compounds 1 and 2 as β-carotene and lycopene, respectively.
Compound 3Red amorphous powder, UV-VIS: λmaxn-hexane 447.5 (ε=78400). IR: νmax (KBr) 1735 cm−1 (ester carbonyl group) IR: νmax (KBr) 1735 cm−1 (ester carbonyl group). Positive HR-FAB-MS (m/z): 763.6395 [M+H]+ (Calcd for C54H83O2: 763.6393): 789.6546 [M+H]+ (Calcd for C56H85O2: 789.6550)=ca. 6 : 1 in intensity.
Alkaline Treatment of Compound 3Compound 3 (65 mg) was treated with 3% NaOH in MeOH at room temperature for 1 h. After neutralization with 1 m HCl–MeOH, the reaction mixture was concentrated to give a residue that was subjected to silica gel chromatography with n-hexane–ethyl acetate=4 : 1 to obtain a carotenoid, compound 6 (16 mg), and fatty acid derivatives, compounds 7 (11 mg) and 7′ (2 mg).
Compound 6Red amorphous powder, UV-VIS: λmaxn-hexane 448.0 (ε=113800). HR-FAB-MS (m/z): 553.4407 [M (C40H56O)+H]+.
Compound 7Colorless oil, HR-FAB-MS (m/z): 243.2323 [C15H30O2+H]+.
Compound 7Colorless oil, HR-FAB-MS (m/z): 269.2482 [C17H32O2+H]+.
Compound 4Redish-orange amorphous powder, UV-VIS: λmaxn-hexane 445.0 (ε=42300). IR: νmax (KBr) 1732 cm−1 (ester carbonyl group). Positive HR-FAB-MS (m/z): 989.8326 [M+H]+ (Calcd for C68H109O4: 989.8326): 1015.8486 [M+H]+ (Calcd for C70H111O4: 1015.8482)=ca. 7 : 1 in intensity.
Alkaline Treatment of Compound 4Compound 4 (55 mg) was treated in a manner similar to that used for compound 3. After neutralization with 1 m HCl–MeOH, the reaction mixture was concentrated and subjected to silica gel chromatography with n-hexane–ethyl acetate=2 : 1 to obtain a carotenoid, compound 8 (13 mg), and fatty acid derivatives, compound 7 (8 mg) and 7′ (1 mg).
Compound 8Redish-orange amorphous powder, UV-VIS: λmaxn-hexane 445.0 (ε=115200). HR-FAB-MS (m/z): 569.4356 [M (C40H56O2)+H]+.
Compound 5Colorless needles, mp >300°C, MS (m/z): 457 (M+H)+. 1H-NMR spectrum (pyridine-d5): δ 0.93 (3H, s, H3-25), 0.98 (3H, s, H3-29), 1.01 (6H, s, H3-24, 30), 1.04 (3H, s, H3-26), 1.26 (3H, s, H3-23), 1.29 (3H, s, H3-27), 3.45 (1H, dd, J=5.3, 15.0 Hz, H-3), and 5.49 (1H, br s, H-12). 13C-NMR spectrum (pyridine-d5): δ 15.5 (C-25), 16.4 (C-24), 17.3 (C-26), 18.6 (C-6), 23.5 (C-30), 23.6 (C-16), 23.7 (C-11), 26.0 (C-27), 28.1 (C-2), 28.5 (C-15), 28.6 (C-23), 30.9 (C-20) 31.9 (C-22), 31.9 (C-7), 33.0 (C-29), 34.0 (C-21), 37.3 (C-10) 38.9 (C-1), 39.3 (C-4), 39.8 (C-8), 41.8 (C-18), 42.0 (C-14), 47.9 (C-9), 47.9 (C-17), 47.9 (C-19), 55.6 (C-5), 77.9 (C-3), 122.9 (C-12), 150.0 (C-13), and 179.7 (C-28).
