Chromatographic Isolation and Spectroscopic Identification of Phytoconstituents of Jujuba Seeds (Zizyphus jujuba Mill.).

BACKGROUND: The seeds of Zizyphus jujuba Mill. (Rhamnaceae) are astringent, aphrodisiac, tonic; used to cure cough, asthma, vomiting, burning sensation, biliousness, leucorrhoea, and eye infections in traditional systems of medicine.
MATERIALS AND METHODS: The methanol extract of seeds of Z. jujuba was partitioned into petroleum ether and water soluble fractions. Isolation of compounds was performed by silica gel column chromatography. The structures of isolated compounds were established on the basis of spectral studies and chemical reactions.
RESULTS: Chromatographic separation of methanolic extract of seeds yielded three new phyto-constituents characterized as 3, 5, 7-trimethoxy-8, 3', 4', 5'-tetrahydroxy flavone-6-oxy hexahydrobisabolene ether (4), 1, 9-dihydroxy tetrahydrogeranyl-8-oxy-O-β-D-glucuronopyranoside (5) and terahydrogeranyl-8-oxy-O-β-D-glucuronopyranosyl (2a→1b)-O-β-D-glucofuranosyl (2b→1c)-O-β-D-glucofuranosyl (2c→1d)-O-β-D-glucopyranosyl (2d→1e)-O-β-D-glucopyranosyl (2c→f)-O-β-D-glucopyranosyl-2f-benzoate (6) along with five known compounds, palmitoyl palmitoleoyl arachidoyl glyceride (1), tetratriacontenoic acid (2), palmitoyl oleoyl linolenoyl glyceride (3), hexanyl tetraglucoside (7) and pentasaccharide (8).
CONCLUSION: This is the first report of saturated monoterpene and sesquiterpene derivatives from jujuba seeds.

INTRODUCTION

Zizyphus jujuba Mill. (Rhamnaceae) is a thorny sub-deciduous plant, cultivated in China for more than 4000 years.[1] It is distributed mainly in tropical and subtropical parts of the world and has been employed as an essential oriental folk medicine for thousands of years.[23] The ripe fruit of Z. jujuba is nutritive, emollient, aphrodisiac, tonic, laxative, antiemetic, and appetizer. Fruits are consumed in Asian countries as a food, food additive and flavoring agent due to its high nutritional value.[4] The seeds of Z. jujuba are tonic, tranquillizer, analgesic, anticonvulsant, aphrodisiac, astringent; and are used to cure cough, asthma, vomiting, burning sensation, biliousness, leucorrhea, and eye infections in traditional systems of medicine.[5] It is one of the most popular traditional Chinese medicines extensively used for the treatment of anxiety, insomnia, dreaminess, and night sweats.[6] Recent pharmacological studies indicated that it possesses anti-inflammatory,[78] hepatoprotective,[9] antimicrobial,[1011] anxiolytic,[1213] and hair growth promoting[14] effects. Bioactive essential oil,[81114] saponins,[151617181920] phenolic compounds,[2122] alkaloids,[23] and polysaccharides[2425] have been reported from the seeds of Z. jujuba. Some analytical methods for the estimation of saponins and fatty acids are also reported.[2627] The present manuscript reports the chromatographic isolation and spectroscopic identification of new flavone bisabolenyl ether and geranyl glycosides along with known glycerides, fatty acid, and a pentasaccharide from the seeds of Z. jujuba of Indian origin.

MATERIALS AND METHODS

General

Melting points were determined on a perfit apparatus and are reported without correction. The infrared (IR) spectra were measured in KBr pellets on a Bio-Rad Fourier transform IR (FT-IR) spectrometer. Ultraviolet (UV) spectra were obtained in methanol with a Lambda Bio 20 spectrometer. 1H (400 MHz) and 13C (100 MHz) Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker spectrospin spectrometer. CDCl3 and DMSO-d (Sigma-Aldrich, Bengaluru, India) were used as solvents and tetramethylsilane as an internal standard. Electrospray ionization mass spectrometry analyses were performed on a Waters Q-TOF Premier (Micromass MS Technologies, Manchester, UK) Mass Spectrometer. Column chromatography separations were carried out on silica gel (Merck, 60-120 mesh, Mumbai, India). Precoated silica gel plates (Merck, Silica gel 60 F254) were used for analytical thin layer chromatography (TLC) visualized by exposure to iodine vapors and UV radiations.

Plant material

The dried fruits of Z. jujuba were procured from the local market of Delhi, and identified by Dr. H. B. Singh, Scientist F and Head, Raw Materials Herbarium and Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. A voucher specimen was deposited in the herbarium of NISCAIR with a reference number NISCAIR/RHMD/Consult/-2011-12/1808/108.

Extraction and isolation

The seeds of Z. jujuba (1 kg) were dried in oven at a temperature below 45°C for 5 h. Seeds were coarsely powdered and extracted with methanol using a Soxhlet apparatus for 48 h. The extract was filtered and the filtrate was concentrated in vacuo at 40°C to yield a yellowish green, viscous mass (18.25%). The extract (100 g) was suspended in distilled water (500 ml) and partitioned with petroleum ether (500 ml) thrice, successively to give petroleum ether (23.18 g) and water soluble (76.82 g) fractions. The petroleum ether soluble fraction was subjected to silica gel column chromatography (column A) eluting with petroleum ether-chloroform (1:1 and 1:3) mixtures to obtain compounds 1-3. The aqueous fraction was dried using rotary evaporator under reduced pressure at 50°C, dissolved in minimum amount of methanol to adsorbed on silica gel (60–120 mesh). It was air dried and subjected to silica gel column loaded in chloroform. The column was eluted with chloroform-methanol (19:1, 9:1 and 7:3) mixtures to obtain compounds 4–8. Analytical TLC was used to check the homogeneity of eluted fractions.

Palmitoyl palmitoleoyl arachidoyl glyceride (1)

Elution of column with petroleum ether-chloroform (1:1) furnished pale yellow mass of 1, 522 mg (0.52%); R 0.68 (CHCl3); m.p. 113–114°C; UV λmax (MeOH): 224, 276 nm; IR vmax (KBr): 2924, 2854, 1739, 1455, 1261, 1162, 1092, 1024, 800, 723 cm−1; 1H NMR (CDCl3): δ 5.33 (1H, m, H-5′), 5.32 (1H, m, H-8′), 5.31 (1H, m, H-6′), 5.29 (1H, m, H-11′), 5.28 (1H, m, H-14′), 5.27 (1H, m, H-9′′), 5.26 (2H, m, H-9′, H-12′), 5.25 (2H, m, H-15′, H-10′′), 5.19 (1H, m, H-2), 4.28 (2H, dd, J = 4.4, 11.6 Hz, H-1a, 3a), 4.10 (2H, dd, J = 5.6, 11.6 Hz, H-1b, 3b), 0.86 (3H, t, J = 6.8 Hz, Me-16′′), 0.84 (3H, t, J = 6.0 Hz, Me-20′), 0.82 (3H, t, J = 6.2 Hz, Me-16′′′); 13C NMR (CDCl3): δ 173.2 (C-1′, C-1′′′), 173.1 (C-1′′), 140.77 (C-5′), 139.17 (C-6′′), 132.41 (C-8′), 130.86 (C-9′′), 130.17 (C-11′), 129.98 (C-14′), 129.28 (C-9′), 128.09 (C-12′), 127.91 (C-15′), 114.09 (C-10′′), 68.91 (C-2), 62.09 (C-1, C-3), 34.18 (C-2′), 34.01 (C-2′′′), 33.96 (C-2′′), 31.92 (3 × CH2), 29.71 (16 × CH2), 29.63 (2 × CH2), 29.53 (3 × CH2), 29.37 (4 × CH2), 28.20 (C-4′, C-16′), 27.22 (C-5”, C-11”), 25.64 (3 × CH2), 24.87 (C-3′, C-3′′, C-3′′′), 22.69 (C-19′, C-15′′, C-15′′′), 14.12 (Me-20′, Me-16′′, Me-16′′′); ES-MS m/z (rel. int.): 850 [M]+ (C55H94O6) (N.O.), 599 (2.1), 303 (3.5), 301 (100), 251 (16.1).

Tetratriacontenoic acid (2)

Elution of column with petroleum ether-chloroform (1:3) afforded yellowish viscous mass of 2, 671 mg (0.67%); Rf0.61 (CHCl3); m.p. 71–73°C; UV λmax (MeOH): 224, 276 nm; IR vmax (KBr): 2924, 2854, 1708, 1455, 1278, 1115, 917, 722 cm−1; 1H NMR (CDCl3): δ 5.34 (4H, m, H-5, H-8, H-11, H-14), 5.07 (2H, m, H-6, H-9), 5.04 (2H, m, H-12, H-15), 4.09 (1H, brs, H-2a), 3.93 (1H, brs, H-2b), 2.78 (2H, m, H2-7), 2.75 (2H, m, H2-10), 2.35 (2H, m, H2-13), 2.33 (2H, m, H2-3), 2.26 (2H, m, H2-4), 2.03 (2H, m, H2-16), 1.28 (32H, brs, 16 × CH2), 0.88 (3H, t, J = 6.8 Hz, Me-34); 13C NMR (CDCl3): δ 180.13 (C-1), 139.23 (C-5), 130.20 (C-8), 130.01 (C-11), 129.87 (C-14), 128.73 (C-6), 127.92 (C-9), 124.01 (C-12), 114.10 (C-15), 34.10 (C-2), 31.94, (C-15), 31.64 (C-4), 29.61 (16 × CH2), 14.13 (C-34); ES-MS m/z (rel. int.): 501 [M + 1]+ (C34H61O2) (100), 457 (98.2).

Palmitoyl oleoyl linolenoyl glyceride (3)

Elution of column with petroleum ether-chloroform (1:3) yielded dark brown semisolid mass of 3, 864 mg (0.86%); Rf 0.52 (CHCl3); m.p. 77–79°C; UV λmax (MeOH): 203, 226 nm; IR vmax (KBr): 2924, 2854, 1708, 1508, 1446, 1376, 1271, 990, 810, 665 cm−1; 1H NMR (CDCl3): δ 5.37 (1H, m, H-6′), 5.35 (1H, m, H-9′), 5.34 (1H, m, H-12′), 5.32 (1H, m, H-9′′), 5.29 (1H, m, H-7′), 5.27 (1H, m, H-10′), 5.26 (1H, m, H-13′), 5.24 (1H, m, H-10′′), 5.20 (1H, brs, H-2), 4.26 (2H, dd, J = 4.5, 10.0 Hz, H-1a, H-3a), 4.06 (2H, dd, J = 5.6, 10.0 Hz, H-1b, H-3b), 0.89 (3H, t, J = 6.5 Hz, Me-18′′), 0.88 (3H, t, J = 6.2 Hz, Me-18′), 0.86 (3H, t, J = 6.2 Hz, Me-16′′′); 13C NMR (CDCl3): δ 178.5 (C-1′), 176.4 (C-1′′), 175.3 (C-1′′′), 142.0 (C-6′), 139.2 (C-9′), 135.9 (C-12′), 135.1 (C-9′′), 130.2 (C-7′), 129.8 (C-10′), 128.0 (C-13′), 123.9 (C-10′′), 170.0 (C-9′′), 130.2 (C-7′), 129.8 (C-10′), 128.0 (C-13′), 123.9 (C-10′′), 70.0 (C-2), 64.4 (C-1), 62.1 (C-3), 33.8 (C-3′, C-3′′′), 33.5 (C-3′′), 31.9 (C-16′, C-16′′, C-14′′′), 31.5 (CH2), 30.17 (CH2), 29.7 (16 × CH2), 24.4 (C-17′, C-17”, C-15′′′), 14.1 (Me-18′, Me-18′′, Me-16′′′); ES-MS m/z (rel. int.): 854 [M]+ (C55H98O6) (N.O.), 297 (11.5), 281 (28.4), 277 (32.4), 255 (30.5).

Zizabisabolanyl flavone (4)

Elution of column with chloroform-methanol (19:1) furnished pale yellow mass of 4, recrystallize from acetone, 44 mg (0.044%); Rf0.22 (CHCl3-MeOH, 9:1); m.p. 134–135°C; UV λmax (MeOH): 227, 275, 341 nm; IR vmax (KBr): 3366, 2925, 2855, 2368, 2363, 1707, 1605, 1513, 1456, 1375, 1274 cm−1; 1H NMR (DMSO-d): δ 7.72 (1H, d, J = 7.2 Hz, H-2′), 7.37 (1H, d, J = 7.2 Hz, H-6′), 3.88 (1H, brs, H-2′′), 3.49 (3H, s, OCH3), 3.16 (3H, s, OCH3), 3.09 (3H, s, OCH3), 2.92 (2H, m, H-5′′), 2.90 (1H, m, H-6′′), 2.48 (1H, m, H-7′′), 2.46 (2H, m, H-1′′), 2.25 (2H, m, H-9′′), 2.02 (2H, m, H-8′′), 1.93 (2H, m, H-4′′), 1.39 (1H, m, H-3′′), 1.73 (2H, m, H-10′′), 1.27 (1H, m, H-11′′), 1.09 (3H, d, J = 5.7 Hz, H-15′′), 0.98 (3H, d, J = 6.2 Hz, H-14′′), 0.74 (3H, d, J = 6.6 Hz, H-12′′), 0.69 (3H, d, J = 6.6 Hz, H-13′′); 13C NMR (DMSO-d): δ 174.59 (C-4), 167.39 (C-7), 166.43 (C-5), 166.38 (C-2), 161.52 (C-4′), 160.43 (C-9), 145.62 (C-3′), 145.62 (C-5′), 138.57 (C-6), 132.91 (C-3), 129.12 (C-2′), 128.61 (C-6′), 119.33 (C-1′), 114.70 (C-10), 108.54 (C-8), 71.20 (C-2′′), 59.74 (OCH3), 55.98 (OCH3), 51.54 (OCH3), 48.66 (C-5′′), 43.96 (C-6′′), 33.72 (C-8′′), 32.58 (C-10′′), 31.38 (C-7′′), 30.44 (C-3′′), 29.04 (C-4′′), 28.80 (C-1′′), 26.71 (C-9′′), 25.61 (C-11′′), 22.18 (C-13′′), 18.92 (C-12′′), 15.01 (C-15′′), 14.13 (C-14′′); ES-MS m/z (rel.int.): 600 [M]+ (C33H44O10) (N.O.), 375 [C18H15O9]+ (4.5), 225 [C15H29O]+ (1.8), 209 (1.1), 185 (2.5), 183 (100), 168 (11.5), 137 (9.4), 124 (45.3).

Geranyl glucoside (5)

Elution of the column with chloroform-methanol (9:1) afforded yellowish mass of 5, recrystallized from acetone-methanol (1:1), 78.02 mg (0.078%); Rf0.75 (CHCl3-MeOH, 1:1); m.p. 72–73°C; UV λmax (MeOH): 232, 275 nm; IR vmax (KBr): 3376, 2934, 1717, 1601, 1509, 1457, 1327, 1267, 1124, 1038, 876 cm−1; 1H NMR (CDCl3): δ 5.07 (1H, d, J = 7.3 Hz, H-1′), 4.47 (1H, m, H-4′), 4.43 (1H, m, H-2′), 4.38 (1H, m, H-5′), 4.25 (1H, m, H-3′), 3.58 (2H, brs, H2-9), 3.54 (2H, brs, H2-1), 2.62 (1H, brs, H-2), 1.69 (2H, brs, H2-3), 1.58 (2H, m, H2-4), 1.35 (4H, m, H2-5, H2-7), 0.91 (3H, d, J = 6.3 Hz, Me-10); 13C NMR (CDCl3): δ 171.21 (C-6′), 107.83 (C-1′), 84.81 (C-2′), 83.04 (C-4′), 77.33 (C-5′), 72.95 (C-3′), 63.54 (C-8), 61.73 (C-1), 59.94 (C-9), 55.98 (C-6), 48.46 (C-2), 31.42 (C-3), 29.48 (C-4), 23.51 (C-5), 21.89 (C-7), 14.43 (C-10); ES-MS m/z (rel. int.): 366 [M]+ (C16H30O9) (2.3).

Geranyl glycosyl benzoate (6)

Elution of the column with chloroform-methanol (9:1) yielded yellow solid mass of 6, recrystallized from acetone-chloroform (1:2), 52.2 mg (0.052%); Rf0.66 (CHCl3-MeOH, 8.5:1.5); m.p. 62–64°C; UV λmax (MeOH): 227, 275 nm; IR vmax (KBr): 3373, 2922, 2853, 1718, 1608, 1509, 1455, 1221, 1039, 653 cm−1; 1H NMR (DMSO-d): δ 7.87 (2H, dd, J = 7.3, 2.1 Hz, H-3′, H-5′), 7.64 (1H, m, H-4′), 6.43 (2H, d, J = 2.1 Hz, H-2′, H-6′), 3.30 (2H, brs, H2-8), 2.85 (1H, m, H-6), 2.62 (1H, brs, H-2), 1.69 (2H, brs, H2-3), 1.58 (2H, m, H2-4), 1.35 (4H, m, H2-5, H2-7), 1.02 (6H, d, J = 6.6 Hz, Me-1, Me-9), 0.91 (3H, t, J = 6.3 Hz, Me-10); 13C NMR (DMSO-d): δ 174.13 (C-6a), 167.93 (C-7′), 145.85 (C-1′), 138.43 (C-2′), 135.41 (C-5′), 123.45 (C-3′), 120.89 (C-6′), 118.51 (C-4′), 110.50 (C-1b), 109.51 (C-1a), 107.82 (C-1c), 104.45 (C-1d), 102.45 (C-1e), 100.55 (C-1f), 83.38 (C-4a), 82.35 (C-4c), 81.80 (C-2a), 81.51 (C-4b), 81.22 (C-2b), 80.81 (C-2c), 79.81 (C-2d), 77.33 (C-2f), 77.03 (C-2e), 76.14 (C-5a), 75.77 (C-5b), 75.66 (C-5c), 72.94 (C-5d), 70.36 (C-5e), 70.28 (C-5f), 69.66 (C-4d), 69.33 (C-4e), 69.06 (C-4f), 64.23 (C-3b), 64.02 (C-3a), 64.01 (C-3c), 63.53 (C-3d), 63.41 (C-3e), 63.37 (C-3f), 62.03 (C-8), 61.72 (C-6b), 61.53 (C-6c), 61.36 (C-6d), 60.51 (C-6e), 59.92 (C-6f), 55.01 (C-6), 34.94 (C-2), 32.00 (C-3), 31.61 (C-4), 30.20 (C-5), 29.35 (C-7), 22.56 (C-1), 20.92 (C-9), 14.42 (C-10); ES-MS m/z (rel. int.): 1248 [M]+ C53H84O33.

Hexanyl tetraglucoside (7)

Elution of the column with chloroform-methanol (7:3) furnished pale yellow amorphous powder of 7, recrystallized from methanol, 59 mg (0.059%); Rf0.72 (C6H6-MeOH; 8.5:1.5); m.p. 132–134°C (decomp.); UV λmax (MeOH): 205, 227, 275 nm; IR vmax (KBr): 3368, 2930, 2360, 1610, 1511, 1414, 1060, 818, 777 cm−1; 1H NMR (DMSO-d): δ 5.07 (1H, m, H-1′), 5.05 (1H, m, H-1′′), 5.01 (1H, m, H-1′′′), 4.94 (1H, m, H-1′′′′), 4.70 (1H, m, H-4′), 4.43 (1H, m, H-2′′), 4.41 (1H, m, H-2′′′), 4.32 (1H, m, H-2′′′′), 3.93 (1H, m, H-5′′), 3.94 (1H, m, H-5′′′), 3.91 (1H, m, H-5′′′′), 3.74 (1H, m, H-2′, H-5′), 3.71 (1H, m, H-4′′), 3.70 (1H, m, H-4′′′), 3.60 (1H, m, H-4′′′′), 3.54 (2H, brs, H-1), 3.52 (1H, m, H-3′), 3.50 (1H, m, H-3′′), 3.48 (1H, m, H-3′′′), 3.37 (1H, m, H-3′′′′), 3.31 (2H, brs, H2-6′), 3.29 (2H, brs, H2-6′′), 3.27 (2H, d, J = 8.4 Hz, H2-6′′′), 3.12 (2H, d, J = 7.5, H-6′′′′), 1.56 (2H, m, H-2), 1.29 (2H, brs, H-3, H-4), 1.25 (2H, m, H-5), 0.94 (3H, t, J = 6.5 Hz, CH3); 13C NMR (DMSO-d): δ 101.85 (C-1′), 97.60 (C-1′′), 96.68 (C-1′′′), 92.13 (C-1′′′′), 81.95 (C-4′), 78.53 (C-2′′), 78.46 (C-2′), 78.10 (C-2′′′), 77.67 (C-2′′′′), 77.17 (C-5′), 76.51 (C-5′′), 75.03 (C-5′′′), 74.59 (C-5′′′′), 73.25 (C-4′′), 72.20 (C-4′′′), 71.31 (C-4′′′′), 70.43 (C-3′′′′), 70.26 (C-3′′′), 70.06 (C-3′′), 69.11 (C-3′), 63.65 (C-6′), 62.89 (C-6′′), 61.99 (C-6′′′), 60.81 (C-6′′′′); 48.94 (C-1), 31.04 (C-2), 29.06 (C-3, C-4), 22.11 (C-5), 14.01 (C-6); ES-MS m/z (rel. int.): 750 [M]+ (C30H54O21) (N.O.), 179 (51.5), 163 (25.1), 158 (27.1), 142 (49.8).

Pentasaccharide (8)

Elution of the column with chloroform-methanol (7:3) yielded pale yellow amorphous mass of 8, recrystallized from methanol-acetone (1:1), 260 mg (0.26%); Rf0.76 (MeOH); m.p. 102°C (decomp.); UV λmax (MeOH): 230, 274 nm; IR vmax (KBr): 3364, 2926, 2119, 1636, 1363, 1058 cm−1; 1H NMR (DMSO-d): δ 5.30 (1H, brs, H-1′′), 5.30 (1H, brs, H-1′′′), 5.04 (1H, m, H-5), 4.94 (1H, brs, H-1′′′′), 4.92 (1H, brs, H-1′), 4.90 (1H, brs, H-1), 4.52 (1H, m, H-4′′′), 4.50 (1H, m, H-4′), 4.36 (1H, m, H-4′′), 4.29 (1H, m, H-4), 4.27 (1H, m, H-4′′′′), 4.01 (1H, m, H-5′), 3.98 (1H, m, H-5′′), 3.95 (1H, m, H-5′′′), 3.91 (1H, m, H-5′′′′), 3.84 (1H, m, H-2), 3.77 (1H, m, H-2′), 3.68 (1H, m, H-2′′), 3.66 (1H, m, H-2′′′), 3.55 (1H, m, H-2′′′′), 3.48 (1H, m, H-3), 3.46 (1H, m, H-3′), 3.40 (1H, m, H-3′′), 3.33 (1H, m, H-3′′′), 3.26 (1H, m, H-3′′′′), 3.17 (2H, d, J = 8.4 Hz, H-6), 3.13 (2H, d, J = 8.7 Hz, H-6′), 3.06 (2H, d, J = 8.1 Hz, H-6′′), 3.00 (2H, d, J = 8.1 Hz, H-6′′′), 2.92 (2H, d, J = 8.1 Hz, H-6′′′′); 13C NMR (DMSO-d): δ 104.32 (C-1′′′′), 102.15 (C-1′′′), 98.25 (C-1′′), 97.03 (C-1′), 92.39 (C-1), 83.05 (C-4′′), 83.01 (C-4′′′), 82.01 (C-4′), 81.02 (C-4), 76.87 (C-4′′′′), 76.77 (C-2), 75.92 (C-2′), 75.83 (C-2′′), 75.44 (C-2′′′), 74.99 (C-2′′′′), 74.65 (C-5′′′), 73.51 (C-5′′′′), 73.25 (C-5), 72.50 (C-5′), 72.11 (C-5′′), 71.07 (C-3′′), 70.70 (C-3), 70.43 (C-3′), 69.38 (C-3′′′), 67.91 (C-3′′′′), 64.49 (C-6), 63.87 (C-6′), 63.24 (C-6′′), 63.11 (C-6′′′), 61.36 (C-6′′′′); ES-MS m/z (rel. int.): 828 [M]+ (C30H52O26) (N.O.), 179 (12.5), 163 (2.5), 158 (47.3), 142 (12.1).

RESULTS AND DISCUSSION

Compounds 1-3, 7 and 8 are known phytoconstituents characterized as palmitoyl palmitoleoyl arachidoyl glyceride, tetratriacontenoic acid, palmitoyl oleoyl linolenoyl glyceride, hexanyl tetraglucoside, pentasaccharide, respectively [Figure 1]. The fatty acids and their glycerides were identified on the basis of spectral comparison with related compounds.[282930]

Figure 1

Structure of phytoconstituents 1-7 isolated from the methanol extract of the jujuba seeds (Zizyphus jujuba Mill.)

Compound 4, designated as zizabisabolanyl flavone, was obtained as pale yellow mass from chloroform-methanol (19:1) eluents. Its FT-IR spectra displayed absorption bands characteristic of hydroxyl group (3366 cm−1), carbonyl group (1707 cm−1) and aromatic moiety (1605, 1513 cm−1). Its UV spectrum showed strong absorption peak at 227 nm and a short neck at 275 characteristic of a flavonoid moiety. On the basis mass spectrum and 13C NMR data, its molecular weight was determined at m/z 600 consistent with the molecular formula C33H44O10. Important fragment peaks at m/z 375 [C18H15O9]+ and 209 (C15H29]+ indicated the presence of a flavonoids and a sesquiterpene in compound 4. The flavonoid moiety displayed typical retro-Diel Alder fragmentation pattern to yield base ion peak at m/z 183 [C9H10O4]+ and subsequent peaks at m/z 168 [(185-OH)]+ and 137 (168-OMe)]+ indicating the presence of three hydroxyl groups in ring C and methoxy group at C-3. The 1H NMR spectrum of 4 displayed two downfield doublets, one proton each, at δ 7.72 and 7.37 (J = 7.2 Hz, each) assignable to ortho-coupled H-2‘ and H-6‘ aromatic protons. A one-proton broad singlet at δ 3.88 was ascribed to H-2” carbniol proton. Three singlets at δ 3.49, 3.16 and 3.09, each integrating for three protons, were ascribed to methyl protons. The remaining methylene protons of the sesquiterpenic unit resonated between δ 2.92 and 1.27. Four three-proton doublets at δ 0.74 (J = 6.6 Hz), 0.69 (J = 6.6 Hz), 0.98 (J = 6.2 Hz) and 1.09 (J = 5.7 Hz) were attributed to Me-12”, Me-13”, Me-14” and Me-15” protons, respectively. The 13C NMR spectrum of 4 exhibited important signals for carbonyl carbon at δ 174.59 (C-4); aromatic carbons from δ 114.70 to 167.39; oxygenated methine carbon at δ 71.20 (C-2”); methoxy carbons at δ 51.54, 55.98, 59.74 and four methyl carbons at 22.18, 18.92, 15.01 and 14.13. On the basis of above spectral evidences the structure of 4 was elucidated as 3, 5, 7-trimethoxy-8, 3‘, 4‘, 5‘-tetrahydroxy flavone-6-oxy hexahydrobisabolene ether [Figure 1]. This is the first report of flavone ether from this plant.

Compound 5, designated as geranyl glucoside, was obtained as yellowish mass from chloroform-methanol (9:1) eluents. Its FT-IR spectrum showed absorption bands characterized of hydroxyl group (3376 cm−1). Its mass spectrum displayed as molecular ion peak at m/z 366 consistent with the molecular formula C16H30O9 of a geranyl glycoside. Its formula indicated the presence of two-double bond equivalents, one of which was adjusted in a sugar ring and other in an ester group. The 1H NMR spectrum of 5 displayed a one-proton doublet at δ 5.07 (J = 7.3 Hz) assigned to anomeric proton H-1′. The remaining sugar proton appeared at δ 4.47 (H-4′), 4.43 (H-2′) and 4.38 (H-5′). Three two-proton broad signals at δ 3.58, 3.28 and 3.54 were ascribed correspondingly to H2-9, H2-8 and H2-1 oxygenated methylene protons. Two one-proton signals at δ 2.85 and 2.62 were attributed to H-6 and H-2 methine protons, respectively. The remaining methylene protons resonated from δ 1.69 to 1.35. A three-proton doublet at δ 0.91 (J = 6.3 Hz) was assigned to Me-10 secondary methyl protons. The 13C NMR spectrum of 5 exhibited important signals for carboxy carbon at δ 171.21 (C-6′) and anomeric carbon at δ 107.83 (C-1‘). The remaining sugar carbons resonated between δ 84.81 and 72.95. The oxygenated methylene carbons of the geranyl moiety resonated at δ 63.54 (C-8), 61.73 (C-1) and 59.94 (C-9) while as the methine carbons appeared at δ 55.98 (C-6), 48.46 (C-2). The remaining methylene carbons appeared at δ 31.42 to 21.89. On the basis of above discussion the structure of 5 was elucidated as 1, 9-dihydroxy tetrahydrogeranyl-8-oxy-O-β-D-glucuronopyranoside [Figure 1]. This is the first report of geranyl glucoside from this plant.

Compound 6, designated as geranyl glycosyl benzoate, was obtained as yellow solid mass from chloroform-methanol (9:1) eluents. It showed UV absorption maxima at 227 nm. Its FT-IR exhibited important absorption bands for hydroxyl groups (3373 cm−1), ester linkage (1718 cm−1) and aromatic moiety (1608, 1509 cm−1). On the basis of mass and 13C NMR spectra the molecular ion peak of 6 was determined at m/z 1248 corresponding to the molecular formula C53H84O33. The 1H NMR spectrum of 6 exhibited two-proton double doublet at δ 7.87 (J = 7.3, 2.1 Hz) assigned to H-3′ and H-5′ aromatic protons. A two-proton doublet at δ 6.43 (J = 2.1 Hz) was attributed to H-2′ and H-6′ aromatic protons. A one-proton multiplet at δ 7.64 was ascribed to H-4′. The signals for the saturated geranyl unit remained similar to compound 5 except that the compound 6 showed the presence of a six-proton doublet at 1.02 (J = 6.6 Hz, Me-1, Me-9) for a typical iso-propyl unit of geranyl unit. The 13C NMR spectra of 6 displayed important signals for carboxylic carbons at δ 174.13 (C-6a) and 167.93 (C-7‘); aromatic carbons between δ 145.85 and 120.89; and six anomeric carbons from δ 109.51 to 100.55. Slightly deshielded signals for C-2a, C-2b, C-2c, C-2d, C-2e and C-2f indicated as 2→1 linkage between sugar units in the compound. On the basis of above the structure of 6 was elucidated as terahydrogeranyl-8-oxy-O-β-D-glucuronopyranosyl (2a→1b)-O-β-D-glucofuranosyl (2b→1c)-O-β-D-glucofuranosyl (2c→1d)-O-β-D-glucopyranosyl (2d→1e)-O-β-D-glucopyranosyl (2c→f)-O-β-D-glucopyranosyl-2f-benzoate [Figure 1]. This is the first report of a geranyl glycosidic ester from the plant.

CONCLUSION

The present work reports the chromatographic isolation and spectroscopic identification of new flavone bisabolenyl ether and geranyl glycosides along with known fatty acid glycerides, fatty acid and a pentasaccharide from the methanolic extract of seeds of Z. jujuba. Compounds 4–6 are unique with respect to formation of ether linkages between saturated sesquiterpenes or monoterpenes and flavonoid or sugar units. This study has enhanced the knowledge base related to phytochemical compositions of jujuba seeds.

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Conflicts of interest

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