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Two New Antiinflammatory Triterpene Saponins

Bull. Korean Chem. Soc. 2009, Vol. 30, No. 8

1811

Two New Antiinflammatory Triterpene Saponins from the Egyptian Medicinal Food Black Cumin (Seeds of Nigella sativa)
Mohamed Elbandy,†,§ Ok-Hwa Kang,‡ Dong-Yeul Kwon,‡ and Jung-Rae Rho†,*

Department of Oceanography, Kunsan National University, Jeonbuk 573-701, Korea. *E-mail: jrrho@kunsan.ac.kr ‡ College of Pharmacy and Wonkwang-Oriental Medicines Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749, Korea § Department of Food Sciences and Technology, Faculty of Environmental Agricultural Sciences, Suez Canal University, Elarish, North Sinai, Egypt Received June 24, 2009, Accepted July 02, 2009
An extensive phytochemical investigation of the polar fractions of a methanolic extract of Egyptian medicinal food, black cumin (seeds of Nigella sativa L.) led to the isolation of two new triterpene saponins, named sativosides A and B (1-2), along with four known saponins (3-6). Sativoside A (1) is the first example of saponins containing 18-ene triterpene aglycon not only in this Nigella genus but also in the family Ranunculaceae. The structure of the new saponins was elucidated mainly by a combination of 1D and 2D NMR data, together with HRFABMS and acid hydrolysis. Three compounds (1-3) showed the significant inhibition effect of phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187-induced production of IL-6 in a human mast cell (HMC-1) line.

Key Words: Nigella sativa L, Sativosides A and B, 18-Ene triterpene, Pro-inflammatory cytokine

Introduction The seeds of Nigella sativa L (black cumin) are an amazing medicinal food. In Arabic countries it is called Al-Haba-ElSauda or Habat-Al-Baraka. Nigella sativa is an annual dicotyledon herbaceous plant of the family Ranunculaceae. It grows in Middle east area and it is believed to be indigenous to the Arabic countries and the Mediterranean region but has been cultivated in other parts of the world including northern Africa 1-2 and parts of Asia. For thousands of years, black cumin has been used in many Asian, Middle Eastern and Far Eastern coun3 4 tries as an additive in spices, flavored and aromatic substances, 1 and food preservative. These seeds are commonly eaten alone or in combination with honey and it is also used as condiments in many food preparations (curries, pastries, bread and Medi5-6 terranean cheese). In addition, hot aqueous extract of seeds is also widely used in Egypt as warm drink. N. sativa extract can be used in the preservation of food and prevention of food poisoning.7 It has long been used in traditional folk medicine as a protective and health remedy for a wide range of illnesses, including headache, bronchial asthma, abdominal pains, hypertension, gastrointestinal problems, diarrhea and rheumatism, 8 especially in the Southeast Asia and Middle East. Black cumin is one of the most extensively pharmacologically studied medicinal foods. Many biological studies on the effect of N. sativa seed extract or its active compounds including antioxidant, antiinflammatory, anticarcinogenic, antidiabetic, antiulcer, antiparasitic, antihistaminic, antiviral, antifungal, antibacterial, antihelminthic, and immunomodulatory effects have been reported. The seeds have very rich and diverse chemicals which contain fixed and volatile oils, proteins, amino acids, alkaloids and 5,9-10 Several constituents were characterized in volatile saponins. oil, including carvone, carvene, α-pinene, p-cymene, and the crystalline active principle, nigellone. Moreover, thymoquinone,

dithymoquinone, thymohydroquinone, and thymol are the pharmacologicaly active components in the volatile oil. Two indazole alkaloids, nigellicine and nigellidine, an isoquinoline alkaloid, 9-10 nigellimine and its N-oxide, and eight new dolabellane-type diterpene alkaloids, nigellamines A1, A2, B1, B2, A3, A4, A5, and C were isolated from black cumin.11-12 Four known hedragenin saponins have been previously reported from the seeds of N. sativa, two monodesmosidic glycosides with disaccharide (α-hederen) and trisacharide chains at C-3 of the aglycon through 13 an ether bond, in addition to two bisdesmosidic saponins with 14 two sugar chains at C-3 and C-28 of the hedragenin triterpene. The polar extracts of the seeds of N. sativa showed many biological activities, including anticancer, antiulcer, antimicro5, 9-10 and saponins bial, anti-inflammatory and spasmolytic effects were the main chemical constituents in the polar fractions of our methanolic extract. Moreover, the importance of saponins is increasing in food, cosmetics, and pharmaceutical sectors because of their physicochemical (surfactant) properties and mounting evidence on their biological activity.15-17 These observations prompted us to continue in our research aimed at finding biologically active and/or novel saponins from plant 18-23 Recently, we investigated the suppression effect of origin. the production of cytokine interleukin (IL)-6 in human mast cell (HMC-1) on the methanolic extract of the seeds of N. sativa, which may contribute to the treatment of asthmatic inflamma1 tion. Through bioactivity and H NMR monitoring guided fractionation of the methanolic extract of the seeds of N. sativa, six triterpene glycosides were isolated composed of two new compounds, sativosides A (1) and B (2) and four known ones. Herein, we report the isolation and structure elucidation of two new triterpene saponins and the evaluation of inhibition effect of the isolated compounds on phorbol 12-myristate 13acetate (PMA) plus calcium ionophore A23187-induced production of cytokine IL-6 in HMC-1 line.

No.040 mm.5. sativoside B (2.0. NMR Spectral Data of the Aglycon Portion of sativosides A (1) and B (2) in CD3OD no 1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16a 16b 17 18 19 20 21a 21b 22a 22b 23 24 25 26 27 28 29 30 a 1 δH 0.6 g) and MeOH (3 × 1000 mL. tR = 24.07 1. MeOH). d 1. IR (film) 3366 (OH).91 3.50a 1. d 37. s a 1.17 (3H. s) a 1.7. t 1. s 2. CH2Cl2 (3 × 1000 mL. and D-glucose yielded consistent peaks. tR = 33. The 70% MeOH (2. t = CH2. Plant material.3.2. 0. q 17.8.2.70a a 1. Each compound (each 5. s 5. to yield 21 fractions (500 mL each). tR = 31.35a a 2 δC 40. Merck) by using a gradient of increasing polarity with CH2Cl2.57a 2. EtOAc. q 17. t 1. This mixture was subjected to a reversedphase flash column chromatography (YMC Gel ODS-A 12 nm S-75 µm) using step mixtures of the H2O/MeOH solvent system (100/0.1.2. q 15. s 49. s) 0. 13 mg.69 2. 1624 (C = C). 11. 18. d 44.30 and δC 49.8.1.2 (c 0. arabinose (tR = 8.1812 Bull.13 (3H. q 178.35 min).06 min).7 g). 1027-1039 (C – O) -1 1 13 cm . L-arabinose.6354). 25 g) at room temperature with constant stirring for 24 h for each solvent.4.60 34.5. yielding a crude saponin mixture (7.0. Optical rotations were measured on a JASCO P-1010 digital polarimeter.13 min). Seeds of N. S-5 µm). 2931 (CH).6346 for [M + Na]+ (Calcd for C64H102O30Na:1373. and MeOH as the solvent.6.7) 42.7. d 43. Soc.70a 31. bs) a 1.3. 2009. d 144. q 26. 1728 (C = O). s 30. t 33. s) 10.6.9.0.9. Coelution experiments with standard sugar samples allowed the identification of rhamnose (tR = 7. Co-injection of each hydrolysate with standard L-rhamnose.8. s) 0. bs) 134. s 1. 8 Experimental Mohamed Elbandy et al.6 (c 0. All solvents were distilled prior to use and authentic sugars were purchased from Fluka and Sigma.0. s) 0.2. s 48.45a a 2. H and C NMR are given in Tables 1 and 2.68a a 1. t 64. q 16.0 as an internal standard.6.1.85 (1H. HRFABMS m/z:1373. .9 min).2. IR spectra were recorded on a JASCO FT/IR-4100 spectrometer. S-5 µm) and the Varian polaris NH2 column (4. s 24. t 35. t 13. s = C. t 34.0.3. and 6 (6 mg. q 48.4.43 1.3. IR (film) 3389 (OH). Vol.33a 1. Then.6 × 250 mm.22 34. sativa (500 g) were extracted successively with hexane (3 × 1000 mL. q Overlapped with other signals. 3 (600 mg.0 mg) in H2O (2 mL) and 2 N CF3COOH (5 mL) were refluxed on a water bath at 75 o C for 6 h.2 min).6510).9.1. s 43.4.05 1. and 0/100).1.5. s 48. supported by Korea basic science Table 1. d. d 37. After solvent removal. Faculty of Environmental Agricultural Sciences.4 min). 16. tR = 28.5.97 (3H. t 82. and then dissolved in H2O (250 mL) and partitioned with water-saturated n-BuOH. 14 mg.0. t 2. t 1. The combined EtOAc extracts were washed with H2O and evaporated to dryness to afford the aglycon.32a a 0.70 9. q 16. q 24. Extraction and isolation. Institute Daegu Center.33 a 3.04a a 33. 27 o Sativoside B (2) : [a] D + 7.91 (3H. 4.5.21a a 1. The dried and powdered seeds of N. D-xylose. s 52.1 min).25 (1H.79 (3H.25 27.91 5.33 1. d 56.6517 (calcd for C64H104O30Na:1375. the reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (3 × 20 mL). 30. t 41. Fractions 14.3 min). Suez Canal University. q 29.94 (3H.89 1. d 1. H and C NMR are given in Tables 1 and 2.7. and 17 were selected by the moderate inhibition effect of IL-6 in the HMC-1 line and combined for further separation.3. s) 1.1 g) subfraction was further purified by reversed-phase semipreparative HPLC eluting with a 65% aqueous MeOH solvent in a flow rate of 2 mL/min to afford sativoside A (1. 12.60a 33.8.01 (3H.81 (3H.0.68 a a δH δC 39.48a a 1. t 82.6. t 1.02a 49. t 26. 4 (60 mg. the residue of the n-BuOH fraction (8. q 177.12 g). The aqueous layer was repeatedly evaporated to dryness with MeOH and the residue was dissolved in CH3CN-H2O (1:1) followed by HPLC analysis. s) 0. Egypt in May 2008.1.9. t a 1. 2929 (CH).2.37a 1. 1027-1039 (C – O) -1 1 13 cm . The MeOH extract (20 g) was fractionated by flash column chromatography (Si gel 60. q = CH3.93 1. 15. bs) 209.8.58a a 1.76 a 1.3. MeOH).93 (3H. EtOAc (3 × 1000 mL. 1717 (C = O). General methods. xylose (tR = 8. The 1D and 2D NMR spectra were obtained on a Varian VNMRS 500 spectrometer 1 13 working at 500 MHz for H and 125 MHz for C. t 41.6. t 1. tR = 18 min).6. 1644 (C = C).0. s 138.77 a 1.89 3.18.86 min). d 1. Korean Chem.8.5 (1H.8. s) 0.015∼0.99 (3H.3) 42.6.36 1.1.1 g) was dissolved in a small amount of MeOH (50 mL) and purified by precipitation with Et2O (2 × 250 mL). using an isocratic elution of CH3CN-H2O (85:15) with RI detection in a flow rate of 0. sativa were purchased from an herbal market in Elarish.73a a 1.13a 47. d 21. and glucose (tR = 11. t 26. d 18.5.22 a 3. 122 g).9. d = CH.2. t 1. s) 0. dd. 5 (15 mg.75a a 1. s 22.3. s 34.18a 30. 27 o Sativoside A (1) : [a] D +24.51 0. tR = 21.9 mL/min. All NMR chemical shifts were referenced to CD3OD at δH 3.87a 1.44a 1.28 (1H. t 123.68 (3H.1. s 33. s) 0.61a 1. HRFAB mass spectra were acquired with a Jeol JMS-700. HRFABMS m/z:1375.14 (1H.45a a 1. HPLC was performed with a Varian RI detector using the YMC ODS-A column (10 × 250 mm. This mixture was concentrated to a brown viscous residue o under reduced pressure at less than 40 C.0. The voucher specimen is at the Department of Food Sciences and Technology.9.22 1. s) 1. s 28. s) 1. t 24.00 (3H. Sugar analysis. 30/70.

3.9. d 72. d. 8 o 1813 C-3 Ara 1 4. Franklin o Lakes.0) for at least 1 h. d 76. d 74. d 3 3.22 (1H.7. d a 75.29 a 78.2) 101.8. This extract was further fractionated by a combination of silica gel and RP-18 flash column chromatography.4.E.3.26 (3H.8. The HMC-1 cells were pretreated with two concentrations of six compounds (5 and 10 µg/mL) for 1 h before PMA plus A23187-stimulation.3.7) 3.27a 3.23 (1H.6. bs) 3. we used a modified ELISA method.2) a 3.49 (1H.3. d 75. bs) 3.8. d.1. t and 10% fetal bovine serum (FBS) at 37 C in 5% CO2 with 95% humidity. 3. d 5 3.1) 17.46 (1H.26 (3H.4) 104.7. bd. NMR Spectral Data of the Sugar Portion of sativosides A (1) and B (2) in CD3OD no 1 δH δC δH 2 δC Bull.6. d 70. and 100 µg/mL) were added to each well and incubated with 10 µL of an MTT o solution (5 mg/mL) for 4 h at 37 C under 5% CO2 and 95% air. Cell culture. bs) a 65. d.07 a 82. d 1.56 (1H.5) 4. sativosides A (1) and B (2). d 4 3.24a 3.7.4) 1.3. bs) 3. 3.2. d. 30. d a 77.87 (1H. After incubation for 2 h. 1.3) 17.9.46 (1H.9.3) C-28 Glc I 1 5.32 (1H.9. and all standards and samples were assayed in duplicate.5.6) 103. d.8.29 a 79.83 72.45 (1H. Accordingly.0. 8. 3.32 a 69. d. 7. d 75.63a a 3. t.0. d 69. a working detector (biotinylated anti-human IL-6 monoclonal antibodies and streptavidin-horseradish peroxidase reagent) was added and incubated for 1 h. 3.7) 70.0.0. 3.9. 5.22 (1H. d 2 3. 50.4) 6 1. Vol.07 (1H. 5.96 (1H.1. t.Two New Antiinflammatory Triterpene Saponins And then each of these elutes were individually collected. d. Results and Discussion The n-butanol fractions obtained from the methanolic extract of the seeds of N.6. The HMC-1 cells were grown in IMDM and supplemented with 100 U/mL penicillin.61(1H.53 a 76. d 3 3. 4. 7.92 (1H. d = CH. sativa were suspended in MeOH and purified by precipitation with Et2O yielding a crude saponin mixture.9. 3. d 3 3. d 76. 3. The HMC-1 cells were treated with new triterpene saponin compounds for 1 h.5) a 73. d 4 3. d. All subsequent steps took place at room temperature. To measure the cytokine IL-6.1) a 3. d 3 3. d 73.1) 3. d.45 (1H. d 4 3. Becton Dickinson Labware.47 (1H. d 76. d. d. The cells were then stimulated with 50 nM of phorbol 12-myristate 13-acetate (PMA) plus 1 o µM of A23187 and incubated at 37 C for 8 h.5) and then washed four times with phosphate buffered saline (PBS) containing 0. 6.8. Then.4) 6 1. d.7. d. d 3. bs) 102. 2. t a 6b 3.87a 69. d. d a 74. d 79. d 4 3. followed by semipreparative reversed-phase HPLC to afford two new saponins.3) 106.7. Denmark).81 6b 4.40 70. The nonspecific protein binding sites were blocked with assay diluent (PBS containing 10% FBS. d 2 3. and Student’s test for single comparisons.8.39 3. The compounds (5. evaporated to dryness and dissolved in H2O and the optical rotation was then recorded at room temperature.7.3) 106.5) 3.39 (1H.05% Tween 20. Soc.5) Glc II 1 4. t 6a 3. the supernatant was removed and the formazone crystals were dissolved by the addition of 100 µL of DMSO.1 M carbonate. Statistical analysis. 8. d 3.8) 104. bs) 3. Cytokine assay.5) 100.4.51 (1H.9.69a 76.9.68 69. d.2. dd. An automatic microplate reader was used to read the absorbance of each well at 590 nm. d.7. d 4 3. d.2.8.50 (1H.7) 3. Then. d. d 3 3.4) 3.62 a 3.3) 95. d 61. d 2 3. 2. d.20 (1H.4) 3. 3.63 (1H. d 67.84 (1H.24 (3H.1. q Xyl 1 4. d. t 104.80 (1H.25 (3H. 2. 2009. Cell viability was examined 5 by an MTT assay. bd.00 (1H. d. 6.51 (1H.3) 5. the test sample or recombinant IL-6 standards were added. d 17. MTT assay for cell viability. d 5 3.63a a 3. 3. t = CH2. d.5. 3.5. We first conducted a sandwich ELISA for IL-6 in duplicate in 96-well ELISA plates (Nunc. d 5 3.6. d 78. The data from the experiments are presented as means ± S. substrate solution (tetramethylbenzidine) was added to the wells and incubated for 30 min in the dark before the reaction was stopped with a solution of 2 N H3PO4. 1.1) 3. The absorbance was recorded at 450 nm.5. d 75. 2.35 (1H.3. q Overlaped with other signals. d 3. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons. d 69. pH 9.7. d 72.54 4 3.82 (1H. 11.78 4. t 5a 3.28 3.3. 3. d 70. d 2 4. d. d 77. q = CH3.7.9) 72.2. Sativoside A (1) was isolated as a white amorphous powder and its molecular formula was established as C64H104O30 on the 102. d 78. No.7.6.65(1H.46 6a 3. d 2 3.9.5) 71.7.9.38a 3.8.6. q a 4. d 75.4.83 72.85 (1H.32 (1H.07 (1H. After HMC-1 aliquots were seeded (3 × 10 ) in microplate wells. bs) 3. pH 7. bd.7) Rha I 1 5.7.38 (1H. 1. d 71.79 Rha II 1 4.51 5b 3. 8. NJ) were coated overnight at 4 C with anti-human IL-6 monoclonal antibody diluted in coating buffer (0. d 4.2.3. d.30a a 78.2. Table 2.1) 17. 6.41 71. d 5 3. After washing the plates again.21 5b 3.6. Korean Chem.0. 6.1.M. ELISA plates (Falcon. dd. the supernatant was decanted into a new microcentrifuge tube and the cytokine was quantitated by ELISA. d a 71. t 5.87a 81. t 5a 3.78a 4. dd.5.53 (1H. together with four known compounds (Figure 1). d 2 3. d 3 3. d 65. 11.47a 3. t 95. 5. m) a 66.2) 61. d.58 (1H.84 (1H. d a 75.31a 3. 7. 10.82 (1H.6. d. We then used the enzymelinked immunosorbent assay (ELISA) method to assay the culture supernatants for the IL-6 protein levels.7. q 4.8.75 (1H. 100 µg/mL streptomycin.95 (1H.23 a 76.70 3. d a 72.9) 69. 3. .

5 ppm) region of the H NMR spectrum deduced compound 1 to be a triterpene glycoside. Based on the evaluation of spin-spin couplings and ROESY data. indicating that the sugar part was composed of rhamnose (L-Rha). In the HMBC experiment (Figure 2). J = 8.3 Hz). and glucose (D-Glu) in a ratio of 2:1:1:2. The relative stereochemistry of all chiral centers in the aglycon part of 1 was achieved by the ROESY experiment. typical of the hydroxyl and carbonyl group. From careful analysis with 1D and 2D NMR data (COSY.33 and 3. 21 22 COOR2 28 COOR2 COOR2 R1O CH2OH R 1O O H R1 O CH 2OH I compounds 1 2 3 4 5 6 aglycon I II III III III III R1 II III R2 α-L-Rha II (1 → 4)-β-D-Glu II (1 → 6)-β-D-Glu I α-L-Rha II (1 → 4)-β-D-Glu II (1 → 6)-β-D-Glu I α-L-Rha II (1 → 4)-β-D-Glu II (1 → 6)-β-D-Glu I α-L-Rha II (1 → 4)-β-D-Glu II (1 → 6)-β-D-Glu I β-D-Glu II (1 → 6)-β-D-Glu I β-D-Glu II (1 → 6)-β-D-Glu I β-D-Xyl (1 → 3)-α-L-Rha I (1 → 2)-α-L-Ara β-D-Xyl (1 → 3)-α-L-Rha I (1 → 2)-α-L-Ara β-D-Xyl (1 → 3)-α-L-Rha I (1 → 2)-α-L-Ara α-L-Rha I (1 → 2)-α-L-Ara β-D-Xyl (1 → 3)-α-L-Rha (1 → 2)-α-L-Ara α-L-Rha (1 → 2)-α-L-Ara Figure 1.33 and 3.50 (d.51 and the carbon at δ 44. An additional HMBC correlation between the H-1 (δ 4. TOCSY.50 (H-1) of the Ara residue. of which C-2 was in turn connected to the H-1 of Rha I via an ether linkage from the correlations between the anomeric proton at δ 5. Soc. Then. The configuration of the glycosylated OH group at C-3 was assigned to be β from the NOE peak between H-3 and H-5. starting from the well-separated anomeric protons at δ 4. And the NOE effect between H3-24 and H3-25 indicated that the methyl group at C-24 was in a β position.99. together with anomeric signals corresponding to six monosaccharide residues in the HSQC spectrum. xylose (D-Xyl).2). The carbonyl group at C-28 was established to be oriented in the β-axial direction based on 24-25 the observation of the NOE peak between H-16b and H-22b.1814 Bull. 13C and DEPT) of 1 displayed the presence of eight methyls.81.1) of Rha I completed the linkage of the trisaccharide chain at C3 of the aglycon part. 0.02 (H-16) and the carbonyl carbon at δ 177. 0. one double bond and one carboxylic group.68. one α-arabinopyranosyl (Ara). Korean Chem.46 (d. 1.3 Hz). . 4. respectively.35) of Glc II OH HO Ara O O O CH2OH HO O HO O HO Rha II O OH Glc II O HO HO HO HO O Rha I HO O O OH Xyl OH HO C O O Glc I OH O OH Figure 2. 23-dihydroxyolean-18-en-28-oic acid. 30.0 (C-19) and of the proton at δ 5. 4.9 (C-17).46) of Glc I and C-28 (δ 177. TOCSY and HSQC spectra.0) of Ara unit.47) of Xyl and C-3 (δ 82. the aglycon part of 1 was revealed to be a triterpene of 3. two diastereotopic protons at δ 3.01. and 1.0 (C-28). J = 7.14 with two carbons at δ 42. and two α-rhamnopyranosyl (Rha) residues were recognized.0∼5.93. H-1 (δ 4. Vol. the downfield shifted carbon at C-3 of the aglycon showed long-range correlation with the proton at δ 4.1 (C-4) and between the proton at δ 2. 0. J = 7. The IR spectrum showed strong absorption bands at 3389 and -1 1728 cm . one β-xylopyranosyl (Xyl).84 (bs). 8 29 19 12 25 1 2 10 3 24 4 5 23 6 11 9 8 7 27 26 14 16 15 13 18 17 20 30 Mohamed Elbandy et al. The linkage of the CH2OH and the carbonyl carbon groups to two quaternary carbons at C-4 and C-17.8 Hz). No. Structures of Saponins 1-6 isolated from Nigella sativa L.3 (C-13) and 49.5 1 ppm) and midfield (3. the sequence of monosaccharide units in the glycosidic part was established from the HMBC and ROESY spectra. 2009.14.35 (d. J = 5. The 1D NMR spectra (1H. arabinose (L-Ara). the 28-O-triglycoside structure of 1 was characterized on the basis of the HMBC cross-peaks between H-1 (δ 5. All signals observed in the upfield (0. respectively.47 (d. The monosaccharide residues in the glycosidic part of 1 were assigned by a combination of COSY. 0. + 5.97 and 0. characteristic of six methyl singlets at δ 0.99 with the olefinic carbon at δ 134.4 Hz). 4.97.51 ascribable to a CH2OH group and a broad singlet at δ 5. the existence of two β-glucopyranosyl (Glc).23 (bd.6510) of HRFABMS and the C NMR spectrum.23 (H-1) of Rha I and C-2 (δ 76. and 5. Followed by the same analysis.6517 [M +Na] 13 (calcd 1375. key HMBC correlations for Sativoside A (1). This is confirmed by both HPLC analysis with authentic sugars after acid hydrolysis of 1 and optical rotation measurement for each isolated residue. The position 18(19) was evident from the HMBC coof the double bond at ∆ rrelations of two methyl protons at δ 0. basis of a combination of a peak at m/z 1375. was also secured by the HMBC correlations between two methylene protons at δ 3. HSQC and HMBC).5∼2.0) of the aglycon part.

the aglycon of 1 possesses a rare olean-18-en moiety.6354) consistent with the molecular formula C64H102O30 in the HRFABMS. Accordingly. a 3-O-[β-D-xylopyranosyl-(1 → 3)-O-[α-Lrhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl] hedragenin 28-O-[β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] ester. Healing Arts Press. This research was financially supported by the Brain Korea 21 (BK21) project.5 7. H-4 (δ 3. 2009. G.. A. 2004. 30. M. Indian Materia Medica. Bioactive Saponins from Plants: An Update. 1749.. Compounds 4-6 were determined as the known triterpene glycosides by analysis of the 2D NMR data by the same procedure as 1. A. M. 7. 2003. Republic of Korea. 869. Vermont. El-Azzouy. Nadkarni.7) of Glc. H. A. A. Anil. Chem. Wary.. their Taxonomy and Agricultural Significance. The feature of 2 indicated the presence of an aldehydic group. Compound 2 may be inferred to be oxidized from 3 in the biosynthesis pathway.. Although weak cytotoxicity at high concentration of 100 µg/mL. This group was connected to the position of C-4 from the HMBC correlation from the downfield shifted aldehydic proton at δ 9. V. Inter.. M. 1208.. Inter. 5.28 However. Willuhn.. M. A. C. Ministry of Education & Human Resources Development. M.6346 [M + Na] (calcd 1373. 5. T. which may be important in developing future anti-inflammatory therapies. K. 13. 275. Phytother. Effect of compounds 1-6 on IL-6 production control PMA + A23187 1 2 3 4 5 6 10 µg/mL 5 µg/mL 10 µg/mL 5 µg/mL 10 µg/mL 5 µg/mL 10 µg/mL 5 µg/mL 10 µg/mL 5 µg/mL 10 µg/mL 5 µg/mL IL-6 (pg/mL) 3±1 679 ± 4 386 ± 8 572 ± 22 265 ± 21 510 ± 7 271 ± 18 422 ± 25 549 ± 15 695 ± 24 678 ± 11 675 ± 6 533 ± 10 674 ± 3 Inhibition IC50 (µg/mL) rate (%) 43 16 61 25 60 38 19 0 0 0 22 0 12. Popular Prakashan Pvt.. 42. Phytother.53) of Glc II and C-1 (δ 102. Turk. Blunden. Ethnopharmacol.. T. Xu. J. Benny. L. Ninomiya. Swamy. F. sativa. Lacaille-Dubois.. compound 2 was identified as a 3-O-[β-D-xylopyranosyl-(1 → 3)-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-Larabinopyranosyl] gypsogenin 28-O-[α-L-rhamnopyranosyl(1 → 4)-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] ester. Asthma. S. K. Ashfaq. No. Kashima. Oil Res. and 6 as a 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl] hedragenin 28-O-[β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] ester. Yoshikawa. H. Hammerschmidt. pp 76-85. 561.. A. J. Black Seed Cumin: the Magical Egyptian Herb for Allergies.6 8.. Bedir. Schleicher. The survey showed that the elucidated structure of 2 was very similar to that of 3 reported recently from the seeds 14 of N. Addis Ababa. I. Khwaja. M. References 1. S.. Ltd. Gilani. Morikawa. confirmed by the signal at δ 9. 27 previously reported from Cephalaria pastricensis. Abou-Gazar. H. Caliskan. and Immune Disorders. This is the first reported occurrence of an olean-18-en type saponin in family Ranunculacea. Treatment of cell with compounds 1-3 (5 and 10 µg/ mL) significantly inhibited IL-6 production in a concentrationdependent and statistically significant manner. A. Res. Morikawa. Res. Acknowledgments. Nawwar. 29.. 29. M.3 (C-4). Condiments and Medicinal Plants in Ethiopia. J.6 30. pp 301-340. Korean Chem. 67. M. 12. the signal assignment of 2 from extensive 1D and 2D NMR spectroscopic studies suggested a bisdesmosidic saponin with two trisaccharides at the C-3 and C-28 position of the aglycon with the olean-12-en type. Khan. Barakat. 921. gypsogenin.. Food Sci. 1998. Planta Med.3 in the C NMR spectrum. H. O. The triterpene glycoside isolated from Oreopanax guatemalensis is the 25 closest relative. K. T. J. Y. the latter saponins 5 and 6 were not previously reported from the Nigella genus. Aboutabl. Org.(1 → 6)-β-D-glucopyranosyl] ester. 17. 4 was suggested to be a 3-O-[α-L-rhamnopyranosyl-(1 → 2)O-α-L-arabinopyranosyl] hedragenin 28-O-[α-L-rhamnopyranosyl-(1 → 4)-O-β-D-glucopyranosyl-(1 → 6)-β-D-gluco14 pyranosyl] ester 5. Rashid. pastricensis and N. G. the levels of IL-6 was considerably increased after stimulation with PMA plus A23187 in HMC-1.5) of Glc II. compared with common bisdesmosidic saponins with trisaccharide chains at C-3 and C-28 of the aglycon.Two New Antiinflammatory Triterpene Saponins Table 3. Essent.50 to the quaternary carbon at δ 56. M. Xu... M. Phytochemistry 1997. 15.4 Bull. 14.. 1976. Merfort. B. 10. Ramadan. Matsuda. As shown in Table 3. E. F. Ninomiya. Sativoside B (2) exhibited a quasimolecular ion peak at m/z + 1373.9 23. and H-1 (δ 4.8) of Rha II. A. A. 4. Salem. glandulifera Freyn. all compounds were found not to affect HMC-1 cells viability at concentrations of 5. Thus.. M. Ali. 8 1815 and C-6 (δ 69. Yoshikawa. 10. To the best of our knowledge. K. 49.. Chem. E. 16. 2. 494. 11. F.. 299. As described previously.50 in the 1 13 H NMR spectrum and by the signal at δ 209. isolated from C. Mahmood. Spices. M. Hatem. Wagner. 2004. Lett. K. H.. S. P. Immunopharmacol. We examined the inhibition effect of the six isolated compounds on PMA plus calcium ionophore A23187-induced production of pro-inflammatory cytokine IL-6 in the HMC-1 line.. H. Bull. 3. 2005. Pharm. Matsuda. H. 359. Soc. K. E. Hanafy. 17. F.. A.. 50 and 100 µg/mL (data not shown). Prog. M. H. A. 23-dihydroxy olean-18en-28-oic acid 28-O-[α-L-rhamnopyranosyl-(1 → 4)-O-βD-glucopyranosyl. K.. the structure of 1 was determined as 3-O-[β-D-xylopyranosyl-(1 → 3)-O-[α-L-rhamnopyranosyl(1 → 2)-O-α-L-arabinopyranosyl] 3β. 4. 6. 46.. 9. 2007. M. In studies in Natural Products Chemistry . Jansen. 1981. 2003. M..84) Rha II and C-4 (δ 79. Taskin.26 And the acid hydrolysis followed by HPLC analysis and optical activity measurement revealed sugar components to be the same with those of 1. 1991. R. Saleh. S. Vol. Rochester. Hussein. 1986. 2001. H. M. The unequivocal sequence of two trisaccharide chains was also supported from ROESY peaks between the anomeric protons and the proton in the adjacent residue. Technol. Center for Agricultural Publishing and Documentation.. Bombay. M. 2005. 6. H.. I. P. 8. 34.

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