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Journal of Chemistry, Vol. 45 (1), P. 120 - 125, 2007 PROTOTANE-TYPE TRITERPENES FROM THE RHIZOMES OF ALISMA PLANTAGO-AQUATICA Received 30 August 2006 CHAU VAN MINH , PHAN VAN KIEM1, PHAM HAI YEN1, TRAN THU HUONG2, ALESSANDRA BRACA3 1 Institute of Natural Products Chemistry, VAST 2 Department of Chemical Technology, Hanoi University of Technology 3 Department of Bioorganic Chemistry and Biopharmaceutics, University of Pisa, Italia 1 SUMMARY Three terpenes with Protostane type were isolated from the rhizomes of Alisma plantagoaquatica. The chemical structures of isolated compounds were characterized as 11 ,23S,24R,25tetrahydroxyprotost-13(17)-en-3-one (alisol A, 1), 11 ,23S,25-trihydroxyprotost-13(17)-en-3one-24R-yl acetate (alisol A acetate, 2), and 11 ,23S,24S-trihydroxyprotost-13(17),25-dien-3-one (alisol G, 3), by detailed analysis of the 1D- and 2D-NMR spectra such as 1H-, 13C-NMR, DEPT 90, DEPT135, HSQC, HMBC, 1H-1H COSY, and by the Electronspray Ionization (ESI) mass spectrum. This is the first report of alisol G from Alisma plantago-aquatica. I - INTRODUCTION The dried rhizome of Alisma plantagoaquatica L. var. orientalis Samuelsson is a crude drug, and has been used as a folk medicine for diabetes and swellings [1]. From the phytochemical investigations including its physiological active principles, it was reported to contain protostane-type triterpenoids, e.g. alisol A and its 24-acetate, alisol B and its 23acetate and alisol C and its 23-acetate [2 - 5] and many other components isolated from fresh rhizome Alismatis orientale and the crude drug Alismatis rhizoma of Japanese and Chinese origins [6]. Here, we report the isolation and structural determination of three terpenes with Protostane type as 11 ,23S,24R,25-tetrahydroxyprotost-13(17)-en-3-one (alisol A, 1), 11 ,23S,25-trihydroxyprotost-13(17)-en-3-one24R-yl acetate (alisol A acetate, 2), and 11 ,23S,24S-trihydroxyprotost-13(17),25-dien120 3-one (alisol G, 3) from the rhizomes of this plant. II - EXPERIMENTAL 1. General experimental procedures The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer using TMS as the internal standard. The Electronspray Ionization (ESI) mass spectrum was obtained using a AGILENT 1100 LC-MSD Trap spectrometer. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70 230 mesh and 230 - 400 mesh, Merck) or YMC RP-18 resins (30 - 50 µm, FuJisilisa Chemical Ltd). Thin layer chromatography (TLC) was performed on DC-Alufolien 60 F254 (Merck 1.05715) or RP18 F254s (Merck) plates. 2. Plant material The rhizomes of Alisma plantago-aquatica L. var. orientalis Samuelsson were collected in Tam Dao Mountain, Vinh Phuc Province in January, 2006 and were identified by Dr Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology. 3. Extraction and isolation Air-dried and powdered rhizomes of Alisma plantago-aquatica L. (6.0 kg) were extracted with methanol to get the residue (150 g), which was then suspended in water and extracted sequentially using hexane, chloroform and ethyl acetate to yield hexane (53 g), CHCl3 (64 g), EtOAc (13 g) extracts, and water layer (20 g). Repeated chromatography of the CHCl3 extract (64 g) on a silica gel or YMC column with the suitable solven systerms to get compounds 1 (250 mg), 2 (130mg) and 3 (54 mg) as white crystals. III - RESULTS AND DISSCUSSION Compounds 1 - 3 were obtained as white crystals from the methanolic extract. The 1HNMR spectrum of 1 showed 7 singlets of the quaternary methyl groups ( 1.00, 1.05, 1.06, 1.07, 1.13, 1.27, 1.21) and a doublet at 1.01 (3H, d, J = 7.0 Hz, H3-21), three protons of the oximethine carbons at 3.88 (1H, ddd, J = 5.8, 10.7, 10.7 Hz, H-11), 3.76 (1H, d, J = 9.0 Hz, H-23) and 3.76 (1H, d, J = 9.0 Hz, H-24). All signals of the 1H-NMR spectrum suggested that 1 is a triterpenoid. The 13C-NMR and DEPT spectra of 1 exhibited the signals of 30 carbons including 8 methyl, 8 methylene, 6 methine and 8 quaternary carbons. The carbonyl group was assigned at 220.5, a double bond without olefinic protons was confirmed at 137.6 and 135.5, four carbons bearing oxygen atom including three oximethine at 69.4, 69.9, 77.6 and a quaternary carbons at 74.1. The side chain of 1 was connected from the spin-system of the 1H-1H COSY and was further confirmed by the long-range correlations in the HMBC spectrum as shown in table 1. All the NMR data suggested the Protostane type of 1 resembling those of alisol A. The hydroxyl group with configuration was at C-11 confirmed by H-C long-range correlations between H-11 and C-13 ( 137.6)/C-9 ( 49.6)/C-10 ( 36.9) in the HMBC spectrum and by the spin-coupling of proton H-11 ( 3.88, ddd, J = 5.8, 10.7, 10.7 Hz) [6]. The carbonyl group was assigned to C3 from the cross peaks of protons H-23/H-24 and carbon C-3 in the HMBC spectrum. Furthermore, the ESI spectrum of 1 exhibited ion peaks at m/z 473 [M-H2O+H]+, 455 [M2H2O+H]+, 437 [M-3H2O+H]+ and 419 [M4H2O+H]+, corresponding to the molecular formula of C30H50O5. Consequently, 1 was identified as 11 ,23S,24R,25-tetrahydroxyprotost-13(17)-en-3-one (alisol A). Table 1: The NMR data of compound 1 C 1 2 3 4 5 6 7 8 9 10 # C 31.3 t 33.9 t 219.2 s 47.1 s 48.8 d 20.3 t 34.6 t 40.8 s 50.0 d 37.2 s a,c C 31.0 t 33.7 t 220.5 s 46.9 s 48.5 d 20.0 t 34.9 t 40.4 s 49.6 d 36.9 s b,c H 2.13 m; 2.26 m 2.34 m; 2.70 m 2.11* 1.32 m; 1.42 m 1.24 m; 2.03 m 1.77 d (10.6) - (J in Hz) HMBC (H to C) 11 121 # C C 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a,c C 70.1 d 34.8 t 137.2 s 57.2 s 30.8 t 29.5 t 135.4 s 23.4 q 25.8 q 28.7 d 20.3 q 40.4 t 69.5 d 77.6 d 74.1 s 27.6 q 26.4 q 29.8 q 20.4 q 24.3 q b,c HMBC (H to C) 9, 10, 13 9, 11, 13, 14 (J in Hz) 3.88 ddd (5.8, 10.7, 10.7) 2.80 dd (5.8, 13.2) 1.34 m; 1.90 m 2.17 m 1.13 s 1.05 s 2.77 m 1.01 d (7.0) 1.39 m; 1.67 ddd (4.2, 9.3, 13,9) 3.76 d (9.0) 3.01 br s 1.27 s 1.21 s 1.07 s 1.06 s 1.00 s H 69.9 d 34.4 t 137.6 s 56.9 s 30.5 t 29.1 t 135.5 s 23.0 q 25.6 q 28.3 d 20.1 q 40.0 t 69.4 d 77.6 d 74.1 s 27.3 q 26.2 q 29.5 q 20.0 q 24.1 q 13 5, 10, 9 17 24, 25 24, 25 4, 3, 5 4, 3, 5 7, 8, 9, 14 # of alisol A [6], a125 MHz, b500 MHz, cMeasured in CDCl3 *Overlap signals, Chemical shift are given in ppm. Assignments were confirmed by COSY, 1D-TOCSY, HMQC, and HMBC experiments. C R 21 20 HO 11 1 10 13 OH 25 27 11 1 10 OH 26 25 27 16 15 18 5 4 23 17 14 8 3 O 7 13 9 2 15 12 30 19 16 24 20 HO OH 7 6 6 29 29 22 26 18 5 4 24 23 17 14 9 8 3 O 12 30 19 2 22 R 21 28 28 1 R = OH; 2 R = OCOCH3 3 R = OH Figure 1: The structures of compounds 1 - 3 The NMR spectra of compound 2 were very similar to those of 1 except for the more appearence of an acetate group in the NMR spectra of 2 ( C 170.8/20.7 and H 2.20). This evidence suggested that 2 was an acetyl derevative of 1. In the other hand, the ESI spectrum of 2 exhibited ion peaks at m/z 515 [M-H2O+H]+, 497 [M-2H2O+H]+, 479 [M-3H2O+H]+, corresponding to the molecular formula of C32H52O6. The NMR assignments of 2 were made from the comparison with those of 1, and were further confirmed by HSQC and HMBC spectra of 2. The H-C long-range correlation were 122 observed between H-24 ( 4.61) and carbon carbonyl C-31 at 170.8, confirming that the acetate group was connected to C-24. All NMR data of 2 were in good agreements with those of alisol A 24-acetate. Accordingly, 2 was determined as 11 ,23S,25-trihydroxyprotost-13(17)-en-3-one-24R-yl acetate (alisol A 24-acetate). Table 2: The NMR data of compound 2 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 # C 31.3 t 34.0 t 219.2 s 47.1 s 48.8 d 20.3 t 34.6 t 40.7 s 50.0 d 37.2 s 70.0 d 34.8 t 137.7 s 57.2 s 30.8 t 29.4 t 135.0 s 23.5 q 25.8 q 28.2 d 20.2 q 40.0 t 69.1 d 78.8 d 73.9 s 27.6 q 26.9 q 29.7 q 20.4 q 24.3 q 170.5 s 21.0 q a,c C 30.9 t 33.7 t 220.5 s 47.0 s 48.6 d 20.0 t 34.3 t 40.4 s 49.5 d 36.9 s 69.8 d 34.3 t 138.3 s 57.0 s 30.4 t 28.9 t 135.0 s 23.0 q 25.5 q 27.8 d 19.9 q 39.6 t 69.0 d 78.7 d 73.9 s 27.2 q 26.7 q 29.5 q 20.0 q 24.1 q 170.8 s 20.7 q b,c (J in Hz) 2.15 m; 2.30 m 2.36 m 2.73 m 2.12* 1.32 m; 1.49 m 1.28 m; 2.05 m 1.77 d (10.6) 3.88 ddd (5.8, 10.7, 10.7) 2.89 dd (5.8, 13.2) 1.35 m; 1.92 m 2.18 m 1.16 s 1.10 s 2.77 m 1.00 d (7.0) 1.39 m; 1.67 ddd (4.2, 9.3, 13.9) 3.88 d 9.0 4.61 br s 1.18 s 1.34 s 1.11 s 1.01 s 1.02 s 2.20 s H HMBC (H to C) 11 9, 11, 13, 14 13 5, 10, 9 17 31 24, 25 24, 25 4, 3, 5 4, 3, 5 7, 8, 9, 14 31 # of alisol A 24-acetate [6], a125 MHz, b500 MHz, cMeasured in CDCl3 *Overlap signals and chemical shift are given in ppm. Assignments were confirmed by COSY, 1D-TOCSY, HMQC, and HMBC experiments. C 123 The NMR spectra of compound 3 were also similar to those of 1, except for the more appearence of the signals of a double bond at C 144.7 (s), 114.1 (t) / H 4.94 (br s) and 4.98 (br s), instead of of the signals of a quaternary carbon at 74.1, and the methyl group at C 26.2/ H 1.21 as shown in the NMR spectra of 1. This evidence suggested that the double bond must be at C-25 and C-27. All the NMR assignments of the Protostane skeleton of 3 were made by comparison with those of 1. In the HMBC spectrum, H-27 H 4.94 (br s) and 4.98 (br s) correlated with C-24 79.9/C-25 ( 144.7)/C-26 ( 17.8) confirming that the double bond was at C-25 and C-27, and that compound 3 must be alisol G. Furthermore, the ESI spectrum of 1 exhibited the ion peaks at m/z 473 [M+H]+, 455 [M-H2O+H]+, 437 [M-2H2O+H]+ and 419 [M-3H2O+H]+, corresponding to the molecular formula of C30H48O4. Oviously, compound 3 was identified as 11 ,23S,24Strihydroxyprotosta-13(17),25-dien-3-one. The stereochemistry of this compound at C-11 was further confirmed by ROESY spectrum. The NOEs correlation between H-11 and H-30 was observed confirming that the hydroxyl group was axial. This is the first report of 3 from Alisma plantago-aquatica L. Table 3: The NMR data of compound 3 C # C a,c C b,c H HMBC (H to C) ROESY 1 31.0 t 31.1 t 2.11 m; 2.25 m 2 33.7 t 33.8 t 2.26 m; 2.69 m 3 220.3 s 220.6 s - 4 5 46.9 s 48.5 d 47.0 s 48.5 d 2.12 m 6 20.0 t 20.1 t 1.30 m; 1.46 m 7 34.3 t 34.3 t 1.25 m; 2.03 m 8 40.6 s 40.6 s - 9 49.6 d 49.6 d 1.75 d (10.5) 10 36.9 s 37.0 s - 11 70.0 d 69.9 d 3.88 ddd (5.8, 10.7, 10.7) H-30 12 34.5 t 34.5 t 2.81 dd (5.8, 13.2); 2.83 m H-11 13 137.7 s 137.9 s - 14 57.0 s 56.0 s - 15 30.6 t 30.6 t 1.23 m; 1.81 m 16 29.1 t 29.1 t 2.16 m 17 135.4 s 135.2 s - 18 23.3 q 23.3 q 1.14 s 8, 13, 14, 15 19 25.7 q 25.6 q 1.05 s 5, 9, 10 20 28.3 d 28.3 d 2.88 m 21 22 20.3 q 38.3 t 20.4 q 38.3 t 1.01 d (7.0) 1.39 m 124 8, 11, 30 17, 20 C # C a,c C b,c H HMBC (H to C) ROESY 23, 26, 27 H-24 H-23 23 24 70.7 d 79.7d 70.8 d 79.9 d 3.49 d (7.5) 3.78 d (7.0) 25 144.6 s 144.7 s - 26 17.9 q 17.8 q 1.67 s 27 113.9 t 114.1 t 28 29.5 q 29.6 q 4.94 br s 4.98 br s 1.07 s 3 29 20.1 q 20.1 q 1.06 s 3 30 24.0 q 24.0 q 1.00 s 24, 25, 27 H-11 # of alisol G [7], a125 MHz, b500 MHz, cMeasured in CDCl3 *Overlap signals and chemical shift are given in ppm. 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