PHYTOCHEMISTRY Phytochemistry 66 (2005) 2304–2308 www.elsevier.

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Cycloartane type triterpenoids from the rhizomes of Polygonum bistorta
Karuppiah Pillai Manoharan a, Tan Kwong Huat Benny b, Daiwen Yang
a b

a,c,*

Department of Chemistry, Faculty of Science, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore Department of Pharmacology, Faculty of Medicine, National University of Singapore, Building MD2, 18 Medical Drive, Singapore 119260, Singapore c Department of Biological sciences, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Singapore Received 5 May 2005; received in revised form 8 July 2005 Available online 19 August 2005

Abstract Two new compounds, 24(E)-ethylidenecycloartanone (1) and 24(E)-ethylidenecycloartan-3a-ol (2) were isolated from the rhizomes of Polygonum bistorta, together with seven known compounds viz., cycloartane-3,24-dione (3), 24-methylenecycloartanone (4), c-sitosterol (5), b-sitosterol (6), b-sitosterone (7), friedelin (8) and 3b-friedelinol (9). All the cycloartane type triterpenoids, compounds 7 and 8 are reported for the first time from this plant. A combination of 1D and 2D NMR spectroscopy and MS were mainly used to elucidate the structures of the new compounds 1 and 2. Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Polygonum bistorta; Cycloartane triterpenoids; b-Sitosterol; b-Sitosterone; Friedelin; 3b-Friedelinol; Cycloartanone

1. Introduction Polygonum bistorta, commonly known as Bistort or Snakeroot, belongs to the Polygonaceae family. It is one of the strongest herb astringents. The ethanolic extract showed strong anti-inflammatory effect (Duwiejua et al., 1994); alnusenone and 3b-friedelinol were identified as active constituents for such effect (Duwiejua et al., 1999). It was also reported that the aqueous extract strongly inhibits the mutagenicity of Trp-P-1 (Miki et al., 1995). In the course of our investigations directed to the search for antitumour natural products from terrestrial plants, we have studied the rhizomes of P. bistorta. Two new and seven known compounds were isolated and characterised by spectral and other data. The known compounds, b-sitosterol (6), friedelin (8)

and 3b-friedelinol (9) were confirmed also through 2D NMR spectroscopy.

2. Results and discussion 2.1. 24(E)-ethylidenecycloartanone (1) The molecular formula of compound 1 was deduced as C32H52O by the molecular ion peak at m/z 452.4019 in the HREIMS and 13C NMR analysis. The IR spectrum of compound 1 showed absorption for ketone functionality (mmax 1712 cmÀ1). Its 13C NMR spectrum exhibited 32 signals including one carbonyl carbon (d 216.4 ppm) of cycloartane type (Cantillo-Ciau et al., 2001) and one ethylidene double bond (d 145.8 and 116.4 ppm). The 1H NMR spectrum of the compound supported the presence of cycloartane type skeleton with typical high-field AB doublets due to the non-equivalent hydrogens at C-19 in the cyclopropane ring (Cantillo-

*

Corresponding author. Tel.: +65 6874 1014; fax: +65 6779 2486. E-mail address: dbsydw@nus.edu.sg (D. Yang).

0031-9422/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.07.008

Precoated silica gel plates (Merck. Experimental 3. However the same compound was reported in pure form (Ohtsu et al.17 ppm. Republic of Singapore. 1998). / Phytochemistry 66 (2005) 2304–2308 2305 Ciau et al.0 ppm. Class II Laser product. Ohtsu et al. We carried out further investigation by purchasing 12 kg of plant material and extracted it as previously. 3. Both extracts were showed significant cytotoxic activity toward the P388 and HL60 cancer cell lines. respectively. The IC50 values of the hexane extract were less than10 and 20 lg/ml toward P388 and HL60. the methyl group at C-31 has E-stereochemistry.25 mm or Baker Si250F. l-Porasil (300 · 5 mm) column with a Shimadzu RID-10A. Fragment peaks at m/z 313 (M À C8H15O) is an indicative of the presence of cycloartane skeleton with C8 side chain. Plant material The plant materials were purchased from local market and a voucher specimen (KMano PB 2003) is deposited in the Department of Biological Sciences.60 ppm as expected for ethylidene group. HREIMS were measured on Finnigan/MAT MAT 95 XL-T mass spectrometers. Fragment peaks at m/z 315 (M À C10H19) is the indicative of cycloartane skeleton with hydroxyl group and C10 side chain. Teres et al.4175 in the HREIMS corresponding to the molecular formula C32H54O. while the chloroform extract had less than 20 lg/ml on both cell lines.P. 1998) but it had inconsistency with our assignment.K.. 1H and 13C NMR spectra were recorded on Bruker. 2. Extraction and isolation The rhizomes of P. The 1H NMR chemical shift values in the latter compound at these positions were d 1. 40–63 lm) was used for separation and/or purification. For a b-orientation.45 (br. NMR assignments were made for this compound and were listed. The chloroform extract was chromatographed over silica gel using n-hexane and fractionated with increasing polarity. HMBC) allowed us to assemble the structure 1. Silica gel 60 (Merck. The residue dissolved in water/methanol mixture (95:5) and was then extracted successively with n-hexane and chloroform. IR spectra were recorded on a Bio Rad.13 ppm. HSQC-DEPT. 0..24-dione (3) Compound (3) has molecular ion peak at m/z 440.. Such correlation also supported the proposed structure as well as used to assign the stereochemistry of compound 1 at C-24. Inspection of its 2D NMR data (1H–1H COSY.3. Kieselgel 60F 254. 2. 9:1) afforded 24-methylenecycloartanone 4 (7 mg) (Ohtsu et al. The hexane extract was chromatographed over silica gel using n-hexane and eluted in a gradient fashion . Excalibur Series. corresponding to the molecular formula C30H48O2. 1994. 1992). In otherwords.. these chemical shift values would be around d 3. 0..57 and 5. Although compound 3 had been reported earlier as an inseparable mixture..1. which is similar to that of fucosterol.28 (m) and d 78. 1998. Fragment peak at m/z 313 (M À C10H19) was also an evidence of the presence of cycloartane skeleton (Ayatollahi et al. 0. 600 Microwatts at 632. General experimental procedures Melting points were determined on a Buchi Melting point B-540 apparatus.. which was confirmed by comparing these values with the related compound (Januario et al. LREIMS were measured on a Finnigan/MAT MAT 95 XL-T or VG Micromass 7035. in particular at the C-3 position. respectively.2. 1997). 1992) with C10 side chain.2. The presence of McLafferty rearrangements product at m/z 354 indicated the presence of a carbonyl group in the side chain at C-24 (Davies et al.. 3. 3. respectively (Greca et al.063– 0. Compound 1 and fucosterol have identical side chain structure. Protons on methyl group at C-32 gave a clearly defined doublet at d 1.. Further purification of the major fraction by HPLC (hexane/chloroform. 1:9) followed by preparative TLC (chloroform/methanol.8 ppm.3655 in the HREIMS.200 m) was used for column chromatography. indicating that the hydroxyl group is a-oriented at this position. 2001). The spectral correlations regarding its side chain structure and NMR chemical shift values for other positions are comparable to compound 1. Lichroprep RP-18 (Merck. The single proton on C-31 was also clearly evident showing the expected quartet at d 5. Cycloartane-3. structure identification was made based on only MS fragmentation pattern of GC-MS analysis (Davies et al... 1987). Spots were detected using UV light or by spraying with 50% H2SO4 and heating at 110 °C for 5 min. Manoharan et al.25 mm) were used for preparative TLC and/or analytical TLC.. The MS fragmentation patterns for compound 3 were identical as reported in the literature (Davies et al. National University of Singapore.8 nm CW. The 1H and 13C chemical shift values at C-3 position were observed as d 3. respectively (Nes et al. 300 or 500 MHz spectrometers. HPLC was carried on a Waters associates. Chemical shifts are reported in parts per million (ppm) with TMS as a reference standard and coupling constants (J) are expressed in hertz. bistorta (600 g) were ground into powder and then extracted with chloroform (3 l · 4) at room temperature. 1966). Bohme et al. 24(E)-ethylidenecycloarta-3a-ol (2) Compound 2 has molecular ion peak at m/z 454.3.. refractive index detector. 1992). 1992. 1992). s) and d 77.

(2003). %): 452 [M]+ (72). 381 (42). m. 1H and 13C NMR data: same as the data reported in Deeb et al. 271 (44). 423 (47).2306 K. 69 (75). 70 eV).10. 1719. 3. Manoharan et al. 1992. 55 (38). 3. 2882. 3.5. 437 (42). 3. 191 (44). 1049 cmÀ1.. 124 (100). %): 412 (90). 175 (51). 315 (72). Deeb et al. 1990). 145 (78).11. 3. 1385 cmÀ1. The chloroform extract was chromatographed over Lichroprep RP-18 and eluted in isocratic fashion with methanol. 271 (20). 370 (60). 1464. 1. 1448. 271 (44). 340 (48). m/z (rel. cycloartane3. 121 (46). 2939. IR (KBr) mmax 3429.4–137. 109 (98). 289 (70).4175 (calcd for C32H54O. MS (EI. 1992. 397 (32). HREIMS m/z 440. 395 (31). 355 (28).p. 231 (38). 1H and 13C NMR data: same as the data reported in Ohtsu et al. m/z (rel. m/z (rel. 300 (39). inten. MS (EI. 1992).4018). m.4019 (calcd. 145 (45).p.24-dione (3) Colorless crystals. 1986. b-sitosterone (ca. (1992). HREIMS m/z 452. / Phytochemistry 66 (2005) 2304–2308 with increasing polarity. MS (EI. 69 (28).8. 70 eV). 0.1 °C. 24(E)-ethylidenecycloarta-3a-ol (2) Colorless solid. inten. IR (KBr) mmax 2946.0 mg) (see Fig. (1998). 135 (87). 342 (30). Nes et al. MS (EI. 147 (49).1–138 °C. 173 (18). 2960. m. 24(E)-ethylidenecycloartanone (7 mg) and 24(E)-ethylidenecycloartan-3a-ol (ca.. 302 (32). 95 (100). 2862. 1376 cmÀ1. 340 (40). 24(E)-ethylidenecycloartanone (580 mg). 452. MS (EI.9. %): 438 (100). 354 (86). 1H NMR data: same as the data reported in Gopalakrishnan et al. 163 (57). 1. 203 (66). 81 (40).. 1H and 13C NMR data: see Table 1. 255 (50). %): 454 [M]+ (72). 313 (72). MS (EI. 379 (19). m/z (rel. 70 eV). m/z (rel. 205 (56). 107 (56). Salazar et al. 216 (78). 95 (100). 175 (84). 69 (32).2 g) (Klass et al. (1990). 3. Purification of eluted fractions afforded friedelin (6 mg) (Klass et al. 271 (42). 95 (68). 302 (59). for C32H52O. IR (KBr) mmax 3040. inten. c-sitosterol (4 mg) (Ulubelen.5 mg). HREIMS m/z 454. b-sitosterol (1. 70 eV). 425 (61). 24-methylenecycloartanone (4) Colorless crystals. 70 eV). %): 414 (64).p. 83 (26). Gopalakrishnan et al. 1H and 13C NMR data: see Table 1. inten.24-dione (ca. 70 eV). 3b-friedelinol (112 mg) (Betancor et al.P. 1442. 219 (34). bistorta.. 327 (28).3654). 55 (43). Biswas and Malik.4174). c-Sitosterol (5) Colorless crystals.. Friedelin (8) Colorless crystals. 2864. MS (EI. 175 (51). 1712. 70 eV). 55 (38) 43 (46). 127 (77). 95 (100). 136. 3. 93 (85).. 13 (100). 149 (64). 216 (78). 1662. 1376. 437 (42). 396 (100). Purification of eluted fractions afforded b-sitosterol (110 mg). 1969. 1H and 13 C NMR data: same as the data reported in Klass et al. 263–265 °C. 55 (54). b-Sitosterone (7) Amorphous solid. 303 (30). 2869. 2000). 2003). %): 440 [M]+ (84). 229 (96). %): 414 (16). 1464. Fig. 313 (100). b-Sitosterol (6) Colorless flakes. MS (EI. 55(15). 135. 354 (78). 231 (40). 440. inten. 55 (27).3655 (calcd for C32H52O.. Cycloartane-3. 273 (54). inten. 1330. . 2939. 341 (12). 2880. 111–113 °C. mp147–148 °C. 814 cmÀ1.5 mg). 24(E)-ethylidenecycloartanone (1) Colorless crystals. 1. 354 (86). 340 (48). inten. 55 (54). IR (KBr) mmax 3046.4. m/z (rel. 1377. 1). 71 (46). 3. m/z (rel. 175 (54). %): 426 [M]+ (28). 187 (30). 313 (75). 407 (21). Structures of compounds isolated from the rhizomes of P.p. 1H and 13C NMR data: see Table 1. 1980. 70 eV). 454. inten. 1712. m/z (rel.6.7. m.

0 37.55 2.88 0.2 18.84 m 1.2 48.5) ddd (13.4. / Phytochemistry 66 (2005) 2304–2308 Table 1 1 H NMR and Position 2307 13 C spectral data of compounds 1.33 1. 2003.12 1.16b 17 18 19a 19b 20 21 22a.87 1.8 45. m/z (rel.14 ddd (16.3 29. Nat. 69 (76). m.61 0. Cantillo-Ciau.5) 1.96 m. J.1 28. C.2 32.8.9.. 1380.16 m 1.24 m 2.S.19 m 1. Sung. Malik.P.5.. R154000187112).04 1. 1.94 0. 159–162.. C. Bohme. Phytochemistry 45.5.16 1. Phytochemistry 31. IR (KBr) mmax 3500. Three triterpenes and other terpenoids from Catha cassinoides.5) 1 dC (ppm) 1a 1b 2a 2b 3 4 5 6a.9 18.5.5).0) m d (5.0. 64. 4. 1986.2 116.9 26. Nat. 1997. 4.4.90 – – m d (6. Abdel-Monem. %): 428 [M]+ (215). 959–962. 1370. 95 (100).8 30.60 d (6.8 21.7 35. T. 220 (42). Prod.0 29.5 28.5.0) (1H) 3.4 215. J..87 1.32 dddd (13.85 1.50 m m m m br. 4.28 1.0.3) 0. 3b-Friedelinol (9) Colorless crystals.58 dd (12.0.7 45. A.11b 12 13 14 15 16a.V.22b 23a 23b 24 25 26 27 28 29 30 31 32 33. a new triterpene from Euphorbia clarkeana. J.4 21.73 1.8 47. .7 32.4..8 26.39 1. 24-Methylenepollinastanone.8 28.06 m.5.7 32. 205 (53).5 28.92 1. related triterprnoids and sterols from Costus tonkinensis.1 34..0.0) 2.M..2 28. 1175.4 21.4.7 19. 413 (27).K.0) d (6.5 41. Triterpenoids from Tillandsia fasciculate.7) ddd (13. 1265.54 2. Triterpenoids in bud exudates of Fijian Gardenia species.57 d (4.56 3. 2 and 3 in CDCl3 2 dH (ppm)...3) m d (6.24 1.M.28 1. 184–191.5) m.. Manoharan et al..78 m (2H) m.3 37..5.. 6.7) (3H) d (6. 2.7) ddd (13.41 0. Cassiadinine. A.70 1.0 22.4 36. Deeb. Salazar.3 – – dH (ppm).0 21. 12.5. 2.5.2 20.94 m. Quijano.61 dd (12. A.58 m m.5 25.06 s l. 1...34 1. Gonzalez.56 2. Pascard.32 m 1. Malik. Miller.. Brito-Loeza. a chromone alkaloid and (+)-6-hydroxymellein.8 149. Prange.7) ddd (13. Z..1 20.3 116.2 48.00 d (6.0 19. Phytochemistry 19.5 35.11 s 0. Cycloclarkeanol.01 0.. Acknowledgements This research has been supported by a grant from the National University of Singapore (Grant No.. K. 125 (98).2 48.4 40.7 35.9 21. 165 (82).37 1. J.92 0..9) 2.0) d (4.5. inten.56 1. Annie Hsu for the technical assistance at the Pharmacology Laboratory. 6.59 m 1.4 216..86 0. Naidu. Prod. 1.33 m m s d (4. 5.8 48. 1.64 t (7.9) 1. 1980.4.6.32 tdd (13. H.9 22.9 26. T.M.5).12.3 48.5.. References Ayatollahi. 1.3 35.92 1. J.13 q (6.S. 13. (2H) 1.W. 4. 1041–1044. R. One of the author.E.45 1.4.33 1. (2H) 1. Betancor.59 1. 12. 4. 177 (57)..9 21.6 18.4) dC (ppm) 27.49 0. 1. K.5) s s s 2. J (Hz) 1.0 52.6b 7a 7b 8 9 10 11a.3 37. Ai-Haidari. Phytochemical and pharmaceutical studies of Maytenus forsskaoliana.68 t (7. 2.3 48. 55(34). 10. 4.1) (1H) 1.92 0.0 39. 800 cmÀ1. 11.5 28.4 50..5. 1. 1989–1993.5 29. 1020. 0. Sotheeswaran.77 m.4 26.8 19. 4. 4.0 19. Rwaida. 1H and 13C NMR data: same as the data reported in Salazar et al.3 36.5) d (4.17 1.09 s 0. 6.36 0. 4. 2.75 1.5 77.57 1.5) (3H) 1.14 ddd (16. 4. 953–955. Freire.93 m dddd (12.6. 231 (52). S.5 25. 2.4) 1.09 0.5 21.80 m m m m 3 dC (ppm) 33. 275 (46) 248 (26). 280–282 °C.12 m m m 1.4 25. Schmidt. 1992. J..72 2.4.5) m. L.9 35.p.8 dH (ppm).5 21. 5. 0. Adam.2) 2.33 1.A.78 d (4.56 1.4) ddd (13.3 29.95 1.5) d (6.39 m 1.80 0. J (Hz) 1. 3.5.85 5. 13. 1.92 s 5.5) m.0 25. Saudi Pharm.03 1.7 1.5.98 1.0 25.G.4 12.2 18. W.8 47.10 m 1.81 0.60 m 2.4) 1.0) s s s q (6. 1. G. Karuppiah Pillai Manoharan thank the National University of Singapore for the financial assistance and to Ms. J (Hz) 1.0 21. 1. Phytochemistry 25. R.10 2.09d (6.72 2.7 19.6 20.7 26. 4.56 0.33 1.3) 1. Biswas. N.5) dddd (12. 2001. (2000). 0. 1727–1730. M.7 45. Z.8 18. Davies.92 m m m m d (6.. F.2 145.80 0. 55.7) ddd (13.4 50. 6. 0.7. Ahmed. 9.1 52. s m m.00 d (6.5.4 216. 4.9 52.2) m.1 20.63 1. 1992.58 1.4 12. a dihydroisocoumarin from Cassia siamea.28 1.9) dd (12.10 m m s d (4. A.96 m m m dd (12.4 18.0 22. J.05 m.32 m (2H) 1.

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