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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.50 n.3 Concepción sep. 2005

http://dx.doi.org/10.4067/S0717-97072005000300011 

 

J. Chil. Chem. Soc., 50, N° 3 (2005), págs: 587-589

 

PENTACYCLIC TRITERPENES FROM Cirsium pascuarense

 

ROSA MARTHA PÉREZ GUTIÉRREZ

Laboratorio de Investigación de Productos Naturales. Escuela Superior de Ingeniería Química e Industrias extractivas IPN. Punto fijo 16, col. Torres Lindavista cp 07708, México D.F. México.

Dirección para correspondencia


INTRODUCTION

The plant Cirsium pascuarense (H.B.K.) Spreng. (Compositae) is commonly know as "cardo santo" . It is a common herb that grows wild and abundantly in the fields of Mexico. A water extract of the fresh leaves has long been used for treating diabetes mellitus and it is generally believed to produce benefical effects (1). Crude extracts of C. pascuarense have shown to possess a hypoglycemic effect in fasted normal mice and also checked hyperglycemia due to the effect of alloxan (2). In this paper, we had report the isolation of two pentacyclic triterpene from C. pascuarense.

MATERIALS AND METHODS

General Experimental Procedures

IR: KBr , MS: 70eV, Silica gel 60 (0.2-0.5mm). All the NMR were measured in CD3OD. Chemical shifts were referenced to residual hydrogen (3.31 ppm) or carbon 49.15 ppm); coupling constants are given in Hz. 1H and 13CNMR spectra were run on a DPX-300 MHz. Mass spectra were recorder on Jeol CC-Mate.

Plant material

Cirsium pascuarense stem was collected in Noxtepec, Mpo. De Tetipac, state of Guerrero. The material was identified in the Department of Botany of ENEP-Iztacala UNAM, and a voucher specimen of the plant (4967) is stored in the herbarium of this Department for reference.

Extraction and separation of compounds

The leaves were dried at room temperature, grounded and then 5 kg were successively extracted with hexane (70 l) and chloroform (70 l). The chloroform eliminated under reduced pressure at 40 C giving an extract (190 g) which was subjected to chromatography on silica gel (500 g; 100 X 4 cm column) with mixtures of chloroform, hexane-acetone (20:1), CH2Cl2-MetOH (10:1). The fraction that produced green colour on Liebermann-Buchars test was purified by chromatographed on Sephadex LH-20 (80 g) with CHCL3 -MeOH (25:3) yielded compound (I) (90 mg) and Compound 2 (60 mg).

Compound (I). white amorphous powder; IR n max cm-1: 3422 (OH), 3050, 2900-2800 (C-H),1370, 1343 (isopropyl group) and 1660 (C=C), 880; 1HNMR (CDCl3, 300 MHz) d 0.84 (3H, s) , 0.86 (3H, s), 0.88(3H, s), 0.98 (3H, s), 1.01 (3H, s), and 1.68 (3H, s), 2.19 (d, J=16.3 Hz, H-22), 2.68 (d, J=10.8 Hz, H-19), 3.34, and 3.85 (each 1H, d, J=10.4 Hz, H-28), 3.94 (t, J=2.6 Hz, H-3), and 5.26 (t, J=.5.3 Hz, H-12); 5.73 (s, H-29); 13CNMR(CDCl3, 75.5 MHz) d 38.9 (C-1), 27.6 (C-2), 78.8 (C-3), 38.5 (C-4), 55.1 (C-5), 18.2 (C-6), 34.9 (C-7), 41.3 (C-8), 50.7 (C-9), 37.6 (C-10), 23.6 (C-11), 126 (C-12), 141 (C-13), 50.1 (C-14), 27.3 (C-15), 29.5 (C-16), 47.1 (C-17), 47.2 (C-18), 48.7 (C-19), 150.1 (C-20), 29.4 (C-21), 34.4 (C-22), 28.2 (C-23), 15.9 (C-24), 16.8 (C-25), 16.7 (C-26), 14.9 (C27), 61.6 (C-28), 109.3 (C-29), 19.9 (C-30); EIMS 70eV, 440.3893 (M+), Calcd. for C30 H48O2; m/z:440 [M+], (C30H48O2), 340 (C24H36O), 326 (C23H34 O), 232 (C16H24O), 219 (C15H23 O), 208 (C14H24 O), 207(C14H23 O), 201 (232-CH2OH), 191 (232-C3H5), 189 (207-H2O).


Compound (2). white amorphous powder; IR n max cm-1: 3430 (OH), 2915 (CH), 1685 (C=0), 1440 (C=C), 1368 (isopropyl group); ElMS (rel. Int) m/z 472.4563 calcd for C30H4804, 472 [M+], 454, 246, 207, 189 (100); 1HNMR (CDCl3, 300 MHz) d 0.83 (m, H-1), 1.88 (m, H-2), 3.48 (br t, J = 8.2 Hz, H-3), 0.83 (m, H-5), 1.38 (m, H-6), 1.49 (m, H-7), 1.26 (t, J = 9.0 Hz, H-9), 1.20 (m, H-11), 1.17 (m, H-12), 2.61 (m, H-13), 2.19 (t, J = 12.1 Hz, H-15), 4.16 (dd, J = 11.4, 4.3 Hz, H-16), 1.83 (m, H-18), 3.49 (m, H-19), 1.69 (m, H-22), 1.24 (s, Me-23), 1.09 (s, Me-24), 0.88 (s, Me-25), 1.18 (s, Me-26), 1.17 (s, Me-27), 4.79 (s, H-29 ), 1.79 (s, Me-30); 13CNMR(CDCl3, 75.5 MHz) d 38.9 (C-1), 28.7 (C-2), 77.1 (C-3), 39.3 (C-4), 56.0 (C-5), 18.6 (C-6), 34.3 (C-7), 41.6 (C-8), 50.1 (C-9), 37.9 (C-10), 20.8 (C-11), 25.0 (C-12), 38.4 (C-13), 44.2 (C-14), 39.9 (C-15), 75.9 (C-16), 62.1 (C-17), 49.0 (C-18), 48.6 (C-19), 149.9 (C-20), 32.3 (C-21), 36.5 (C-22), 28.5 (C-23), 16.2 (C-24), 16.5 (C-25), 16.6 (C-26), 16.1 (C-27), 178.1 (C-28), 110.4 (C-29), 19.4 (C-30),


RESULTS AND DISCUSSION

Structures determination

The compound 1 was obtained as a white amorphous powder. The high resolution mass spectrum established the molecular weight with the molecular ion at m/z 440 and the molecular formula as C30 H48 O2 . The 1HNMR data indicated that the compound was a terpenoid with five tertiary methyl groups at d 0.84-1.01 (s, 3H each) , and two olefinic protons d 5.73, (H-29) and 5.26 (H-12), an isoprenyl group (d 1.68) is confirmed by the 13CNMR signals at d 19.9 (C-30), 109.3 (C-29) and 150.1(C-20). Analysis of the IR spectrum suggested that it contained a hydroxyl group (3422 cm-1), a terminal double bond (3050, 1660, and 880 cm-1 ). The 13CNMR-DEPT spectrum exhibited the presence of thirty carbon atoms incluiding six methyl, eleven methylene, six methine and seven quaternary carbons. This data suggested that the compound might have a lupane-type triterpene (3) . The carbon signals observed at d 78.8, and 61.6 indicated the presence of a secondary, and a primary hydroxyl group, respectively, also this spectra revealed characteristic signals of an additional primary hydroxyl group d3.34 and 3.85 (each 1H, d, J=10.4 Hz), (4). This CH2OH group was assigned to C-28 based on comparison of 1 with betulin (3). A detailed structural elucidation of compound was carried out using HMBC (Fig. 1) and NOESY spectrum. The NOESY spectrum showed, the connectivities of H-5 with H-9, H-23 and H6a with H-23, H-3, H-27 showed that the primary hydroxyl-group at C-3 is b-oriented (equatorial). The H-20 with H-18 and H-19, demonstrated that isopropyl group has a-dispositions.


 
Figure 1. 1H-13C long-range correlation by HMBC of lupa-12,20(29)- dien-3b,28-diol.

In the mass spectrum of 1 the significant fragments at m/z 232 and 207 derived from the characteristic retro-Diels-Alder cleavages of the molecule, and the ion at m/z 191 [232- isoprenyl group]+, strongly indicated that the compound is of a lup-12-ene type (5). Further, the base peak at m/z 201 [232- CH2OH]+,instead of m/z 232 allowed the location of the second hydroxyl group at C-28. Based on the data spectral comparison of proton (6), and 13CNMR (7) of 1 with oleanderol and betulin, the compound 1 was identified as lupa-12,20(29)-dien-3b, 28-diol.

The compound 2 had molecular formula C30H48O4 as determined by HREIMS and confirmed by l3C NMR and DEPT analysis. The IR spectrum of 2 exhibited hydroxyl (3430 cm-l), carbonyl (1685 cm-l) absorptions. The lHNMR spectrum showed five tertiary methyls d 1.24 (Me-23), 1.09 (Me-24), 0.88 (Me-25), 1.18 (Me-26), 1.17 (Me-27), and one vinylic methyl at d1.79 (Me-30), two protons of an isopropenyl moiety at d 4.79. The l3C NMR spectra of compound 2 revealed 30 carbon signals, which were assigned by DEPT as six methyl, ten methylene, seven methine, six quaternary, one carboxylic acid. This data suggested that the compound might have a lupane-type triterpene (3). The data indicated that 2 was 16b-Hydroxybetulinic acid (8). The spectral data (IR, 1HNMR, 13CNMR and EM) for 2 are in agreement with literature values and betulinic acid parent structure as aliphitolic acid, platanic acid and betulinic acid isolated from the leaves of Syzigium claviflorum (9).

 

REFERENCES

1. Villamar, A.A., Asseleih, L.M.C., Rodarte, M.E. 1994. Atlas de las plantas de la medicina tradicional Mexicana. Instituto Nacional Indigenista México. I: 335.         [ Links ]

2. R.M. Perez G., E. Ramirez L., R. Vargas S. 2001. Effect of Cirsium pascuarense on blood glucose levels of normoglycaemic and alloxan-diabetic mice. Phytotherapy Research 15: 1-3.         [ Links ]

3. Kuo, Y., Chang, C., Kuo, Y,H. 1997.Triterpenes from Diospyros maritima. Phytochemistry 46: 1135-1137.         [ Links ]

4. Yuruker, A., Oriala, J., Sticher, O., Rali, T. 1998.Triterpenes from Rhus taitensis. Phytochemistry 48: 863-866.         [ Links ]

5. Wyllie, S.G. and Djerassi, C. 1968. Mass spectrometry in structural and stereochemical problems CXLVI. Mass spectrometric fragmentation typical of sterols with unsaturated side chains, 33: 305-313.

6. Siddiqui, S., Hafeez, F., Begum, S., Siddiqui, B. 1988. Oleanderol, a new pentacyclic triterpene from the leaves of Nerium oleander. J. Nat. Products 51, 229-233.         [ Links ]

7. Sholichin, M., Yamasaki, K., Kasai, R., Tanaka, O. 1980. 13C Nuclear Resonance of lupane-type triterpenes, lupeol, betulin and betulinic acid Chem. Pharm. Bull. 28: 1006-1008.         [ Links ]

8. Ye, Y., Kinoshita, K., Koyama, K., Takahashi, K., Kondo, N., Yuasa, H. 1998. New Triterpenes from Machaerocereus eruca.J. Nat. Prod. 61: 456-460.         [ Links ]

9. Fujioka , T., Kashiwada, Y., Kilkuskie, R., Cosentino, M., Ballas, L., Jiang, B., Janzen, W. 1994. Anti-Aids agents 11. Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids. J. Nat. Prod. 57: 243-247.         [ Links ]

 

Send information to:
Dra. Rosa Martha Perez Gutierrez
Punto Fijo 16 Col. Torres Lindavista
Cp 07708, Mexico D.F.
E mail: rmpg@.prodigy.net.mx

 

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