Chymotrypsin Inhibitory Triterpenoids from Silybum marianum

Abstract

Silybum marianum GAERTH is an erect stout biennial, herb belonging to the family Compositae which comprises about 1000 genera and 20000 species. S. marianum is common in Kashmir at an altitude of 1800-4000 m and is cultivated for ornaments in gardens. The plant is used in the treatment of hepatitis, cirrhosis, regeneration from liver diseases, and liver poisoning.1) A decoction of this plant is also used by the local physicians for the treatment of a variety of viral diseases.1) A literature survey revealed that twenty compounds have so far been reported from S. marianum. The major constituents are, however, flavonoids, flavanolignans, diterpenes and flavanoglycosides.2,3) The ethnopharmacological and chemotaxonomic importance of the genus Silybum prompted us to investigate the chemical constituents of S. marianum. A methanolic extract of this plant showed strong toxicity in the brine shrimp lethality test, revealing the possible presence of bioactive compounds. Further pharmacological screening revealed significant inhibitory activity against the enzyme chymotrypsin. The inhibitory activity was most pronounced in the chloroform-soluble fraction. This prompted us to carry out bioassay-guided isolation of bioactive compounds from this fraction. As a result of these studies we have isolated marianine, a new lanostane-type triterpene (1) and marianosides A (2) and B (3), two new triterpenoidal glucosides, respectively. All three showed significant chymotrypsin inhibiting activity. This is the first report of the natural occurrence of lanostane-type triterpenes in the genus Silybum. Results and DiscussionMarianine (1) was isolated from the chloroform fraction of S. marianum as colorless crystals. It gave color reactions of triterpene and the molecular formula was determined to be C 31 H 50 O 3 by [M] ϩ peak in high-resolution ( H correlation spectroscopy (COSY) with two other protons limiting its location to either C-1 or C-3. However, it could be assigned to the usual C-3 position on the basis of heteronuclear multiple-bond connectivity (HMBC) as illustrated in Fig. 2. The larger coupling constant of this proton allowed us to assign b and equatorial configurations to the hydroxyl group which was substantiated by nuclear overhaueser effect (NOE) between H-3 and H-28. The loss of the side chain resulted in a fragment ion at m/z 329, showing the presence of one hydroxyl and an olefinic moiety in the side chain. Since both methyl groups at C-25 were observed downfield as singlets, the hydroxyl group could be referred to C-25 and confirmed by the HMBC correlations (Fig. 2). The methylene group could be assigned to C-24 Marianine, the new lanostane triterpene (1) and marianosides A (2) and B (3) have been isolated from the whole plant of Silybum marianum. Their structures were elucidated on the basis of extensive analysis of their one dimensional and two dimensional nuclear magnetic resonance (1D, 2D-NMR) spectral data. All inhibited chymotrypsin in a concentration-dependent manner.

Chemical Constituents from Euphorbia kansui

“…The relative configuration of 1 was determined by the 1 H-NMR ( Table 1 ) and NOESY data. 1 H-NMR chemical shift of CH 3 -21 (δ H 0.94, d, J = 6.6 Hz) confirmed that compound 1 was classified as the tirucallane series [ 11 , 12 , 13 ]. The large coupling constants H-C(3) (δ H 3.31, J = 9.5, 6.4 Hz) obviously indicated that the 3-OH group was in equatorial β-position [ 10 , 14 ].…”

“…The comparison of 1 H-NMR, 13 C-NMR ( Table 1 and Table 2 ) and NOESY data ( Figure 2 ) showed that compound 2 and the known Compound 1 [ 10 ] were semblable in structure, except that a Hydrogen at C-11 of Compound 1 was superseded by a hydroxyl group in compound 2 , which was proven by correlations of H-11 (δ H 4.69, t, J = 8.2 Hz) with C-8 (δ C 140.44), C-9 (δ C 161.30) and C-12 (δ C 42.83) in HMBC spectrum ( Figure 3 ). The large coupling constants H-C(3) (δ H 3.31, J = 9.5, 6.4 Hz) obviously indicated that the 3-OH group was in equatorial β-position [ 11 , 12 ]. The NOESY correlations ( Figure 2 ) H-C(3) (δ H 3.31)/H-C(5) (δ H 1.62–1.68), H-C(3) (δ H 3.31)/CH 3 -28 (δ H 1.01), H-C(11) (δ H 4.69)/CH 3 -18 (δ H 0.73), CH 3 -30 (δ H 1.16)/H-C(17) (δ H 1.55–1.60), CH 3 -29 (δ H 0.93)/CH 3 -19 (δ H 1.27), CH 3 -18 (δ H 0.73)/CH 3 -19 (δ H 1.27), showed that H-C(3), H-C(5), H-C(17), and CH 3 -28 were all in α -orientation, whereas 11-OH, CH 3 -19, CH 3 -30 and CH 3 -29 were in β-orientation, and no correlations between CH 3 -21/CH 3 -18 [ 10 , 15 ] indicated that it belonged to the euphane-type triterpenes.…”

Chemical constituents of Euphorbia kansui

“…All 12 of the known compounds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were identified according to the spectroscopic data ( 1 H-NMR, 13 7) [4], kansenone (8) [10], epi-kansenone (9) [10], kansuinin A (10) [4,17], kansuinin D (11) [4], kansuinin E (12) [4], euphol (13) [4,18], and tirucallol (14) [4,19].…”

“…The comparison of 1 H-NMR, 13 C-NMR (Tables 1 and 2) and NOESY data (Figure 2) showed that compound 2 and the known Compound 1 [10] were semblable in structure, except that a Hydrogen at C-11 of Compound 1 was superseded by a hydroxyl group in compound 2, which was proven by correlations of H-11 (δ H 4.69, t, J = 8.2 Hz) with C-8 (δ C 140.44), C-9 (δ C 161.30) and C-12 (δ C 42.83) in HMBC spectrum (Figure 3). The large coupling constants H-C(3) (δ H 3.31, J = 9.5, 6.4 Hz) obviously indicated that the 3-OH group was in equatorial β-position [11,12]. The NOESY correlations (Figure 2 17), and CH 3 -28 were all in α-orientation, whereas 11-OH, CH 3 -19, CH 3 -30 and CH 3 -29 were in β-orientation, and no correlations between CH 3 -21/CH 3 -18 [10,15] indicated that it belonged to the euphane-type triterpenes.…”

Four New Lanostane Triterpenoids from Euphorbia humifusa

“…The large coupling constant (J ¼ 11.0 Hz) between HÀC(3) and H b ÀC(2) clearly indicated that the 3-OH group was in equatorial b-position [7]. The NOESY correlations ( showed that 1 and 2 were very closely related to each other, including configuration, except for the monounsaturated side-chain moiety.…”