Article pubs.acs.org/jnp
Anti-inflammatory Ursane- and Oleanane-Type Triterpenoids from Vitex negundo var. cannabifolia Man-Man Li,†,‡ Xiao-Qin Su,†,‡ Jing Sun,†,‡ Yu-Fan Gu,†,‡ Zheng Huang,†,‡ Ke-Wu Zeng,⊥ Qian Zhang,† Yun-Fang Zhao,† Daneel Ferreira,§ Jordan K. Zjawiony,§ Jun Li,*,† and Peng-Fei Tu*,† †
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China ⊥ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, People’s Republic of China § Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *
ABSTRACT: Six new polyoxygenated triterpenoids, cannabifolins A−F (1−6), and eight known triterpenoids, 7−14, were isolated from the leaves of Vitex negundo var. cannabifolia. The absolute configuration of cannabifolin A (1) was determined by single-crystal X-ray crystallographic analysis. Compounds 1 and 2 represent a class of rare natural pentacyclic triterpenoids bearing cis-fused C/D rings and are the first examples of 12,19epoxy ursane- and oleanane-type triterpenoids. Compounds 3, 7, 8, and 14 exhibited inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages with IC50 values in the range 24.9−40.5 μM.
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to treat colds, cough, asthma, stomachache, diarrhea, and beriberi. In turn, the roots are utilized for the treatment of colds, headache, toothache, malaria, and rheumatic arthralgia,5 while the essential oil from the leaves is used to treat chronic bronchitis.6 Previous phytochemical studies on the different parts of V. negundo var. cannabifolia have resulted in the isolation of diterpenoids,7,8 flavonoids,7−10 iridoid glycosides,7,9 lignans,9 and phenolic glycosides.9,10 In the course of a search for anti-inflammatory agents from medicinal plants of the genus Vitex, the EtOAc-soluble fraction from a 95% EtOH extract of the leaves of V. negundo var. cannabifolia was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages (89% inhibition at 40 μg/mL). Subsequent isolation of the bioactive fraction afforded six new polyoxygenated triterpenoids, cannabifolins A−F (1−6), together with eight known triterpenoids (7−14). Compounds 1 and 2 are rare natural pentacyclic triterpenoids with cis-fused C/D rings and represent the first examples of 12,19-epoxy ursane- and oleanane-type triterpenoids. Herein, the isolation and structural elucidation of the new compounds as well as an evaluation of their in vitro anti-inflammatory activities are described.
he genus Vitex (Verbenaceae) consists of small trees and shrubs, with about 250 species distributed mainly in tropical and subtropical regions. About 14 species, seven varieties, and three forms are found in mainland China.1 Many Vitex plants have been used in traditional medicine worldwide to treat a wide range of ailments, such as menstrual disorders, premenstrual dysphoric disorders, corpus luteum insufficiency, colds, cough, asthma, rheumatism, inflammatory joint conditions, allergy, venereal diseases, malaria, wounds, skin diseases, snake bite, and body pain.2,3 More than 30 species of Vitex have been investigated biologically and phytochemically in the past decades. Crude extracts and pure compounds from Vitex species have been reported to exhibit a wide array of bioactivities including antibacterial, antimalarial, antifeedant, antioxidant, antiviral, antiproliferative, anti-inflammatory, hepatoprotective, antiaging, skin-whitening, antipyretic, analgesic, and potential effects on menopausal symptoms. Phytochemical investigations have indicated the presence of flavonoids, diterpenoids, phytoecdysteroids, iridoid glycosides, triterpenoids, phenylpropanoids, phenolic glycosides, and essential oils in Vitex plants.2−4 Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. (syn.: Vitex cannabifolia Sieb. et Zucc.), a shrub or small tree, is mainly distributed in the Yangzi River basin of the People’s Republic of China. Different parts of V. negundo var. cannabifolia are used in traditional Chinese medicine for the treatment of various diseases. Its fruits, leaves, and stems have been used © XXXX American Chemical Society and American Society of Pharmacognosy
Received: June 22, 2014
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dx.doi.org/10.1021/np500509q | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 1
Table 1. 1H NMR Data of Compounds 1−6 (δ in ppm, J in Hz, 500 MHz, in Pyridine-d5)
a
position
1a
2a
1a 1b 2 3 5 6a 6b 7a 7b 9 11a 11b 12 13 15a 15b 16a 16b 18 19a 19b 20 21a 21b 22a 22b 23 24 25 26 27 29 30
2.06, dd (12.0, 4.5) 1.85, m 4.29, dt (11.0, 3.5) 3.74, d (2.0) 1.70, d (12.5) 1.50, m 1.50, m 1.50, m 1.50, m 2.10, d (11.5) 2.28, d (12.5) 1.81, d (12.5)
2.08, m 1.88, m 4.29, m 3.76, br s 1.71, d (10.5) 1.51, m 1.51, m 1.53, m 1.53, m 2.09, m 2.26, d (13.0) 1.87, m
2.21, d (14.5) 1.89, m 1.08, dt (14.5, 6.5) 2.34, dt (14.0, 6.0) 1.56, m 2.58, d (14.5)
2.13, d (15.5) 1.89, m 1.11, dt (14.5, 5.5) 2.39, dt (14.0, 6.0) 1.56, m 2.92, dd (15.0, 8.5) 3.98, d (8.5)
2.13, m 4.64, t (5.5)
4.32, d (5.5)
2.56, d (12.0) 1.73, d (12.0, 5.5) 1.26, s 0.92, s 0.96, s 1.21, s 1.22, s 1.54, s 1.25, d (8.0)
2.60, d (12.5) 1.81, dd (12.5, 6.0) 1.27, s 0.92, s 0.95, s 1.20, s 1.23, s 1.19, s 1.23, s
3a
4a
5a
6a
2.09, m 1.76, d (12.0) 4.30, m 3.78, br s 1.63, d (12.0) 1.50, m 1.41, m 1.52, m 1.23, d (12.5) 1.67, d (12.0) 2.21, dd (12.0, 5.0) 2.02, m 4.27, m
1.84, m 1.72, m 4.29, dt (12.0, 2.5) 3.77, br s 1.62, d (12.0) 1.53, m 1.47, m 1.52, m 1.21, m 2.01, t (9.5) 2.73, d (9.5) 2.73, d (9.5)
1.86, m 1.72, m 4.29, d (11.0) 3.76, br s 1.61, d (11.5) 1.52, m 1.46, m 1.52, m 1.30, m 1.85, dd (14.5, 3.5) 2.83, t (14.5) 2.64, dd (14.5, 3.0)
1.93, m 1.79, m 4.33, d (10.5) 3.79, br s 1.66, d (12.0) 1.54, m 1.39, m 1.56, m 1.33, m 1.94, m 2.03, m 2.03, m 5.51, br s
2.09, m 1.16, m 2.10, m 1.33, m 2.74, d (11.5) 1.79, d (11.5)
1.86, m 1.18, m 2.08, dt (13.5, 6.0) 1.32, m 2.58, d (11.5) 1.72, d (11.5)
1.98, dd (14.5, 6.5) 1.22, m 2.08, dt (13.0, 6.0) 1.25, m 2.78, t (14.0) 1.72, m 1.72, m
1.83, m 1.18, m 2.20, m 2.03, m 3.46, d (14.5) 2.11, m 1.51, m
0.88, m 1.42, m 1.34, m 1.89, m 1.56, m 1.28, s 0.91, s 0.95, s 1.35, s 1.12, s 1.29, d (5.5) 0.88, overlapped
0.88, m 1.44, m 1.33, m 1.91, m 1.56, m 1.28, s 0.93, s 0.93, s 1.24, s 1.07, s 0.97, d (6.0) 0.84, d (5.5)
1.33, m 1.16, m 1.77, dd (14.0, 4.5) 1.70, m 1.27, s 0.92, s 0.95, s 1.37, s 0.87, s 0.90, s 0.91, s
1.84, m 1.42, m 2.19, m 1.96, m 1.29, s 0.93, s 0.99, s 1.04, s 1.23, s 3.60, br s 1.24, s
Assignments were based on HSQC and HMBC experiments.
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RESULTS AND DISCUSSION A 95% EtOH extract of the leaves of V. negundo var. cannabifolia was suspended in H2O and extracted successively with petroleum
ether, EtOAc, and n-BuOH. The bioactive EtOAc-soluble fraction was subjected repeatedly to silica gel, Sephadex LH-20, and Lichroprep RP-C18 gel column chromatography, followed by B
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Table 2. 13C NMR Data of Compounds 1−6 (δ in ppm, 125 MHz, in Pyridine-d5)
a
position
1a
2a
3a
4a
5a
6a
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
44.0, CH2 66.5, CH 79.7, CH 39.4, C 49.7, CH 18.2, CH2 36.2, CH2 41.8, C 51.0, CH 39.3, C 34.6, CH2 106.1, C 56.3, CH 41.8, C 31.9, CH2 30.9, CH2 45.3, C 51.7, CH 78.7, C 41.4, CH 82.2, CH 29.2, CH2 29.8, CH3 22.5, CH3 18.4, CH3 22.4, CH3 31.8, CH3 179.9, C 35.2, CH3 15.7, CH3
44.0, CH2 66.5, CH 79.6, CH 39.4, C 49.6, CH 18.2, CH2 36.3, CH2 41.8, C 51.0, CH 39.3, C 33.7, CH2 108.5, C 53.7, CH 41.5, C 31.8, CH2 30.1, CH2 44.9, C 44.7, CH 79.9, CH 36.9, C 86.8, CH 31.1, CH2 29.9, CH3 22.5, CH3 18.4, CH3 22.2, CH3 32.0, CH3 180.0, C 29.3, CH3 23.8, CH3
43.8, CH2 66.5, CH 79.7, CH 39.3, C 49.0, CH 18.3, CH2 34.7, CH2 43.6, C 50.3, CH 39.0, C 29.8, CH2 69.5, CH 95.4, C 44.3, C 28.4, CH2 23.4, CH2 46.0, C 52.9, CH 39.2, CH 40.5, CH 31.5, CH2 32.8, CH2 29.9, CH3 22.4, CH3 18.3, CH3 19.4, CH3 17.8, CH3 180.1, C 16.9, CH3 19.9, CH3
42.9, CH2 66.2, CH 79.6, CH 39.2, C 48.8, CH 18.1, CH2 33.2, CH2 42.4, C 48.7, CH 38.9, C 37.4, CH2 206.3, C 90.6, C 43.8, C 26.5, CH2 23.1, CH2 45.7, C 54.3, CH 38.4, CH 40.6, CH 31.3, CH2 32.3, CH2 29.7, CH3 22.3, CH3 17.1, CH3 18.9, CH3 17.1, CH3 178.4, C 19.1, CH3 19.8, CH3
43.3, CH2 66.3, CH 79.5, CH 39.2, C 48.6, CH 17.4, CH2 33.5, CH2 43.0, C 51.5, CH 39.3, C 38.1, CH2 206.6, C 91.4, C 44.3, C 26.4, CH2 21.4, CH2 44.5, C 44.7, CH 37.7, CH2 32.0, C 34.7, CH2 28.3, CH2 29.7, CH3 22.3, CH3 18.0, CH3 19.2, CH3 18.4, CH3 178.5, C 33.4, CH3 24.2, CH3
43.2, CH2 66.5, CH 79.7, CH 39.2, C 49.2, CH 18.9, CH2 33.2, CH2 40.3, C 48.4, CH 39.1, C 24.3, CH2 122.6, CH 145.6, C 42.6, C 28.8, CH2 24.3, CH2 47.6, C 41.8, CH 41.8, CH2 37.0, C 29.9, CH2 33.6, CH2 29.6, CH3 22.7, CH3 17.0, CH3 18.0, CH3 26.5, CH3 180.8, C 74.3, CH2 20.2, CH3
Assignments were based on HSQC and HMBC experiments.
semipreparative RP-HPLC, to afford six new (1−6) and eight known (7−14) triterpenoids. Cannabifolin A (1) was obtained as colorless plates via crystallization from MeOH−H2O (95:5), [α]21D −14. Its molecular formula, C30H46O6, was deduced from the HRESIMS (m/z 525.3200 [M + Na]+, calcd for C30H46O6Na, 525.3187) and 13C NMR spectroscopic data, indicating eight indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3423 cm−1) and carbonyl (1783 cm−1) functionalities. The 1H NMR data (Table 1) displayed characteristic resonances for six tertiary methyl (δH 0.92, 0.96, 1.21, 1.22, 1.26, and 1.54, each 3H, s), one secondary methyl [δH 1.25 (3H, d, J = 8.0 Hz)], and three oxygenated methine [δH 3.74 (1H, d, J = 2.0 Hz), 4.29 (1H, dt, J = 11.0, 3.5 Hz), 4.64 (1H, t, J = 5.5 Hz)] groups. The 13C NMR data of 1 (Table 2) showed 30 carbon resonances comprising seven methyl, seven methylene, eight methine (three oxygenated), and eight quaternary carbons (one oxygenated, one hemiacetal, and one carbonyl). These NMR spectroscopic data were characteristic for an ursane-type triterpenoid skeleton. Comparison of the NMR data of 1 with those of 2α,3αdihydroxyurs-12,20(30)-dien-28-oic acid (7)11 and 2α,3α,19αtrihydroxyurs-12-en-28-oic acid (12)12 suggested that they share the same A- and B-ring substitution patterns. This was supported by the HMBC correlations between H2-1 and C-2/C-3/C-5/ C-10; H-3 and C-1/C-2/C-4/C-5/C-23/C-24; H3-24 and C-3/ C-4/C-5/C-23; and H3-25 and C-1/C-5/C-9/C-10 (Figure 1). The doubly oxygenated nature of C-12 (δC 106.1, hemiacetal carbon) was evident on the basis of HMBC correlations between
Figure 1. Selected HMBC (arrows point from protons to carbons) correlations of compounds 1 and 2.
H-9 and C-11/C-12; H2-11 and C-12/C-13; and H-13 and C-11/C-12. The significantly deshielded C-19 (δC 78.7) and C-21 (δC 82.2) resonances as well as the HMBC correlations between H-18/H-20/H-21/H3-29/H3-30 and C-19 and between H-20/H2-22/H3-30 and C-21 (Figure 1) indicated that C-19 and C-21 are oxygenated. In the 1H NMR spectrum of 1, the deshielded H-21 resonance (δH 4.64, t, J = 5.5 Hz) suggested that the C-21 hydroxy group is acylated, with therefore a γ-lactone moiety between C-21 and C-28 likely. This was supported by the HMBC correlation between H-21 and the C-28 carbonyl carbon at δC 179.9. In view of the mono- and doubly oxygenated nature of C-19 and C-12, respectively, an ether bridge between C-12 and C-19, forming a tetrahydrofuran ring, was proposed to satisfy the eight indices of hydrogen deficiency required by the molecular formula. This arrangement was confirmed by single-crystal X-ray crystallographic analysis. The relative configuration of 1 was established on the basis of the C
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confirmed by the HMBC correlations between H2-1 and C-2/ C-3/C-5/C-10; H-3 and C-1/C-2/C-5; H-5 and C-6/C-10/ C-24; H3-24 and C-3/C-23; H3-25 and C-1/C-5/C-9/C-10; H-9 and C-12/C-25/C-26; H2-11 and C-12/C-13; H-13 and C-12; H-16 and C-28; H-18 and C-13/C-14/C-17/C-19/C-20/C-28; H-19 and C-12/C-13/C-17/C-29/C-30; H-21 and C-17/C-19/ C-28/C-29; and H2-22 and C-17/C-18/C-20/C-21/C-28 (Figure 1). The relative configurations at C-2, -3, -5, -8, -9, -10, -13, -14, -17, -18, and -21 were the same as assigned for 1 on the basis of the NOESY data. The NOE correlations of H-19/H-18 and H3-30 indicated that H-19 is β-oriented. Therefore, the structure of cannabifolin B was defined as shown in 2. It is worth emphasizing that trans fusions of rings A/B, B/C, and C/D and a cis junction of the D/E rings are usually prevalent for the pentacyclic triterpenoids. Ursane- and oleanane-type triterpenoids possessing cis fusion of the C/D rings such as in compounds 1 and 2 are rarely encountered.13,14 The 12,19epoxide moiety and acetalization at C-12 in pentacyclic triterpenoids have never been reported. Thus, compounds 1 and 2 represent the first examples of ursane- and oleanane-type triterpenoids possessing the unprecedented 12,19-epoxy functionality. Cannabifolin B (3) gave the molecular formula of C30H48O5 on the basis of its 13C NMR and HRESIMS data (m/z 489.3578 [M + H]+, calcd for C30H49O5, 489.3575), indicating seven indices of hydrogen deficiency. The IR absorption bands at 3427 and 1763 cm−1 were found to be characteristic of hydroxy and γ-lactone functionalities. The 1H NMR spectroscopic data (Table 1) revealed the presence of five tertiary methyl (δH 0.91, 0.95, 1.12, 1.28, 1.35, each 3H, s), two secondary methyl (δH 0.88, 1.29), and three oxygenated methine (δH 3.78, 4.27, 4.30) groups. The 13C NMR data (Table 2) showed 30 carbon resonances comprising seven methyl, eight methylene, eight methine (three oxygenated), and seven quaternary carbons (one carbonyl and one oxygenated). These data indicated that 3 is an ursane-type triterpenoid. Comparison of the 1H and 13C NMR spectroscopic data (Tables 1 and 2) with those of 2α,3αdihydroxyurs-12-en-28-oic acid (10)15 suggested that the structure of 3 is similar to that of 10. The main differences were the absence of the C-12−C-13 trisubstituted double bond and the presence of an oxygenated methine (δH 4.27, δC 69.5) and an oxygenated quaternary carbon (δC 95.4) in 3. These changes indicated that the additional methine and quaternary carbons are located at C-12 and C-13, respectively, as confirmed by HMBC correlations between H-9 and C-12; H2-11 and C-12/ C-13; H-18 and C-13; and H3-27 and C-13 (Figure 4). In view of
Figure 2. Key NOE correlations of compound 1.
NOESY data (Figure 2). NOE correlations of H-2/H3-24 and H3-25, H-3/H3-23 and H3-25, H3-25/H3-26, H-5/H-9, H3-24/ H3-25, H3-23/H-5, H-11α/H-9, and H-11β/H3-26 indicated that the A/B and B/C rings are trans-fused, and the relative configurations at C-2, -3, -5, -8, -9, and -10 of 1 are the same as those of 2α,3α,19α-trihydroxyurs-12-en-28-oic acid (12).12 The NOEs of H-13/H3-27, H-18/H3-26, H-13/H-22α, and H-16β/ H-18 suggested the α-orientation of H-13 as well as the cis fusion of the C/D and D/E rings. In turn, the NOE correlations of H-18/H3-29 and H-20/H3-29 indicated that these hydrogens are cofacial and β-oriented, while the correlations of H-13/H3-30 and H-21/H3-30 suggested that these protons are α-cofacially oriented (Figure 2). The absolute configuration of 1 was defined as (2R,3S,5S,8R,9R,10S,12R,13S,14R,17R,18S,19R,20R,21S) by single-crystal X-ray diffraction analysis (Figure 3).
Figure 3. ORTEP drawing of compound 1 (the ORTEP diagram is one of four independent molecules of compound 1).
Cannabifolin B (2) was obtained as a white, amorphous powder, [α]21D +37. It had the same molecular formula of C30H46O6 as 1 according to 13C NMR spectroscopic data and the observed pseudomolecular ion at m/z 501.3226 [M − H]− (calcd for C30H45O6, 501.3222) in the negative-ion HRESIMS. Comparison of the 1D NMR spectroscopic data of 2 (Tables 1 and 2) with those of cannabifolin A (1) suggested that an oxygenated quaternary carbon (δC 78.7, C-19) and a methine (δH 2.13, m; δC 41.4, C-20) in 1 were replaced by an oxygenated methine (δH 3.98, d, J = 8.5 Hz; δC 79.9, C-19) and a quaternary carbon (δC 36.9, C-20) in 2, respectively. This is in agreement with an oleanane-type triterpenoid skeleton for 2, which was supported by the presence of a methyl singlet in 2 instead of a doublet for H3-30 in 1. The molecular structure of 2 was
Figure 4. Selected HMBC (arrows point from protons to carbons) correlations of compounds 3 and 4.
the seven indices of hydrogen deficiency, there must be an additional ring in the structure of 3. A γ-lactone moiety between C-13 and C-28 was proposed on the basis of the deshielded C-13 (δC 95.4) resonance.16,17 The molecular structure of 3 was D
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(Table 1) displayed singlets for six tertiary methyl groups at δH 0.93, 0.99, 1.04, 1.23, 1.24, and 1.29, as well as the resonances of an olefinic proton [δH 5.51 (1H, br s)], two oxygenated methines [δH 3.79 (1H, br s), 4.33 (1H, d, J = 10.5 Hz)], and an isolated oxygenated methylene [δH 3.60 (2H, br s)]. The 13C NMR spectrum showed 30 carbon resonances including six methyl, 10 methylene (one oxygenated), six methine (one olefinic and two oxygenated), and eight quaternary carbons (one carbonyl and one olefinic). These spectroscopic data revealed that compound 6 is an oleanane-type triterpenoid. The 13C NMR data of 6 (Table 2) were nearly superimposable on those of 3-epimaslinic acid (8),18 except for the significantly deshielded C-29 (δC 74.3; ΔδC +40.7) and C-20 (δC 37.0; ΔδC +5.7) resonances and the shielded C-19 (δC 41.8; ΔδC −5.1), C-21 (δC 29.9; ΔδC −4.7), and C-30 (δC 20.2; ΔδC −4.0) resonances. This is in agreement with the replacement of a 29-CH3 in 8 by a hydroxymethylene functionality in 6,19 which was supported by the HMBC correlations between H2-29 and C-19/C-20/ C-21/C-30 and between H3-30 and C-19/C-20/C-21/C-29 (Figure 6). The relative configurations at C-2, -3, -5, -8, -9, -10,
confirmed by the HMBC correlations as shown in Figure 4. The relative configurations at C-2, -3, -5, -8, -9, -10, -14, -17, -18, -19, and -20 were the same as those for 10 on the basis of NOESY data (Figure 5). The NOE correlations of H-12/H-9, H3-27, and
Figure 5. Key NOE correlations of compound 3.
H3-29 and of H3-27/H-12 and H-19 indicated the β-orientations of 12-OH and the γ-lactone moiety. Thus, the structure of 3 was characterized as 2α,3α,12β-trihydroxyursan-13β,28-olide and was named cannabifolin C. Compound 4 was obtained as a white, amorphous powder, [α]21D −15. Its molecular formula was determined as C30H46O5 from the 13C NMR and negative-ion HRESIMS data (m/z 531.3317 [M + HCOO]−), indicating the molecular mass of 4 to be two mass units less than that of 3. The IR spectrum of 4 showed absorption bands for hydroxy (3441 cm−1), γ-lactone (1781 cm−1), and carbonyl (1719 cm−1) groups. The 1 H and 13C NMR spectroscopic data of 4 were similar to those of 3, implying that these compounds share the same carbon skeleton. Analysis of its 1D (Tables 1 and 2) and 2D NMR data (Figure 4) indicated that the C-12 hydroxymethine function in 3 is replaced by a carbonyl group (δC 206.3) in 4. The relative configurations at C-2, -3, -5, -8, -9, -10, -13, -14, -17, -18, -19, and -20 were the same as assigned for 3 on the basis of NOESY data. Therefore, the structure of compound 4 was established as 2α,3α-dihydroxy-12-oxo-urs-13β,28-olide and was named cannabifolin D. Cannabifolin E (5) exhibited the same molecular formula C30H46O5 as 4, as established by the 13C NMR and negative-ion HRESIMS data (m/z 531.3318 [M + HCOO]−). Analysis of the 1 H and 13C NMR spectroscopic data of 5 (Tables 1 and 2) showed a close structural resemblance to 4. The major difference was that two methine functionalities (δH 1.72, δC 38.4; δH 0.88, δC 40.6) in 4 were replaced by a methylene (δH 1.72, δC 37.7) and a quaternary carbon (δC 32.0) in 5, suggesting that 5 is an oleanane-type triterpenoid. This was supported by the presence of two methyl singlets for H3-29 and H3-30 in 5 instead of two doublets in 4, as well as the HMBC correlations between H3-29 and C-19/C-20/C-21/C-30 and between H3-30 and C-19/ C-20/C-21/C-29. The relative configurations at C-2, -3, -5, -8, -9, -10, -14, -17, and -18 as assigned via NOE associations were the same as defined for 4. Accordingly, the structure of 5 (cannabifolin E) was established as 2α,3α-dihydroxy-12-oxoolean-13β,28-olide. Cannabifolin F (6) was obtained as a white, amorphous powder, [α]21D −7. Its negative-ion HRESIMS data showed a pseudomolecular ion at m/z 487.3416 [M − H]−, which, in conjunction with the 13C NMR data, established a molecular formula of C30H48O5, with seven indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3426 cm−1) and carbonyl (1697 cm−1) functionalities. The 1H NMR data
Figure 6. Selected HMBC (arrows point from protons to carbons) and NOE correlations of compound 6.
-14, -17, and -18 were the same as those of 8 on the basis of NOESY data (Figure 6). The NOE correlations of H3-30/H-18 and H-22β and of H2-29/H-19α, H-19β, H-21α, and H-21β indicated the α-orientation of the C-29 hydroxymethylene group. Thus, the structure of 6 (cannabifolin F) was defined as 2α,3α,29-trihydroxyolean-12-en-28-oic acid. By comparing spectroscopic and specific rotation data with literature values, the eight known compounds were identified as 2α,3α-dihydroxyurs-12,20(30)-dien-28-oic acid (7),11 3-epimaslinic acid (8),18 ursolic acid (9),20 2α,3α-dihydroxyurs-12-en28-oic acid (10),15 2α-hydroxyursolic acid (11),21 2α,3α,19αtrihydroxyurs-12-en-28-oic acid (12),12 2α,3α,24-trihydroxyolean12-en-28-oic acid (13),18 and tormentic acid (14).21 Compounds 1−14 were evaluated for their inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages using the Griess assay.22−25 As shown in Table 3, compounds 3, 7, 8, 13, and 14 moderately inhibited NO production with IC50 values of 34.0, 26.1, 27.7, 40.5, and 24.9 μM, respectively. Compounds 1, 2, 4, 6, and 12 were inactive (
