Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
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Grayanane Diterpenoid Glucosides from the Leaves of Rhododendron micranthum and Their Bioactivities Evaluation Na Sun,† Yan Zhu,‡ Haofeng Zhou,† Junfei Zhou,† Hanqi Zhang,† Mengke Zhang,† Hong Zeng,† and Guangmin Yao*,† †
J. Nat. Prod. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 11/19/18. For personal use only.
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ School of Chemistry and Pharmaceutical Engineering, Taishan Medical University, Tai-An 271016, People’s Republic of China S Supporting Information *
ABSTRACT: Thirteen new grayanane diterpenoid glucosides, 3-epi-grayanoside B (1), micranthanosides A−E (2−6), 7αhydroxygrayanoside C (7), micranthanoside F (8), 14β-acetyoxymicranthanoside F (9), micranthanoside G (10), 14-Oacetylmicranthanoside G (11), 14β-hydroxypieroside A (12), and micranthanoside H (13), and six known analogues (14−19) were isolated from the leaves of Rhododendron micranthum. The structures of 1−19 were elucidated based on spectroscopic analysis, comparison with literature, and chemical methods. The absolute configurations of 3-epi-grayanoside B (1) and micranthanosides A (2) and C (4) were defined by single-crystal X-ray diffraction analysis. This is the first report of the crystal structures of grayanane diterpenoid glucosides. 3-epi-Grayanoside B (1) represents the first example of a 3α-oxygrayanane diterpenoid glucoside, and micranthanosides A−D (2−5) are the first examples of 5α-hydroxy-1-βH-grayanane diterpenoids. In addition, micranthanosides C−F (4−6 and 8) and 14β-acetyoxymicranthanoside F (9) represent the first examples of grayanane glucosides with the glucosylation at C-16. All the grayanane diterpenoid glucosides 1−19 were assayed for their antiinflammatory, antitumor, and PTP1B inhibitory activities, but did not show significant activities at 40 μM. Grayanane diterpenoid glucosides 1−18 were evaluated for their antinociceptive activity, and compounds 2, 3, 7−10, 12, 13, and 16 showed significant antinociceptive effects with percentage inhibitions in excess of 50%.
G
Rhododendron micranthum Turcz., mainly growing in north and northeast China, as well as North Korea, is a semievergreen shrub belonging to the Ericaceae family. As a folk medicine, the leaves and branches of R. micranthum are used to treat rheumatoid arthritis, puerperal arthrodynia, menoxenia, hypertension, fracture, dysmenorrhea, and chronic tracheitis, in China.18 Previous phytochemical investigations revealed the presence of micranthane,19 grayanane, and leucothane type diterpenoids,20 and flavonoids in R. micranthum.21,22 However, there is no report of diterpenoid glucosides from R. micranthum. In the course of exploring new diterpenoids from the Ericaceae plants,23−26 the n-BuOH fraction of R. micranthum, which were re-collected in July 2012 at Mountain Tai, Shandong, People’s Republic of China, was investigated, leading to the isolation of 19 grayanane diterpenoid glucosides,
rayanane diterpenoids, featuring a 5/7/6/5 tetracyclic diterpene carbon skeleton and four methyls, are also known as B-homo-A-nor-ent-kaurane diterpenoids. Grayanane diterpenoids exist exclusively in the Ericaceae plants, such as Craibiodendron, Kalmia, Leucothoe, Lyonia, Pieris, and Rhododendron genera.1 To date, more than 100 grayanane diterpenoids have been reported from nature; however, grayanane diterpenoid glucosides are relatively rare. Since the first grayanane diterpenoid glucoside, grayanoside A, was isolated from Leucothoe grayana in 1978,2 only a total of 23 grayanane diterpenoid glucosides have been reported from the Ericaceae plants, namely, grayanosides A−D,2−5 craiobiosides A and B,6 rhodomosides A−F,7,8 1β-rhodomoside B,9 rhojaponin VI-3-glucoside,8,10 pierosides A 11 and C,12 pierisformosides A−C,13,14 pierisformosides H and I,15 3β(β-D-glucopyranosyloxy)-5β,6β,10α,16β-tetrahydroxygrayan9(11)-ene,16 and rhododeoside I.17 Among them, craiobioside A and rhojaponin VI-3-glucoside were reported to show significant antinociceptive activity.8 © XXXX American Chemical Society and American Society of Pharmacognosy
Received: June 18, 2018
A
DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Chart 1
similarities. However, the major differences were that the C-3 resonance in 1 (δC 85.9) was shifted upfield compared to that (δC 89.1) in 18, while the H-3 resonance in 1 (δH 4.24) was shifted downfield compared to that (δH 3.79) in 18. Thus, compound 1 should be a 3-epimer of grayanoside B. The NMR data [H-1 (δH 3.13, t), C-1 (δC 43.1)] indicated that H-1 was α-oriented.20 The α-orientations of H-6 and H319 were assigned by the NOESY correlations (Figure 1) of H1α to H-6 and H3-19. The strong NOESY correlation between H3-18β and H-3 and the weak NOESY correlation between H3-19α and H-3 indicated the β-orientation of H-3. To determine the absolute configuration of the glucose moiety, 3-epi-grayanoside B (1) was hydrolyzed and the trimethylsilylthiazolidine derivative of the hydrolysate of 1 was prepared.27,28 The hydrolysate derivative of 1 showed a similar retention time (tR = 14.8 min) to that of the standard Dglucose, but differing from that (tR = 15.3 min) of the standard L-glucose. Thus, the absolute configuration of the glucose in 3epi-grayanoside B (1) was determined to be D. The large coupling constant of H-1′ (J = 7.6 Hz) revealed the βglucopyranosyl linkage in 1.27,28 Finally, single-crystal X-ray diffraction analysis confirmed the structure of 1 as 3α-(β-D-glucopyranosyloxy)-5β,6β,16αtrihydroxygrayan-10(20)-ene (Figure 2), and the calculated Flack parameter of 0.00(7)29 permitted assignment of its absolute configuration. This is the first report of the crystal structure of a grayanane diterpenoid glucoside. More importantly, 3-epi-grayanoside B (1) represents the first example of a 3α-oxygrayanane diterpenoid glucoside, although the first 3α-oxygrayanane diterpenoid aglycone, pierisformosoid E, was recently reported.30 Micranthanoside A (2), colorless prisms (MeOH), mp 133− 134 °C, showed an [M + Na]+ ion at m/z 505.2762 in the HRESIMS. Using the 13C NMR data, the molecular formula of micranthanoside A (2) was determined to be C26H42O8 (calcd for C26H42O8Na, 505.2777), which has one less oxygen atom than the known compound grayanoside C (15).4 The NMR
1−19, including the 13 new compounds 3-epi-grayanoside B (1), micranthanosides A−E (2−6), 7α-hydroxygrayanoside C (7), micranthanoside F (8), 14β-acetyoxymicranthanoside F (9), micranthanoside G (10), 14-O-acetylmicranthanoside G (11), 14β-hydroxypieroside A (12), and micranthanoside H (13). 3-epi-Grayanoside B (1) represents the first example of a 3α-oxygrayanane diterpenoid glucoside, and micranthanosides A−D (2−5) are the first examples of 5α-hydroxy-1-βHgrayanane diterpenoids. In addition, micranthanosides C−F (4−6 and 8) and 14β-acetyoxymicranthanoside F (9) are the first examples of grayanane glucosides with glucosylation at C16. Herein, the extraction, isolation, structural determination, and antinociceptive, antitumor, anti-inflammatory, and PTP1B inhibitory activities of the grayanane diterpenoid glucosides 1− 19 are reported.
■
RESULTS AND DISCUSSION 3-epi-Grayanoside B (1) was isolated as colorless prisms (MeOH), mp 230−231 °C. Its molecular formula was assigned as C26H42O9 by the 13C NMR data and the HRESIMS ion at m/z 521.2725 [M + Na]+ (calcd for C26H42O9Na, 521.2727). The 1H NMR data of 3-epi-grayanoside B (1) (Table 1) exhibited resonances assignable to three methyl singlets (δH 1.04, H3-19; 1.16, H3-18; 1.34, H3-17), two oxymethines (δH 4.24, H-3; 3.88, H-6), two olefinic protons (δH 5.04, s; 4.90, s, H2-20), and a glucopyranosyl group (δH 4.25, d; 3.18, t; 3.34, dd; 3.33, t; 3.24, ddd; 3.69, dd; 3.86, dd).27,28 Combined with the DEPT and HSQC data, 26 carbon signals in the 13C NMR spectrum were assigned to be an exocyclic double bond (δC 113.2, C-20; 153.6, C-10), a glucopyranosyl unit (δC 103.4, 78.4, 78.0, 75.3, 71.8, 62.9),27,28 three methyls (δC 25.4, 21.9, 19.1), six methylenes (δC 63.2, 46.5, 35.2, 36.0, 26.4, 24.4), five methines (δC 85.9, 71.8, 55.6, 48.1, 43.1), two oxygenated tertiary carbons (δC 81.5, C-16; 83.0, C-5), and two quaternary carbons (δC 51.2, C-4; 45.1, C-8) (Table 2). 3-epi-Grayanoside B (1) has the same molecular formula as known compound 18 (grayanoside B),3 and their NMR data also showed some B
DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 1. 1H NMR [δ, mult. (J in Hz)] Spectroscopic Data for Compounds 1−7 in Methanol-d4 (400 MHz) no.
1
1α 1β 2α
3.13, dd (9.8, 8.9)
2β 3α 3β 6α 6β 7α 7β 9β 11α 11β 12α 12β 13α 14α 14β 15α 15β 17 18 19 20a 20b 1′ 2′ 3′ 4′ 5′ 6′a 6′b
1.87, ddd (13.0, 9.8, 6.6) 2.12, dt (13.0, 8.9)
2
3
2.99, t (10.2) 2.21, ddd (14.1, 9.7, 4.8) 1.82, ddd (14.1, 9.7, 4.8) 4.15, dd (9.7, 4.8)
3.00, t (10.1) 2.20, dt (14.2, 10.1)
4.24, dd (8.9, 6.6) 3.88, dd (10.4, 1.3) 1.29, m 1.94, m 1.39, dd (14.0, 1.3) 1.51, m 2.02, dd (14.0, 1.70, m 10.4) 2.46, t (7.1) 2.07, d (5.5) 1.66, m 1.80, m 1.52, m 1.90, m 1.58, m 1.59, overlap 1.43, m 1.77, overlap 1.90, m 1.85, m 1.76, d (11.4) 1.55, m 1.84, dd (11.4, 3.1) 1.77, overlap 1.70, d (14.6) 1.87, d (14.2) 1.83, d (14.6) 1.58, d (14.2) 1.34, s 1.34, s 1.16, s 1.04, s 1.04, s 0.96, s 5.04, s 5.21, s 4.90, s 5.11, s 4.25, d (7.6) 4.28, d (7.8) 3.18, t (7.6) 3.17, dd (8.4, 7.8) 3.34, dd (9.2, 7.6) 3.32, t (8.4) 3.33, t (9.2) 3.29, dd (9.3, 8.4) 3.24, ddd (9.2, 5.3, 3.24, ddd (9.3, 5.4, 2.2) 2.2) 3.86, dd (11.8, 2.2) 3.84, dd (11.9, 2.2) 3.69, dd (11.8, 5.3) 3.67, dd (11.9, 5.4)
4
5
6
7
1.81, ddd (14.2, 10.1, 4.9) 4.14, dd (10.1, 4.9)
2.99, t (10.1) 2.97, t (10.1) 2.77, t (9.3) 2.20, dt (13.9, 10.1) 2.20, dt (15.7, 10.1) 2.24, ddd (14.3, 9.3, 4.5) 1.53, ddd (13.9, 1.51, ddd (15.7, 1.86, ddd (14.3, 10.1, 4.8) 10.1, 4.8) 9.3) 4.12, dd (10.1, 4.8) 4.12, dd (10.1, 4.8) 3.70, d (4.5)
2.73, t (9.1) 2.26, ddd (14.1, 9.1, 4.8) 2.16, dd (14.1, 9.1)
1.26, 1.95, 1.48, 1.77,
m m m m
1.32, 1.92, 1.74, 1.63,
3.89, d (8.8)
1.99, 1.91, 1.81, 1.80, 1.62,
d (5.7) m overlap m overlap
2.08, br. s 1.77, m 1.77, m 1.77, overlap 1.60, overlap 2.14, m 1.45, d (11.3) 1.86, d (11.3, 4.7) 1.75, d (14.0) 1.95, d (14.0) 1.39, s 0.96, s 0.86, s 5.21, s 5.09, s 4.40, d (7.8) 3.11, dd (8.8, 7.8) 3.36, t (8.8) 3.28, dd (9.4, 8.8) 3.22, ddd (9.4, 5.4, 2.3) 3.80, dd (11.9, 2.3) 3.62, dd (11.9, 5.4)
1.47, d (11.3) 1.73, dd (11.3, 2.1) 1.66, d (14.6) 1.84, d (14.6) 1.19, s 1.04, s 0.95, s 5.22, s 5.13, s 4.27, d (7.8) 3.17, dd (8.8, 7.8) 3.32, t (8.8) 3.29, dd (9.3, 8.8) 3.23, ddd (9.3, 5.1, 2.9) 3.84, dd (12.0, 2.9) 3.67, dd (12.0, 5.1)
m m m m
1.28, 1.92, 1.51, 1.78,
m m overlap m
2.01, 1.94, 1.86, 2.03, 1.73,
d (7.0) overlap m m m
1.65, d (11.6) 1.96, overlap 1.63, d (14.4) 1.80, d (14.4) 1.27, s 0.96, s 0.86, s 5.23, s 5.11, s 4.52, d (7.8) 3.18, dd (9.1, 7.8) 3.35, t (9.1) 3.26, dd (9.4, 9.1) 3.26, ddd (9.4, 5.0, 1.8) 3.82, dd (11.8, 1.8) 3.63, dd (11.8, 5.0)
3.55, d (10.5) 1.36, d (13.9) 2.63, dd (13.9, 10.5) 2.04, d (6.3) 1.76, overlap 1.62, m 1.79, m 1.57, m 2.22, m 1.75, d (11.3) 1.92, dd (11.3, 4.3) 1.64, d (14.6) 1.94, d (14.6) 1.39, s 0.96, s 1.20, s 5.19, s 5.19, s 4.42, d (7.8) 3.13, dd (8.7, 7.8) 3.37, t (8.7) 3.28, dd (9.5, 8.7) 3.23, ddd (9.5, 5.4, 2.2) 3.81, dd (11.8, 2.2) 3.63, dd (11.8, 5.4)
3.86, d (4.8)
3.39, d (8.8) 2.10, d (7.2) 1.79, m 1.72, m 1.92, m 1.64, m 1.92, m 2.11, d (11.1) 1.40, dd (11.1, 4.4) 1.56, d (14.2) 2.40, d (14.2) 1.35, s 1.03, s 1.29, s 5.25, s 5.20, s 4.36, d (7.8) 3.18, dd (8.9, 7.8) 3.34, dd (8.9, 7.9) 3.28, dd (9.5, 7.9) 3.26, ddd (9.5, 5.1, 2.0) 3.85, dd (11.8, 2.0) 3.67, dd (11.8, 5.1)
The 13C NMR and HRESIMS data assigned the molecular formula of micranthanoside B (3) as C26H42O9, possessing one more oxygen atom than micranthanoside A (2). The NMR data of 3 and 2 were closely comparable, the difference being the deshielding of C-13 (δC 79.2) in 3 compared to that (δC 49.4) in 2 (Tables 1 and 2). Thus, 3 is a 13-hydroxy derivative of micranthanoside A (2). The HMBC correlation from H3-17 (δH 1.19, s) to C-13 (δC 79.2) supported this deduction (Figure S2, Supporting Information). Similar to 2, the βorientation of H-1 in 3 was deduced from the chemical shift of C-1,20 and the β-orientation of H-9 was indicated by the NOESY correlation between H-1β (δH 3.00, t) and H-9 (δH 1.99, d). The α-orientation of 13-OH and the β-orientation of H3-17 were established by the NOESY correlations from H-9β to H-15β and from H-12β to H3-17. Thus, the structure of 3 was defined as 3β-(β-D-glucopyranosyloxy)-5α,13α,16α-trihydroxy-1-βH-grayan-10(20)-ene. Micranthanoside C (4), colorless prisms, possesses the same molecular formula (C26H42O8) as micranthanoside A (2), which was deduced from the 13C NMR data and the HRESIMS ion at m/z 505.2790 (calcd for C26H42O8Na, 505.2777). The NMR data of 4 were similar to those of 2 (Tables 1 and 2), except that C-3 in 4 (δC 80.2) was shifted upfield compared to that (δC 89.4) in 2, while C-16 in 4 (δC
data of 2 closely resembled those of compound 15 (Tables 1 and 2), and the major difference was the shielding of C-6 (δC 33.1) in 2 compared to that (δC 71.7) in 15. Thus, micranthanoside A (2) may be a 6-deoxy derivative of 15. HSQC, 1H−1H COSY, and HMBC data of 2 supported the above conclusion (Figure S1, Supporting Information). The deshielded C-1 (δC 51.8) suggested the β-orientation of H-1 in 2.20 However, C-5 (δC 83.3) and C-1 (δC 51.8) in 2 were shielded compared to those (δC 87.0, C-5; 59.8, C-1) in 15, indicating the α-orientation of 5-OH in 2. NOESY correlations (Figure S1, Supporting Information) from H-1β (δH 2.99, t) to H3-18 (δH 1.04, s) and H-9 (δH 2.07, d) established the βorientations of H-9 and H3-18. A strong NOESY correlation between H-3 (δH 4.15, dd) and H3-19α (δH 0.96, s) revealed the α-orientation of H-3. Similar to 1, comparison of the GC retention time of the derivative of the hydrolysate of 2 with those of the standards established the D-absolute configuration of the glucose moiety in 2. The large coupling constant of H-1′ (J = 7.8 Hz) revealed the β-glucopyranosyl linkage in 2. Finally, the structure of micranthanoside A (2) was proved to be 3β-(β- D -glucopyranosyloxy)-5α,16α-dihydroxy-1-βHgrayan-10(20)-ene by single-crystal X-ray diffraction, and the calculated Flack parameter of −0.02(9)29 permitted assignment of its absolute configuration (Figure 3). C
DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Table 2.
13
Article
C NMR Spectroscopic Data for Compounds 1−13 in Methanol-d4 (100 MHz)
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ Ac
43.1 35.2 85.9 51.2 83.0 71.8 46.5 45.1 55.6 153.6 24.4 26.4 48.1 36.0 63.2 81.5 25.4 21.9 19.1 113.2 103.4 75.3 78.4 71.8 78.0 62.9
51.8 35.4 89.4 51.3 83.3 33.1 36.8 48.0 55.5 151.8 25.4 26.7 49.4 40.9 58.9 81.6 24.2 20.9 19.4 111.6 106.2 75.7 78.4 71.8 77.9 62.9
51.1 35.4 89.4 51.9 83.3 33.2 41.1 44.2 54.1 151.3 26.4 33.6 79.2 42.1 56.8 81.8 21.0 20.9 19.4 111.8 106.2 75.7 78.4 71.8 77.9 62.9
51.3 36.2 80.2 51.6 83.7 33.7 40.3 48.1 55.5 151.9 25.6 26.6 47.7 36.5 56.7 90.2 21.1 20.5 19.1 111.4 99.3 75.3 78.5 71.9 77.8 63.0
51.7 36.2 80.1 51.1 83.8 33.5 41.0 44.9 54.2 150.8 26.3 31.4 80.0 39.7 56.5 88.7 22.0 20.5 19.1 111.9 99.5 75.4 78.3 71.8 77.9 62.8
59.2 36.9 85.0 51.5 87.6 72.0 48.2 46.4 56.7 154.4 27.2 27.7 47.9 35.7 55.3 89.2 21.3 23.0 20.6 111.5 99.4 75.4 78.4 71.9 77.8 63.0
59.0 35.6 93.5 53.1 86.2 71.6 73.6 52.2 54.1 154.1 27.9 27.6 49.7 28.6 52.0 80.4 24.4 23.6 21.0 111.5 105.4 75.7 78.3 71.8 78.1 62.8
44.3 39.1 82.6 51.4 83.7 72.4 45.8 45.1 55.7 153.0 24.2 26.9 46.0 35.7 61.3 90.1 22.5 24.9 19.5 113.6 99.5 75.4 78.4 71.9 77.8 63.0
44.6 39.4 82.4 51.2 83.6 71.5 40.1 48.1 56.0 151.4 24.7 24.7 48.6 83.9 61.2 88.4 24.5 24.8 19.4 114.2 99.6 75.4 78.4 72.2 78.1 63.5 174.8 22.2
51.8 34.9 92.7 53.3 84.8 75.0 44.0 53.0 56.7 79.6 22.7 27.4 56.2 80.1 60.1 81.3 23.4 24.0 19.9 27.7 105.6 75.9 78.3 72.0 78.1 63.0
51.4 34.9 92.7 53.1 84.6 74.7 43.8 51.7 55.6 79.3 22.7 27.8 56.7 83.1 60.8 79.9 24.0 23.9 19.8 27.7 105.5 75.9 78.3 72.0 78.1 63.0 172.6 21.5
44.1 38.1 90.7 52.0 83.5 71.7 37.6 52.8 50.7 152.0 27.9 23.8 52.5 79.3 135.0 142.9 15.4 26.6 20.2 114.1 105.7 75.8 78.1 71.9 78.1 63.0
44.9 36.3 88.0 49.9 85.3 68.8 41.2 57.2 138.1 125.5 27.2 26.7 54.1 89.1 58.7 83.9 23.6 26.2 18.1 20.0 105.1 75.8 78.1 72.0 77.9 63.0
Figure 1. 1H−1H COSY, key HMBC, and NOESY correlations of 3epi-grayanoside B (1). Figure 3. ORTEP drawing of micranthanoside A (2).
Flack [0.0(2)] parameters permitted assignment of its absolute configuration (Figure 4). Micranthanoside D (5) has the same molecular formula (C26H42O9) as micranthanoside B (3), which was established by the 13C NMR data and the HRESIMS data (m/z 521.2704) (calcd for C26H42O9Na, 521.2727). The NMR data of 5 were Figure 2. ORTEP drawing of 3-epi-grayanoside B (1).
90.2) was shifted downfield compared to that (δC 81.6) in 2. Thus, the glucosylation in 4 should be at C-16. This deduction was supported by the HMBC correlations (Figure S3, Supporting Information) from H-1′ (δH 4.40, d) and H3-17 (δH 1.39, s) to C-16 (δC 90.2). Finally, the structure of micranthanoside C (4) was defined as 16α-(β-D-glucopyranosyloxy)-3β,5α-dihydroxy-1-βH-grayan-10(20)-ene by single-crystal X-ray diffraction, and the Hooft31 [0.04(9)] and
Figure 4. ORTEP drawing of micranthanoside C (4). D
DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 3. 1H NMR [δ, mult. (J in Hz)] Spectroscopic Data for Compounds 8−13 in Methanol-d4 (400 MHz) no.
8
1α 2α
2.92, t (9.5) 2.31, ddd (16.4, 9.5, 6.6) 1.78, ddd (16.4, 9.5, 2.7) 3.61, dd (6.6, 2.7) 3.77, dd (10.0, 2.4) 1.42, dd (14.2, 2.4) 2.06, dd (14.2, 10.0) 2.44, t (7.2) 1.61, m 1.48, m 1.50, m 1.40, m 2.21, m 1.68, d (11.3) 1.97, dd (11.3, 4.0) 1.68, d (15.3) 2.04, d (15.3) 1.40, s 1.02, s 1.17, s 5.04, s 4.93, s 4.37, d (7.8) 3.11, dd (9.0, 7.8) 3.34, dd (9.0, 8.5) 3.27, dd (9.5, 8.5) 3.21, ddd (9.5, 5.0, 2.0) 3.80, dd (11.8, 2.0) 3.63, dd (11.8, 5.0)
2β 3α 6α 7α 7β 9β 11α 11β 12α 12β 13α 14α 14β 15α 15β 17 18 19 20a 20b 1′ 2′ 3′ 4′ 5′ 6′a 6′b Ac
9
10
11
2.78, t (9.8) 2.35, ddd (14.3, 9.8, 7.0) 1.76, ddd (14.3, 9.8, 3.0) 3.61, dd (7.0, 3.0) 3.66, dd (10.1, 1.1) 1.64, dd (14.3, 1.1) 2.03, dd (14.3, 10.1) 2.71, d (6.4) 2.32, m 1.73, m 2.13, m 1.59, m 2.70, overlap 5.01, s
2.53, dd (11.7, 4.4) 2.32, dd (15.4, 4.4)
2.60, dd (11.7, 4.4) 2.31, dd (15.3, 4.4)
2.22, ddd (15.4, 11.7, 4.6) 3.69, d (4.6) 3.88, dd (11.8, 4.4) 2.12, dd (13.6, 4.4) 1.87, dd (13.6, 11.8) 2.02, m 1.67, m 1.50, m 1.65, m 1.50, m 1.78, overlap 4.24, s
2.20, ddd (15.3, 11.7, 4.3) 3.68, d (4.3) 3.56, dd (11.0, 5.1) 1.95, dd (13.2, 5.1) 1.92, dd (13.2, 11.0) 2.15, brs 1.75, m 1.56, m 2.13, m 1.59, m 1.84, m 5.46, s
1.95, d (13.8) 2.35, d (13.8) 1.47, s 0.98, s 1.15 s 5.05, s 4.94, s 4.35, d (7.8) 3.08, dd (9.2, 7.8) 3.19, dd (9.2, 8.6) 3.16, dd (9.7, 8.6) 3.35, ddd (9.7, 5.1, 2.2) 3.79, dd (12.2, 2.2) 3.58, dd (12.2, 5.1) 2.08, s
1.79, 1.94, 1.29, 1.07, 1.26, 1.36,
d (14.8) d (14.8) s s s s
1.86, 2.02, 1.33, 1.01, 1.24, 1.37,
d (14.9) d (14.9) s s s s
4.36, 3.14, 3.35, 3.24, 3.25, 3.87, 3.66,
d (7.8) dd (8.9, 7.8) t (8.9) dd (9.6, 8.9) ddd (9.6, 5.0, 2.0) dd (11.8, 2.0) dd (11.8, 5.0)
4.35, 3.13, 3.33, 3.25, 3.24, 3.87, 3.66, 2.10,
d (7.8) dd (9.1, 7.8) t (9.1) t (9.1) ddd (9.1, 5.0, 1.9) dd (11.8, 1.9) dd (11.8, 5.0) s
12
13
2.83, t (9.6) 2.27, ddd (14.3, 9.6, 7.0) 2.08, ddd (14.3, 9.6, 4.0) 3.81, dd (7.0, 4.0) 4.07, dd (10.7, 2.2) 1.60, dd (13.8, 2.2) 1.90, dd (13.8, 10.7) 2.57, m 1.83, m 1.51, overlap 1.91, m 1.51, overlap 2.20, m 4.29, s
3.28, t (8.3) 2.28, ddd (13.8, 8.3, 7.3) 2.15, ddd (13.8, 8.3, 4.6) 3.82, dd (7.3, 4.6) 4.01, dd (5.6, 1.5) 2.20, dd (15.5, 5.6) 2.29, dd (15.5, 1.5)
5.13, q (1.3)
2.00, 2.31, 1.33, 0.95, 1.04, 1.69,
1.73, d (1.3) 1.14, s 1.25, s 4.94, s 4.96, s 4.30, d (7.8) 3.20, dd (8.8, 7.8) 3.35, dd (8.8, 8.3) 3.29, dd (9.2, 8.3) 3.26, ddd (9.2, 5.3, 2.1) 3.85, dd (11.8, 2.1) 3.67, dd (11.8, 5.3)
1.67, overlap 1.54, m 2.51, m 1.92, m 1.99, overlap 3.53 s d (15.8) d (15.8) s s s s
4.29, d (7.7) 3.19, dd (8.9, 7.7) 3.33, dd (8.9, 8.3) 3.28, dd (9.5, 8.3) 3.24, ddd (9.5, 5.4, 2.1) 3.86, dd (11.8, 2.1) 3.67, dd (11.8, 5.4)
Information). Therefore, the structure of micranthanoside E (6) was defined as 16α-(β-D-glucopyranosyloxy)-3β,5β,6βtrihydroxy-1-βH-grayan-10(20)-ene. The 13C NMR data and [M + Na]+ HRESIMS ion at m/z 537.2668 permitted assignment of a molecular formula of C26H42O10 (calcd for C26H42O10Na, 537.2626) to 7αhydroxygrayanoside C (7), having one more oxygen atom than the known grayanoside C (15).4 The NMR data of 7 (Tables 1 and 2) were also similar to 15,4 except for an oxygenated methine (δH 3.39, d, H-7; δC 73.6, C-7) in 7, replacing the methylene (δH 2.18, dd, 2.11, dd, H2-7; δC 35.6, C-7) in 15. Thus, compound 7 is a 7-hydroxy derivative of 15, as confirmed by the 1H−1H COSY correlation between H-7 (δH 3.39, d) and H-6 (δH 3.89, d) and the HMBC correlations from H-7 (δH 3.39, d) to C-8 (δC 52.2) and C-5 (δC 86.2). The large coupling constant of H-7 with H-6 (J = 8.8 Hz) established its β-orientation,32 which was supported by the NOESY correlation between H-7 and H-15α (Figure S6, Supporting Information). Thus, the structure of 7α-hydroxygrayanoside C (7) was defined as 3β-(β-D-glucopyranosyloxy)5β,6β,7α,16α-tetrahydroxy-1-βH-grayan-10(20)-ene. Micranthanoside F (8) had the same molecular formula of C26H42O9 as the known grayanoside B (18)3 based on the HRESIMS ion at m/z 521.2750 (calcd for C26H42O9Na, 521.2727) and 13C NMR data. The NMR data (Tables 2 and 3) were similar to 18,3 except that C-3 (δC 82.6) in 8 was shifted 7.7 ppm upfield compared to that (δC 90.3) of 18, and C-16 (δC 90.1) was shifted 8.7 ppm downfield compared to
similar to those of 3 (Tables 1 and 2), and the differences were that C-16 in 5 (δC 88.7) was shifted downfield compared to that (δC 81.8) in 3. In contrast, C-3 (δC 80.1) in 5 was shifted upfield compared to that (δC 89.4) in 3. Thus, the glucosylation in 5 should be at C-16 instead of at C-3 as in compound 3. The cross-peaks from H-1′of the glucose (δH 4.52) and H3-17 (δH 1.27, s) to C-16 (δC 88.7) in the HMBC spectrum of 5 proved this conclusion (Figure S4, Supporting Information). The chemical shifts of C-9 (δC 54.2), C-5 (δC 83.8), and C-1 (δC 51.7) in 5 are close to those in compound 4, suggesting the α-orientation of 5-OH and the β-orientations of H-9 (δH 2.01, d) and H-1 (δH 2.97, t). The α-orientation of 13-OH and the β-orientation of H3-17 were established by the NOESY correlations from H-12β to H3-17 and from H-9β to H-15β. Thus, the structure of micranthanoside D (5) was established as 16α-(β-D-glucopyranosyloxy)-3β,5α,13α-trihydroxy-1-βH-grayan-10(20)-ene. Using the 13C NMR and HRESIMS data (m/z 521.2696) established the molecular formula of micranthanoside E (6) as C26H42O9 (calcd for C26H42O9Na, 521.2727). The NMR data (Tables 1 and 2) closely resembled those of the known grayanoside C (15).4 However, compared to those (δC 93.8, C-3; 80.6, C-16) of 15, the C-3 resonance (δC 85.0) of 6 was shifted upfield by 8.8 ppm and the C-16 resonance (δC 89.2) was shifted downfield by 8.6 ppm, indicating the glucosylation at C-16 in 6. This was supported by the HMBC correlations of the anomeric proton H-1′ of the glucose moiety (δH 4.42) and H3-17 (δH 1.39, s) to C-16 (δC 89.2) (Figure S5, Supporting E
DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 5. Antinociceptive activities of compounds 1−18 in the acetic-acid-induced writhing test. Morphine (morph) was used as a positive control. Each bar and vertical line represents the mean ± SEM of the values obtained from 10 Kunming mice. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant differences between the values obtained in animals treated with tested compounds or control drugs and mice treated with vehicle (veh) (one-way ANOVA followed by the Bonferroni test).
Using the 13C NMR data, micranthanoside G (10) was assigned a molecular formula of C26H44O11 (calcd for C26H44O11Na, 555.2781) by the HRESIMS ion at m/z 555.2786. The NMR data (Tables 2 and 3) of 10 were similar to those of the known rhodomoside A (16),7 except for an oxygenated tertiary carbon (δC 79.6, C-10) and a methyl group (δC 27.7, C-20; δH 1.36, s, H3-20) in 10, replacing the exocyclic double bond (δH 5.03, s; 5.01, s, H2-20; δC 152.4, C-10; 113.8, C-20) in 16. Thus, micranthanoside G (10) is a hydrated derivative of 16.7 This was confirmed by the HMBC correlations from H3-20 to C-9 (δC 56.7), C-1 (δC 51.8), and C-10 (δC 79.6) (Figure S9, Supporting Information). Thus, the structure of micranthanoside G (10) was established as 3β-(β-D-glucopyranosyloxy)-5β,6β,10β,14β,16α-pentahydroxygrayanane. 14-O-Acetylmicranthanoside G (11) was obtained as a colorless oil. Using the 13C NMR data, the molecular formula was determined to be C28H46O12 (calcd for C28H46O12Na, 597.2887) by the HRESIMS ion at m/z 597.2860. The NMR data (Tables 2 and 3) of 11 resembled micranthanoside G (10), and the major difference was the presence of an acetyl group (δC 172.6, 21.5; δH 2.10, s, 14-OAc) in 11. Thus, 11 is an acetylated derivative of 10. The HMBC correlation from H14 to the acetyl carbonyl suggested acetylation at OH-14. Therefore, the structure of 14-O-acetylmicranthanoside G (11) was defined as 3β-(β-D-glucopyranosyloxy)-5β,6β,10β,16αtetrahydroxy-14β-acetoxygrayanane by chemical methods and 2D NMR data analysis (Figure S10, Supporting Information). 14β-Hydroxypieroside A (12) possessed a molecular formula of C26H40O9 (calcd for C26H40O9Na, 519.2570), as revealed by the 13C NMR data and HRESIMS ion at m/z 519.2552. Comparison of the NMR spectroscopic data (Tables 2 and 3) of 12 with those of pieroside A (19)3 suggested their structural similarity, except for an oxygenated methine (δC 79.3, C-14; δH 4.29, s, H-14) in 12 replacing the methylene (δH 1.53, dd, 2.19, d, H2-14; δC 39.6, C-14) in 19. Thus, compound 12 is a 14-hydroxy derivative of 19, which was supported by the HMBC correlations from H-14 (δH 4.29, s) to C-13 (δC 52.5), C-15 (δC 135.0), and C-16 (δC 142.9). The NOESY correlation between H-14 (δH 4.29, s) and H-1α (δH 2.83, t) established the β-orientation of OH-14 (Figure S11,
that (δC 81.4) of 18. Thus, micranthanoside F (8) is a grayanane diterpenoid glucoside with the glucosidation site at C-16. The HMBC correlation of H-1′ (δH 4.37) to C-16 (δC 90.1) (Figure S7, Supporting Information) supported this conclusion. Therefore, compound 8 was defined as 16α-(β-Dglucopyranosyloxy)-3β,5β,6β-trihydroxygrayan-10(20)-ene. Based on the 13C NMR data and HRESIMS ion at m/z 579.2779 [M + Na]+, a molecular formula of C28H44O11 (calcd for C28H44O11Na, 579.2781) was assigned to 14β-acetyoxymicranthanoside F (9). Analysis of their NMR data (Tables 3 and 2) indicated that compound 9 resembled micranthanoside F (8), and the major differences were the presence of an acetyl group (δC 174.8, 22.2; δH 2.08, s) and an oxygenated methine (δH 5.01, s, H-14; δC 83.9, C-14) in 9, instead of a methylene (δH 1.97, dd, 1.68, d, H2-14; δC 35.7, C-14) in 8. Thus, compound 9 is the 14-acetoxy derivative of 8. The HMBC correlation from H-14 (δH 5.01, s) to the acetoxy carbonyl (δC 174.8) defined the location of the acetoxy group at C-14. NOESY correlation between H-14 (δH 5.01, s) and H-1α (δH 2.92, t) established the β-orientation of the acetoxy group (Figure S8, Supporting Information). Thus, the structure of 14β-acetyoxymicranthanoside F (9) was defined as 16α-(β-Dglucopyranosyloxy)-3β,5β,6β-trihydroxy-14β-acetoxygrayan10(20)-ene. Grayanane diterpenoids with H-1α are common in the Ericaceae plants; however, H-1β-grayanane analogues are relatively rare, and only five H-1β-grayanane diterpenoid glucosides were reported so far.4,8,15 Micranthanosides E (6) and 7α-hydroxygrayanoside C (7) are the sixth and seventh examples of H-1β-grayanane diterpenoid glucosides. In addition, in the grayanane diterpenoids, 5-OH is usually βorientated, while 5α-OH analogues are rare. Principinols A and B33 and rhododecorumin VI17 are the first examples of 5αOH-1-αH-grayanane diterpenoids. To date, there is no report of 5α-OH-1-βH-grayanane diterpenoids. Micranthanosides A− D (2−5) represent the first examples of 5α-hydroxy-1-βHgrayanane diterpenoids. In addition, micranthanosides C−F (4−6, 8) and 14β-acetyoxymicranthanoside F (9) are the first examples of grayanane glucosides with glucosylation at C-16, although grayanane glucosides with glucosylation at C-3 are common in the Ericaceae plants.1 F
DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Supporting Information). Thus, the structure of 14βhydroxypieroside A (12) was defined as 3β-(β-D-glucopyranosyloxy)-5β,6β,14β-trihydroxygrayan-10(20),15(16)-diene. Using the 13C NMR data, the HRESIMS ion at m/z 537.2652 assigned a molecular formula of C26H42O10 (calcd for C 26 H42O 10Na, 537.2676) to micranthanoside H (13). Comparison of their NMR data (Tables 2 and 3) revealed that micranthanoside H (13) differed from the known rhodomoside A (16)7 by the presence of a tetrasubstituted double bond (δC 125.5, C-10; 138.1, C-9) in 13, instead of the exocyclic double bond (δH 5.03, s; 5.01, s, H2-20; δC 113.8, C20; 152.4, C-10) in 16. The HMBC correlations from H3-20 (δH 1.69, s) to C-9 (δC 138.1), C-10 (δC 125.5), and C-1 (δC 44.9) revealed the position of the tetrasubstituted Δ9(10) double bond. Thus, the structure of compound 13 was defined as 3β-(β-D-glucopyranosyloxy)-5β,6β,14β,16α-tetrahydroxygrayan-9(10)-ene. By NMR data analysis and comparison with the reported spectroscopic data, six known compounds (14−19) were identified as grayanoside D (14),5 grayanoside C (15),4 rhodomoside A (16),7 grayanosides A (17)2 and B (18),3 and pieroside A (19).3 The isolation of compounds 1−19 expanded the chemical diversity of the grayanane diterpenoid glucosides. Grayanane diterpenoid glucosides 1−19 were evaluated for their antitumor, anti-inflammatory, and PTP1B inhibitory activities, but did not exhibit significant activities at 40 μM. Owing to the exhaustion of sample in the anti-inflammatory, antitumor, and PTP1B inhibitory activities evaluation, pieroside A (19) could not be evaluated for its antinociceptive activity. Grayanane diterpenoid glucosides 1−18 were evaluated for their antinociceptive activity in an acetic-acidinduced writhing test.8 As shown in Figure 5, compared with the vehicle, compounds 2, 3, 7−10, 12, 13, and 16 significantly reduced the writhing numbers (p < 0.001) with inhibition rates in excess of 50%. In particular, compound 2 exhibited significant antinociceptive activities at doses of 0.2, 1.0, and 5.0 mg/kg with inhibition rates of 46.1%, 58.7%, and 82.3%, respectively, and compounds 3 and 7 also showed significant antinociceptive activity even at a low dose of 1.0 mg/kg. In addition, compounds 1, 5, 6, 14, 17, and 18 showed moderate antinociceptive activity, and compounds 4, 11, and 15 expressed weak antinociceptive activity at a dose of 5.0 mg/ kg. Analysis of their structures and antinociceptive activity revealed that 3α-oxygrayanane glucoside 1 exhibited more potent antinociceptive activity than 3β-oxygrayanane glucoside 18, indicating the importance of the 3α-oxy orientation to the antinociceptive activity. Interestingly, 5α-OH-1-βH-grayanane glucosides with glucosidation at C-3 (2 and 3) showed more potent antinociceptive activity than those with glucosidation at C-16 (4 and 5). Compound 7, possessing a 7α-hydroxy group, exhibited more significant antinociceptive activity than compound 15, suggesting that the 7α-hydroxy group is essential for activity in the 5α-OH-1-βH-grayanane glucosides. Micranthanoside G (10) showed more potent antinociceptive activity than 14-O-acetylmicranthanoside G (11), indicating that the 14-acetoxy group may be deactivating in the grayanane glucosides without an exocyclic Δ10(20) double bond. The water-solubility of grayanane diterpenoid glucosides is better than their aglycones. Thus, this study not only enriches the chemical diversity of grayanane glucosides but also provides a new source to develop novel antinociceptive drugs.
Article
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were measured on a Beijing TechX-5 microscopic melting point apparatus (uncorrected). The optical rotations were determined with a Rudolph Research Analytical Autopol IV automatic polarimeter. HRESIMS data were recorded on a Bruker micrOTOF II spectrometer or a Thermo Fisher LC-LTQ-Orbitrap XL in a positive ion mode. NMR spectra were acquired on a Bruker AM-400 spectrometer, and the chemical shifts were referenced to the residual peaks of methanol-d4 at δH 3.31 and δC 49.15, respectively. The crystallographic data were obtained on a Bruker SMART APEX-II CCD diffractometer equipped with graphite-monochromatized Cu Kα radiation (λ = 1.541 78 Å). Samples were purified over an HPLC column (5 μm, 10 × 250 mm, Welch Ultimate XB-C18) at a flow rate of 1.5 mL/min using an Agilent 1200 or Dionex P680 quaternary system HPLC with a UV detector. GC was analyzed using an Agilent 7820A GC with a Welch WM-1 capillary column (30 m × 0.25 mm × 0.5 μm). Plant Material. The leaves of R. micranthum Turcz. were collected in July 2012 at Mountain Tai, Tai-An, People’s Republic of China, and was authenticated by one of the authors, Y.Z. A voucher specimen (No. 20120701) was deposited at the School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology. Extraction and Isolation. The air-dried leaves of R. micranthum (30.1 kg) were powdered and soaked with 95% ethanol (120.0 L × 4) at room temperature. Evaporation of the solvent under reduced pressure gave a crude dark green residue (4100 g), which was suspended in H2O (16.0 L) and excessively extracted with petroleum ether, EtOAc, and n-BuOH. The n-BuOH extract (1556 g) was subjected to an AB-8 macroporous resin column chromatograph (CC) to remove the sugars and polar water-soluble substances with H2O and then eluted successively with 30%, 50%, 70%, and 95% EtOH. The 30% EtOH fraction (356 g) was fractionated by silica gel CC (CHCl3−MeOH, 15:1 to 2:1) to afford 10 fractions, A−J. Fraction I was fractionated using Sephadex LH-20 CC (100% MeOH) to provide five subfractions, I1−I5. Fraction I1 was fractionated by RP C18 CC (from 10% MeOH/H2O to 100% MeOH) to give five subfractions, I1a−I1e. Subfraction I1b was purified by RP HPLC (MeOH−H2O, 30:70) to give compounds 1 (tR = 40.7 min, 4.8 mg) and 10 (tR = 36.5 min, 4.2 mg). Fraction H was fractionated by RP C18 CC eluting with a gradient of MeOH− H2O from 0:100 to 20:80 to afford four subfractions, H1−H4. Fraction H1 was fractionated by a Sephadex LH-20 column (MeOH) to afford three subfractions, H1a−H1c, and fraction H1a was purified by RP HPLC (MeOH−H2O, 15:85) to produce compound 13 (5.4 mg, tR = 28.8 min). In the same way, compound 6 (tR = 33.5 min, 6.0 mg) was obtained from fraction H2 by RP C18 HPLC (MeOH−H2O, 20:80), and 16 (8.2 mg, tR = 33.6 min) was isolated from fraction H3 by RP HPLC (MeOH−H2O, 23:77). Fraction G was fractionated on silica gel CC eluting with a gradient of CHCl3−MeOH−H2O from 15:1:0.01 to 2:1:0.1, followed by Sephadex LH-20 CC (100% MeOH) to give three subfractions, G1−G3. Fraction G1 was separated by RP C18 CC (MeOH−H2O, from 0:100 to 30:70) to give compound 11 (3.2 mg). Fraction F was chromatographed on Sephadex LH-20 CC (100% MeOH) to yield two subfractions, F1 and F2. Fraction F1 was purified by RP C18 HPLC (CH3CN−H2O, 20:80) to afford compound 7 (tR = 43.2 min, 4.2 mg). Fraction D was fractionated by silica gel CC to give five subfractions, D1−D5, and fraction D3 was separated by Sephadex LH-20 CC to yield three subfractions, D3a−D3c. Finally, compound 4 (tR = 29.7 min, 10.6 mg) was obtained by RP HPLC (MeOH−H2O, 30:70) from fraction D3a. Similarly, compound 5 (tR = 33.2 min, 6.8 mg) was obtained from fraction D3b. Fraction D4 was fractionated by Sephadex LH-20 CC (100% MeOH) to afford three subfractions, D4a−D4c, and fraction D4a was purified by RP HPLC (MeOH−H2O, 15:85) to yield compounds 2 (tR = 41.2 min, 8.7 mg) and 15 (tR = 43.5 min, 15.5 mg). Compounds 17 (80.4 mg) and 18 (105.2 mg) were obtained from fraction D4b by RP C18 CC (MeOH−H2O, 0:100 to 50:50). Fraction E was fractionated by RP C18 CC (MeOH−H2O, 0:100 to 40:60) to give five subfractions, E1−E5, and fraction E2 was G
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Article
0.0392 (wR2 = 0.0810) for 17 084 reflections collected. Flack parameter: −0.02(9) for 1652 quotients. Crystallographic data of micranthanoside C (4): 2(C26H42O8)· CH3OH·6(H2O), M = 1105.33, T = 298(2) K, V = 2873.4(2) Å3, Dcalcd = 1.278 mg/m3, Z = 2, monoclinic, P2(1), a = 6.3352(3) Å, b = 39.6779(16) Å, c = 11.4316(5) Å, α = γ = 90°, β = 90.621(2)°, F(000) = 1204, F2 = 1.061, 3.34° < θ < 67.51°, −7 ≤ h ≤ 7, −47 ≤ k ≤ 47, −13 ≤ l ≤ 13, final R indices R1 = 0.0594 (wR2 0.1552) [I > 2σ(I)] for 10 000 independent reflections [R(int) = 0.0514], R indices (all data) R1 = 0.0723 (wR2 = 0.1670) for 40 859 reflections collected. Flack parameter: 0.0(2) for 4740 Friedel pairs; Hooft parameter: 0.04(9) for 4747 Bijvoet pairs. The crystallographic data of 3-epi-grayanoside B (1, CCDC 1848856) and micranthanosides A (2, CCDC 1848857) and C (4, CCDC 1848855) have been deposited in the Cambridge Crystallographic Data Centre. Determination of the Absolute Configuration of the Glucose. Compounds 1−13 were hydrolyzed with trifluoroacetic acid (2.0 mL) at 65 °C, and the trimethylsilylthiazolidine derivatives of the hydrolysates of compounds 1−13 and the standards, D- and Lglucose, were prepared.27 The trimethylsilylthiazolidine derivatives in n-hexane solution were analyzed by an Agilent 7820A gas chromatograph with a Welch WM-1 capillary column. The derivatives of the hydrolysates of compounds 1−13 and the standards, D- and L-glucose, showed GC retention times at 14.841, 14.850, 14.859, 14.850, 14.844, 14.855, 14.867, 14.844, 14.844, 14.838, 14.851, 14.874, 14.857, 14.846, and 15.316 min (Figures S117−S131, Supporting Information), respectively, suggesting the absolute configuration of the glucose moieties in compounds 1−13 was D. Anti-inflammatory Activity Evaluation. The anti-inflammatory activities of 1−19 were evaluated in vitro.35 Cytotoxicity Evaluation. The cytotoxicity of 1−19 against five human cancer cell lines (breast cancer MCF-7, colon cancer SW480, hepatocellular carcinoma SMMC-7721, lung cancer A549, and myeloid leukemia HL-60) was evaluated in vitro.36 PTP1B Inhibitory Activity Evaluation. The PTP1B inhibitory activities of 1−19 were evaluated in vitro.25 Antinociceptive Activity Evaluation. The antinociceptive activities of 1−19 were evaluated in an acetic-acid-induced writhing model,8 and morphine (morph) was used as a positive control drug. The Laboratory Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (approval number 2018-S748), approved the animal experiments. The male and female Kunming mice (6 weeks old, 18−22 g) were ordered from the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology. The license number for the use of experimental animals is No. SCXK (Hubei) 2016-0057, and the license number of experimental animal production is No. SCXK (Hubei) 2016-0009.
purified by semipreparative RP HPLC (MeOH−H2O, 30:70) to yield compound 3 (tR = 35.2 min, 3.2 mg). Fraction E3 was fractionated by Sephadex LH-20 CC (100% MeOH) to afford four subfractions, E3a−E3d, and fraction E3a was purified by RP HPLC (MeOH−H2O, 35:65) to give compounds 12 (tR = 28.9 min, 2.1 mg) and 8 (tR = 36.8 min, 10.4 mg). E3b was purified by RP HPLC (MeOH−H2O, 32:68) to yield compound 9 (tR = 37.5 min, 5.8 mg). Compounds 14 (tR = 36.5 min, 15.8 mg) and 19 (tR = 41.5 min, 3.3 mg) were obtained from fraction E4 by RP HPLC (MeOH−H2O, 30:70). 3-epi-Grayanoside B (1): [α]20D −43 (c 0.3, MeOH); colorless prisms (MeOH), mp 230−231 °C; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 521.2725 (calcd for C26H42O9Na, 521.2727). Micranthanoside A (2): [α]20D −9 (c 0.8, MeOH); colorless prisms (MeOH), mp 133−134 °C; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 505.2762 (calcd for C26H42O8Na, 505.2777). Micranthanoside B (3): [α]20D −33 (c 0.2, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 521.2679 (calcd for C26H42O9Na, 521.2727). Micranthanoside C (4): [α]20D −23 (c 0.7, MeOH); colorless prisms (MeOH), mp 149−150 °C; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 505.2790 (calcd for C26H42O8Na, 505.2777). Micranthanoside D (5): [α]20D −21 (c 0.7, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 521.2704 (calcd for C26H42O9Na, 521.2727). Micranthanoside E (6): [α]20D +13 (c 0.6, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 521.2696 (calcd for C26H42O9Na, 521.2727). 7α-Hydroxygrayanoside C (7): [α]20D −1 (c 0.4, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 537.2668 (calcd for C26H42O10Na, 537.2626). Micranthanoside F (8): [α]20D −15 (c 1.1, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 521.2750 (calcd for C26H42O9Na, 521.2727). 14β-Acetyoxymicranthanoside F (9): [α]20D −9 (c 0.7, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 579.2779 (calcd for C28H44O11Na, 579.2781). Micranthanoside G (10): [α]20D −25 (c 0.3, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 555.2786 (calcd for C26H44O11Na, 555.2781). 14-O-Acetylmicranthanoside G (11): [α]20D −12 (c 0.3, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 597.2860 (calcd for C28H46O12Na, 597.2887). 14β-Hydroxypieroside A (12): [α]20D −12 (c 0.2, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 519.2552 (calcd for C26H40O9Na, 519.2570). Micranthanoside H (13): [α]20D +51 (c 0.5, MeOH); white, amorphous powder; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 537.2652 (calcd for C26H42O10Na, 537.2676). Single-Crystal X-ray Diffraction Analysis and Crystallographic Data. The crystallographic data for 3-epi-grayanoside B (1) and micranthanosides A (2) and C (4) were acquired on a Bruker SMART APEX-II CCD diffractometer, and their crystal structures were solved by the methods described in a previously published paper.34 Crystallographic data of 3-epi-grayanoside B (1): C26H42O9· H2O, M = 516.61, T = 173(2) K, V = 1284.11(7) Å3, Dcalcd = 1.336 mg/m3, Z = 2, monoclinic, P2(1), a = 12.2958(4) Å, b = 6.6149(2) Å, c = 15.8066(5) Å, α = γ = 90°, β = 92.7890(10)°, F(000) = 572, F2 = 1.046, 4.67° < θ < 66.56°, −14 ≤ h ≤ 14, −7 ≤ k ≤ 7, −18 ≤ l ≤ 18, data/restraints/parameters 4531/3/346, final R indices R1 = 0.0391 (wR2 = 0.1060) [I > 2σ(I)] for 4531 independent reflections [R(int) = 0.0415], R indices (all data) R1 = 0.0422 (wR2 = 0.1084) for 15 221 reflections collected. Flack parameter: 0.00(7) for 1794 quotients. Crystallographic data of micranthanoside A (2): C26H42O8· CH3OH, M = 514.64, T = 173(2) K, V = 2649.02(13) Å3, Dcalcd = 1.290 mg/m3, Z = 4, orthorhombic, P2(1), a = 6.9345(2) Å, b = 13.2666(4) Å, c = 28.7946(8) Å, α = β = γ = 90°, F(000) = 1166, F2 = 1.032, 3.069° < θ < 66.666°, −7 ≤ h ≤ 8, −15 ≤ k ≤ 15, −34 ≤ l ≤ 34, final R indices R1 = 0.03355 (wR2 = 0.0782) [I > 2σ(I)] for 4672 independent reflections [R(int) = 0.0415], R indices (all data) R1 =
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00490. 1 H−1H COSY, key NOESY, and HMBC correlations of 2−13; gas chromatogram of the trimethylsilythiazolidine derivative of the standards, D- and L-glucose, and the hydrolysates of 1−13; HRESIMS and NMR spectra for 1−13 (PDF) Crystallographic data for 3-epi-grayanoside B (1) (CIF) Crystallographic data for micranthanoside A (2) (CIF) Crystallographic data for micranthanoside C (4) (CIF)
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Corresponding Author
*E-mail:
[email protected]. H
DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
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(25) Zhou, J.; Sun, N.; Zhang, H.; Zheng, G.; Liu, J.; Yao, G. Org. Lett. 2017, 19, 5352−5355. (26) Zhou, J.; Zhan, G.; Zhang, H.; Zhang, Q.; Li, Y.; Xue, Y.; Yao, G. Org. Lett. 2017, 19, 3935−3938. (27) Teng, Y.; Zhang, H.; Zhou, J.; Zhan, G.; Yao, G. Phytochemistry 2018, 151, 32−41. (28) Teng, Y.; Zhang, H.; Zhou, J.; Li, Y.; Yao, G. Youji Huaxue 2017, 37, 2416−2422. (29) Parsons, S.; Flack, H. D.; Wagner, T. Acta Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater. 2013, 69, 249−259. (30) Niu, C.-S.; Li, Y.; Liu, Y.-B.; Ma, S.-G.; Liu, F.; Cui, L.; Yu, H.B.; Wang, X.-J.; Qu, J.; Yu, S.-S. Tetrahedron 2018, 74, 375−382. (31) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr. 2008, 41, 96−103. (32) El-Naggar, S. a. F.; Doskotch, R. W.; Odell, T. M.; Girard, L. J. Nat. Prod. 1980, 43, 617−631. (33) Liu, C. C.; Lei, C.; Zhong, Y.; Gao, L. X.; Li, J. Y.; Yu, M.-H.; Li, J.; Hou, A. J. Tetrahedron 2014, 70, 4317−4322. (34) Shu, P.; Wei, X.; Xue, Y.; Li, W.; Zhang, J.; Xiang, M.; Zhang, M.; Luo, Z.; Li, Y.; Yao, G.; Zhang, Y. J. Nat. Prod. 2013, 76, 1303− 1312. (35) Zhan, G.; Zhou, J.; Liu, R.; Liu, T.; Guo, G.; Wang, J.; Xiang, M.; Xue, Y.; Luo, Z.; Zhang, Y.; Yao, G. J. Nat. Prod. 2016, 79, 760− 766. (36) Zhan, G.; Qu, X.; Liu, J.; Tong, Q.; Zhou, J.; Sun, B.; Yao, G. Sci. Rep. 2016, 6, 33990.
Guangmin Yao: 0000-0002-8893-8743 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Drs. X. Bao and L. Li at the Instrumental Analysis Center of Shanghai Jiao Tong University for X-ray crystallographic data collection and analysis, and the staff at the Analysis and Measurement Centre of Huazhong University of Science and Technology for spectroscopic data collection. This work was financially supported by the National Natural Science Foundation of China (U1703109 and 81001368) and the Fundamental Research Funds for the Central Universities (HUST: 2016YXMS148).
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DOI: 10.1021/acs.jnatprod.8b00490 J. Nat. Prod. XXXX, XXX, XXX−XXX
