Antinociceptive Grayanoids from the Roots of Rhododendron molle

Article pubs.acs.org/jnp

Antinociceptive Grayanoids from the Roots of Rhododendron molle Yong Li, Yun-Bao Liu, Jian-Jun Zhang, Yang Liu, Shuang-Gang Ma, Jing Qu, Hai-Ning Lv, and Shi-Shan Yu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new grayanoids (1−9), together with 11 known compounds, were isolated from the roots of Rhododendron molle. The structures of the new compounds (1−9) were determined on the basis of spectroscopic analysis, including HRESIMS, and 1D and 2D NMR data. Compounds 4, 6, 12, and 14−20 showed significant antinociceptive activities in an acetic acid-induced writhing test. In particular, 14 and 15 were found to be more potent than morphine for both acute and inflammatory pain models and 100-fold more potent than gabapentin in a diabetic neuropathic pain model.

88.0, and 88.2), and six quaternary carbons (two olefinic at δC 124.0 and 137.5; two oxygenated at δC 82.0 and 84.7). The 1 H−1H COSY and HSQC spectra revealed the presence of the following fragments: −CH−CH(OH)−CH(OH)−, −CH(OH)−CH2−, and −CH2−CH2−CH−CH(OH)−. These structural features are consistent with a grayanane diterpenoid skeleton with six sites of oxygenation.4 A comparison between the 1H and 13C NMR data of 1 with those of rhodojaponin VI (15) showed that both compounds share some common features. However, 1 lacks an oxygenated quaternary carbon and has an extra double bond, which caused obvious differences between the NMR spectra of these two compounds. The double bond in 1 was determined from the HMBC correlations from H-1, H2-7, and H-14 to C-9 (δC 137.5) and was located between C-9 and C-10. Further HSQC and HMBC experiments allowed for the full assignments of the 1H and 13C NMR spectra of 1 to be made. NOE enhancements observed between H-1/H-3, H-1/H-6, H-1/H3-18, and H-1/H-14 supported the configurations of H-1, H-3, H-6, H3-18, and H-14 as being αoriented, while a NOE correlation between H-2/H3-19 suggested they are β-oriented (Figure 1). Therefore, 1 was established as 2α,3β,5β,6β,14β,16α-hexahydroxygrayan-9(10)ene and was given the trivial name rhodomollein XXVI. Compound 2 was obtained as a white powder. Its molecular formula was assigned as C20H30O5 based on the HRESIMS data. The 1H NMR spectrum showed resonances attributable to two tertiary methyls at δH 1.53 and 1.72, four oxygenated methines at δH 4.20, 4.61, 4.75, and 5.00, and four olefinic protons at δH 5.04, 5.11, 5.26, and 5.50. This evidence

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raditional Chinese medicine (TCM) has been used clinically to relieve pain for thousands of years. Among the plants used, Rhododendron molle G. Don (Ericaceae) is one of the most potent medicines for pain relief.1,2 It has been used traditionally as an anodyne and anesthetic and is recorded in the “Divine Farmer’s Herb-Root Classic”, which is the earliest known Chinese herbal manual dating back to the first century A.D. Prior investigations have shown that Rhododendron species are sources of diterpenoids,3−7 triterpenoids, flavonoids, and lignans, which are well known for their broad range of bioactivities, including sodium channel modulating, insect antifeedant, and antioxidant effects.8,9 However, the analgesic components of R. molle have not been well-defined. To investigate further the analgesic components of this herb, a chemical study of the roots was carried out. As a result, nine new grayanoids (1−9), together with 11 known compounds (10−20), were isolated. This report provides details on the isolation, structure elucidation, and antinociceptive activities of these compounds.

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RESULTS AND DISCUSSION The molecular formula of compound 1 was determined to be C20H32O6 based on the [M + Na]+ ion peak at m/z 391.2101 (calcd 391.2091) in the HRESIMS, indicating five indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy group (3386 cm−1) and double-bond (1647 cm−1) absorptions. Its 1H NMR spectrum (Table 1) displayed characteristic signals for four methyl groups (δH 1.15, 1.29, 1.55, and 2.21) and four oxygenated methines (δH 4.02, 4.07, 4.44, and 5.09). In the 13C NMR (DEPT) spectrum (Table 3), 20 carbon signals were observed, including four methyls, four methylenes, six methines (four oxygenated at δC 67.2, 82.2, © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 21, 2015

A

DOI: 10.1021/acs.jnatprod.5b00456 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR Spectroscopic Data of Compounds 1−5 in Pyridine-d5 (δ in ppm, J in Hz) no.

1a

2a

3b

1

4.02, d (9.0)

3.31, d (9.0)

2

5.09, dd (9.0, 2.5) 4.07, d (2.5)

5.00, m 4.20, d (3.0)

3.18, d (10.4) 5.11, dd (10.4, 4.0) 4.02, d (4.0)

4.75, brd (8.5) 2.44, dd (14.0, 1.5) 2.59, dd (14.0, 8.5)

4.72, dd (9.6, 1.6) 2.42, dd (13.6, 1.6) 2.52, dd (13.6, 9.6)

2.93, m

3 4 5 6 7

4.44, brs 2.85, m

8 9 10 11

12 13 14 15

4.02, d (6.5) 2.69, d (15.0) 2.83, d (15.0)

16 17

1.55, s

18 19 20

1.15, s 1.29, s 2.21, s

1′ 2′ 3′ 4′ 5′ 6′

a

2.09, m 2.58, dd (13.5, 6.0) 1.45, m 1.71, m 2.39, brs

4b 2.83, brs 4.19, brd (3.2) 3.24, d (3.2)

5b 3.24, d (10.4) 4.89, dd (10.4, 4.0) 3.97, d (4.0)

4.49, brt (8.0) 2.63, m

2.44, m

2.86, dd (13.6, 4.0)

2.89, dd (12.8, 4.0)

2.95, t (7.2)

2.10, brd (7.2)

2.03, m

1.63, m 2.00, m

1.56, m 1.90, m

2.02, m 2.61, m

1.46, m 2.05, m

1.66, m 2.17, m 2.89, brd (6.0) 4.61, brs 2.48, d (15.5) 2.93, d (15.5)

1.70, m 1.91, m 2.35, brd (6.4) 4.61, brs 2.15, d (14.4) 2.46, d (14.4)

5.29, m 3.06, brd (2.4) 4.82, d (7.2) 2.20, d (14.4) 2.33, d (14.4)

1.67, m 2.58, m 2.51, brs 5.02, d (7.2) 2.04, d (14.4) 2.23, d (14.4)

5.04, 5.11, 1.53, 1.72, 5.26, 5.50,

1.52, s

2.05, s

1.49, s

1.45, s 1.68, s 5.21, brs 5.56, brs 5.01, d (8.0) 4.17, m 4.26, m 4.03, m 4.13, m 4.15, m 4.71, brd (9.0)

1.25, s 1.60, s 1.94, s

1.14, s 1.82, s 1.93, s

brs brs s s brs brs

suggested a grayanane skeleton with two exocyclic double bonds. The HMBC correlations from δH 5.04 and 5.11 (H2-17) to C-15 (δC 50.5), C-13 (δC 50.9), and C-16 (δC 158.6) and from δH 5.26 and 5.50 (H2-20) to C-1 (δC 53.3), C-9 (δC 55.1), and C-10 (δC 150.5) were used to establish the location of the two double bonds at Δ10,20 and Δ16,17, respectively. The NMR data of the remaining part of the molecule closely resembled those of rhodojaponin VI (15). Further HSQC and HMBC experiments enabled the full assignments of the 1H and 13C NMR spectra of 2 to be carried out. Thus, 2 (rhodomollein XXVII) was assigned as 2α,3β,5β,6β,14β-pentahydroxygrayan10(20),16(17)-diene. Compound 3 exhibited a molecular formula of C26H42O11, as deduced from its HRESIMS data. Analysis of the NMR data clearly indicated that 3 is related closely to rhodomollein I (10), but is glycosidic. Six carbons at δC 106.6 (C-1′), 75.4 (C2′), 78.7 (C-3′), 72.8 (C-4′), 78.2 (C-5′), and 63.2 (C-6′) were attributed to a glucose unit. The large coupling constant (8.0 Hz) of the anomeric proton at δH 4.94 (H-1′) revealed that the glucose is in the β-configuration. Acid hydrolysis and GC analysis were used to establish the D-configuration of the glucose. The location of the sugar unit was shown to be at the C-3 position based on glycosylation shifts of C-2 (3, δC 80.8; 10, δC 83.9) and C-3 (3, δC 98.4; 10, δC 88.8) and on the HMBC correlation from H-1′ of the glucose unit to C-3 of the aglycone moiety. Thus, the structure of compound 3 (rhodomoside C) was determined as 3β-[(β-D-glucopyranosyl)oxy]-2α,5β,6β,14β,16α-pentahydroxygrayan-10(20)-ene. Compound 4 was assigned a molecular formula of C20H32O7 based on its HRESIMS data. An epoxy group was identified by the NMR signals at δH 4.19 (δC 60.1) and 3.24 (δC 64.1). This epoxy group was located at C-2 and C-3 by the HMBC correlations from H3-18 and H3-19 to C-3 and from H-1 to C-2 and C-3. The 1H and 13C NMR data of 4 were very similar to those of a known diterpenoid, rhodojaponin III (14). The only difference was that 4 was found to have an additional oxygenated methine (H-12, δH 5.29; C-12, δC 69.6) instead of a methylene as in rhodojaponin III (H2-12, δH 1.68 and 2.50; C-12, δC 27.0). This suggested that 4 should have a hydroxy group at the C-12 position. NOESY correlations from H-12 to H-14, H-13, and OH-10α revealed OH-12 to be β-oriented. Thus, 4 (rhodomollein XXVIII) was determined as 2β,3βepoxy-5β,6β,10α,12β,14β,16α-hexahydroxygrayanane. The HRESIMS of compound 5 indicated a molecular formula of C26H44O12. The 1H and 13C NMR spectroscopic

4.40, m

5.04, d (8.0) 4.10, m 4.04, m 4.21, m 4.25, m 4.35, m 4.56, brd (12.0)

Recorded at 500 MHz. bRecorded at 800 MHz.

B



Article

Table 2. 1H NMR Spectroscopic Data of Compounds 6−9 and 17 in Pyridine-d5 (δ in ppm, J in Hz) 6a

no. 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′ a

7b

8a

9a

17a

2.84, m 4.79, dd (7.2, 4.5) 3.90, brs

2.79, d (8.8) 4.76, brs 4.17, m

3.15, d (7.5) 4.92, dd (7.5, 4.5) 3.96, d (4.5)

3.05, d (8.5) 4.83, m 4.22, m

2.76, d (10.5) 5.19, dd (10.5, 6.0) 3.83, d (6.0)

4.32, brd (10.0) 2.82, m 3.02, dd (13.5, 10.5)

4.26, brd (9.6) 2.84, brd (14.4) 3.07, dd (14.4, 9.6)

4.17, brd (10.0) 2.69, brd (14.0) 2.93, dd (14.0, 10.0)

4.12, brd (10.5) 2.71, brd (13.5) 2.96, dd (13.5, 10.5)

4.62, brd (8.0) 2.54, m 2.87, dd (14.0, 2.5)

2.11, brd (8.0)

2.11, brd (8.0)

2.37, brd (6.0)

2.36, brd (7.0)

2.20, brd (7.0)

1.64, m 2.68, dd (15.0, 5.5) 1.80, m

m dd (15.2, 6.4) m m brs d (7.2) d (14.4) d (14.4)

1.54, 1.69, 1.72, 1.92, 2.40, 4.52, 1.81, 2.17,

m m m m brs brs d (14.5) d (14.5)

1.55, 1.71, 1.73, 1.93, 2.41, 4.54, 1.81, 2.17,

m m m m brs m d (14.5) d (14.5)

1.57, 2.08, 1.67, 2.55, 2.48, 5.08, 2.27, 2.34,

m dd (14.0, 6.0) m m brd (3.0) d (7.0) d (14.5) d (14.5)

1.42, 1.16, 1.74, 5.24, 5.50,

s s s brs brs

1.43, 1.19, 1.87, 5.25, 5.47, 5.12, 4.03, 4.05, 4.18, 4.24, 4.35, 4.57,

s s s brs brs d (8.0) m m m m dd (11.5, 5.5) brd (11.5)

1.53, 1.44, 1.53, 1.78,

s s s s

4.94, 4.10, 4.25, 4.00, 4.12, 4.18, 4.70,

d (8.0) m m m m m brd (10.0)

2.42, 4.63, 1.90, 2.24,

brs d (6.0) d (14.5) d (14.5)

1.57, 2.71, 1.75, 1.82, 2.42, 4.65, 1.91, 2.24,

1.48, 1.14, 1.76, 1.62,

s s s s

1.48, 1.17, 1.89, 1.60,

s s s s

5.11, 3.92, 4.02, 4.17, 4.22, 4.36, 4.56,

d (8.0) m ddd (8.0, 5.4, 2.4) m m dd (11.2, 4.8) brd (11.2)


δC 73.6; 6, δC 74.7) and C-3 (7, δC 91.0; 6, δC 83.3) and the HMBC correlation from H-1′ of the sugar unit to C-3 of the aglycone moiety. Thus, the structure of 7 (rhodomoside E) was i d en t ifi e d a s 1 -e pi -3 β -[(β - D - g l u c o p y r a n o s y l ) o x y ] 2β,5β,6β,10β,14β,16α-hexahydroxygrayanane. Compound 8 gave a molecular formula of C20H32O6, as determined by HRESIMS. The NMR spectra of 8 and 6 were closely comparable, with the major differences observed for the signals of C-1, C-9, C-10, C-11, and C-20. In the 1H NMR spectrum of 8, instead of a methyl proton (δH 1.62) ascribed to H3-20 of compound 6, a pair of exocyclic double-bond signals appeared at δH 5.24 (brs) and 5.50 (brs), which showed HMBC correlations with C-1 (δC 67.6) and C-9 (δC 56.7). These observations suggested that 8 is a C-10(20) dehydrated derivative of 6. Thus, compound 8 (rhodomollein XXX) was identified as 1-epi-2β,3β,5β,6β,14β,16β-hexahydroxygrayan-10ene. The molecular formula of compound 9 was deduced as C26H42O11 by HRESIMS. Comparison of the NMR spectroscopic data of 9 with those of 8 indicated an extra glucose unit to be present. This extra unit was found to be attached at the C3 position of the aglycone on the basis of the glycosylation shifts of C-2 (9, δC 72.8; 8, δC 73.8) and C-3 (9, δC 92.4; 8, δC 84.2) and the key HMBC correlation from H-1′ of the sugar unit to C-3 of the aglycone moiety. Thus, the structure of 9 (rhodomoside F) was identified as 1-epi-3β-[(β-D-gluco-

data of 5 and 17 were found to be quite comparable, with the major differences focused on signals of the A ring. The C-2 (δC 73.9) signal of 5 was shifted upfield by 5.6 ppm when compared with that of 17 (δC 79.5), suggesting a β-configuration of OH-2. This was confirmed by observation of a NOE correlation in 5 between H-2 and H3-18. Acid hydrolysis of 5 afforded Dglucose, which was identified by GC analysis. Thus, the structure of 5 (rhodomoside D) was deduced as 3β-[(β-Dglucopyranosyl)oxy]-2β,5β,6β,10α,14β,16α-hexahydroxygrayanane. The HRESIMS of compound 6 gave a molecular formula of C20H34O7. The 1H and 13C NMR data of this compound closely resembled analogous data for rhodomollein XVIII.10 However, the C-1 (δC 70.7) signal of 6 was shifted downfield by 18 ppm when compared with the latter compound (δC 52.4), suggesting an A/B cis configuration. This was also supported by the observation of NOE correlations between H1/H-9 and H-1/H3-19 in 6 instead of H-1/H-14. In addition, NOE interactions between H-2/H3-18, H-3/H3-18, and H-6/ H3-18 suggested the α-orientation of H-2, H-3, H-6, and H3-18 (Figure 1). Therefore, 6 (rhodomollein XXIX) was determined as 1-epi-2β,3β,5β,6β,10β,14β,16β-heptahydroxygrayanane. Compound 7 provided the molecular formula C26H44O12, as revealed by HRESIMS. It was identified as a glucoside of 6 by comparing the NMR data. The glucose was connected to the C-3 position on the basis of the glycosylation shifts of C-2 (7, C



Article

Table 3. 13C NMR Spectroscopic Data of Compounds 1−9 and 17 in Pyridine-d5 (δ in ppm)

a

no.

1a

2a

3b

4b

5b

6a

7b

8a

9a

17a

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′

52.4 82.2 88.0 46.8 84.7 67.2 41.3 56.6 137.5 124.0 25.9 26.5 53.6 88.2 59.5 82.0 23.9 25.0 17.8 18.9

53.3 83.3 89.0 48.7 83.6 71.2 40.5 48.9 55.1 150.5 24.8 32.5 50.9 77.0 50.5 158.6 106.5 27.0 20.7 113.5

50.0 80.8 98.4 48.2 81.5 70.2 41.6 49.9 55.4 149.1 24.2 24.1 53.5 79.6 62.1 81.2 25.5 28.6 20.1 113.4 106.6 75.4 78.7 72.8 78.2 63.2

54.6 60.1 64.1 47.8 79.9 73.7 43.7 51.8 53.8 76.8 32.0 69.6 63.5 78.2 60.9 80.2 26.2 21.2 20.6 30.9

52.5 73.9 92.0 48.5 83.5 74.7 44.2 52.8 56.4 78.1 21.4 27.2 56.5 79.7 60.3 79.4 23.9 23.5 20.2 30.9 104.1 75.3 78.9 71.5 78.6 62.4

70.7 74.7 83.3 47.9 85.9 70.0 41.3 52.2 61.4 78.0 19.0 27.6 54.9 78.2 59.3 79.5 24.1 21.7 21.5 25.7

70.5 73.6 91.0 48.7 84.8 70.2 41.1 52.2 60.6 77.6 18.8 27.5 54.9 78.0 59.3 79.4 24.1 22.3 21.6 26.0 104.4 75.1 78.9 71.5 78.6 62.5

67.6 73.8 84.2 48.4 86.0 70.4 40.8 50.5 56.7 152.3 26.0 27.2 55.2 77.7 56.1 79.9 24.0 21.9 21.0 109.8

67.9 72.8 92.4 49.3 85.1 71.0 40.8 50.7 56.2 152.1 26.3 27.4 55.5 77.8 56.4 80.0 24.2 23.0 21.4 110.1 104.6 75.2 78.9 71.7 78.6 62.6

54.3 79.5 96.2 48.7 81.4 71.1 44.8 52.2 54.0 78.0 21.8 27.2 55.9 80.3 61.0 80.5 24.0 28.7 20.1 29.2 106.1 75.4 78.7 72.6 78.2 63.1


pyranosyl)oxy]-2β,5β,6β,14β,16α-pentahydroxygrayan-10(20)ene. The HRESIMS of compound 17 indicated a molecular formula of C26H44O12. The NMR data showed 17 to closely resemble 15, a known compound isolated in this investigation, except for an additional sugar moiety. On the basis of the chemical shifts of the sugar unit carbons as well as the coupling constants of the anomeric proton, a β-glucose substituent was identified. The glycosylation shifts of C-2 (17, δC 79.5; 15, δC 80.7) and C-3 (17, δC 96.2; 15, δC 86.7) and the HMBC correlation from H-1′ of the glucose unit to C-3 of the aglycone moiety indicated the connectivity of the glucose to the C-3 position. Acid hydrolysis of 17 afforded D-glucose, which was

Figure 1. Selected 1H−1H COSY, HMBC, and NOE correlations of compounds 1 and 6.

Figure 2. Antinociceptive activities of the purified samples from the roots of R. molle (acetic acid-induced writhing test). Ibuprofen (ib) and morphine (morph) were used as control drugs. Each bar and vertical line represents the mean ± SEM of the values obtained from 12 mice. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant differences between test compound or control drug groups and vehicle group (veh) (one-way ANOVA followed by the Bonferroni test). D



Article

Figure 3. Influence of 14 and 15 on chemical and thermal stimuli pain models. (a) Antinociceptive effects of various concentrations of 14, 15, and morphine (morph) (ip) in the acetic acid-induced writhing test (n = 12). (b) Influence of compounds 14, 15, and morphine (morph) (ip) in the hot-plate test (n = 12). Each bar and vertical line represents the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant differences between test compound or control drug groups and vehicle (veh) group (one-way ANOVA followed by the Bonferroni test).

Figure 4. Antinociceptive properties of 14 and 15. (a) Antinociceptive effects of 14, 15, and morphine (morph) (ip) with or without naloxone pretreatment (n = 12). (b) Effects of 14 and 15 (ip) on phases I and II of the formalin test (n = 10). (c) Oral administration of both 14 and 15 reverses mechanical hyperalgesia in STZ-diabetic mice (n = 8). Morphine (morph), ibuprofen (ib), and gabapentin (gbp) were used as control drugs. Each bar and vertical line represents the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant differences between the naloxonepretreated group (5 mg/kg, ip) and vehicle-pretreated group (in A); test compound or control drug groups and vehicle (veh) groups (in B and C) (one-way ANOVA followed by the Bonferroni test).

XIX (18),17 craiobiotoxin VIII (19),18 and craiobioside A (20).18 The acetic acid-induced writhing test, a known sensitive and predictive animal model for acute pain, was used to evaluate the antinociceptive activities of the isolated compounds. In a preexperiment, the doses used for oral administration (po) were about 10-fold more than that used by intraperitoneal administration (ip) to achieve a comparable effect. For the sake of saving test materials, ip administration was used in most of the antinociceptive tests. As shown in Figure 2, the ip administration of 4, 6, 12, and 14−20 significantly reduced writhing when compared with the vehicle-injected mice (p

Antinociceptive Grayanoids from the Roots of Rhododendron molle

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