Micranthanosides i and II, two novel 1,10-secograyanane diterpenoids and their antinociceptive analogues from the leaves and twigs of: Rhododendron micranthum

Article abstract of DOI:10.1039/c9ra01736d

Micranthanosides I and II (1-2), two diterpenoid glucosides featuring a new 1,10-secograyanane skeleton, thirteen new diterpenoid glycosides (3-15), and 21 known analogues were obtained from the ethanol extract of the leaves and twigs of Rhododendron micranthum. Micranthanoside XII (12) represent the first example of 3,5-epoxy-4,5-seco-ent-kaurane diterpenoid. The structures of these compounds were determined by spectroscopic data analysis and quantum chemical calculations. To clarify the chemical basis and provide reference for rational use of this medicinal plant, the antinociceptive and the anti-inflammatory activities of the compounds were tested. In the acetic acid-induced writhing test, compounds 17 and 19 showed significant antinociceptive activity at a dose of 3 mg kg^-1 and compounds 2, 6 and 32 showed significant antinociceptive activity at a dose of 10 mg kg^-1. Toxic reactions such as nausea and convulsion were observed when 17, 19, 29, and 31 at a dose of 10 mg kg^-1 or 30 and 33 at a dose of 1 mg kg^-1 were administered. The anti-inflammatory activities of the isolated compounds were evaluated by measuring the inhibitory effects of LPS-induced NO production in BV2 cells. At 10 μM, micranthanoside IX (9) and rhodomicranoside F (26) showed moderate anti-inflammatory activities with inhibition rates of 56.31% and 72.43%, respectively.

Full text of DOI:10.1039/c9ra01736d

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Micranthanosides I and II, two novel 1,10-
secograyanane diterpenoids and their
antinociceptive analogues from the leaves and
twigs of Rhododendron micranthum†
Cite this: RSC Adv., 2019, 9, 18439
Yuxun Zhu,‡ Huimin Yan,‡ Xiaojing Wang, Zhaoxin Zhang, Huanping Zhang,
*
Lisha Chai, Li Li,
Jing Qu and Yong Li
Micranthanosides I and II (1–2), two diterpenoid glucosides featuring a new 1,10-secograyanane skeleton,
thirteen new diterpenoid glycosides (3–15), and 21 known analogues were obtained from the ethanol
extract of the leaves and twigs of Rhododendron micranthum. Micranthanoside XII (12) represent the
first example of 3,5-epoxy-4,5-seco-ent-kaurane diterpenoid. The structures of these compounds were
determined by spectroscopic data analysis and quantum chemical calculations. To clarify the chemical
basis and provide reference for rational use of this medicinal plant, the antinociceptive and the anti-
inflammatory activities of the compounds were tested. In the acetic acid-induced writhing test,
compounds 17 and 19 showed significant antinociceptive activity at a dose of 3 mg kgꢀ1 and
compounds 2, 6 and 32 showed significant antinociceptive activity at a dose of 10 mg kgꢀ1. Toxic
reactions such as nausea and convulsion were observed when 17, 19, 29, and 31 at a dose of 10 mg kgꢀ1
or 30 and 33 at a dose of 1 mg kgꢀ1 were administered. The anti-inflammatory activities of the isolated
compounds were evaluated by measuring the inhibitory effects of LPS-induced NO production in BV2
cells. At 10 mM, micranthanoside IX (9) and rhodomicranoside F (26) showed moderate anti-inflammatory
activities with inhibition rates of 56.31% and 72.43%, respectively.
Received 7th March 2019
Accepted 22nd May 2019
DOI: 10.1039/c9ra01736d
rsc.li/rsc-advances
antinociceptive and anti-inammatory effects as well as its
toxicity is still not well understood.
Introduction
Previously, we have reported antinociceptive grayanane
diterpenoids with structural diversity from R. molle, R. decorum,
and Pieris formosa. Among them, rhodojaponin III and VI,
craiobiotoxin IX, and pieristoxin N and P were found to be
highly potent in several antinociceptive models.9–12 In addition,
micranthanoside A, grayanotoxins I, and III in R. micranthum
were reported to have signicant antinociceptive activity at
a dose of 0.2 mg kgꢀ1 in the acetic acid-induced writhing
test.13,14 In order to provide chemical evidence for rational
application of “zhaoshanbai” and discover structurally inter-
esting lead compounds, leaves and twigs of R. micranthum
(107.5 kg) were collected from Shandong Province. An ethanol
extract of the leaves and twigs of R. micranthum was investigated
and afforded 36 diterpenoids, including 15 new analogues (1–
15) (Fig. 1). The isolation, structural elucidation, anti-
nociceptive activities, and anti-inammatory activities of these
compounds are described herein.
Natural products have been important sources for drug
discovery.1,2 Evidence suggests that natural products continue
to play a signicant role in the area of genomics.3 Rhododendron
micranthum Turcz. (Ericaceae), commonly known as “zhaosh-
anbai”, is widely distributed in northern China. This plant is
used traditionally as a medicine for the treatment of post-
partum arthralgia and chronic bronchitis.4 The extract of R.
micranthum has also been developed as a drug for clinical use.
Meanwhile, cases of clinical poisoning with “zhaoshanbai” have
occasionally been reported.5,6 In these cases, some patients were
found to have side effects and toxic symptoms such as nausea,
epigastric pain and burning, hypotension, bradycardia, and
dizziness.7 Although andromedotoxin from R. micranthum has
been reported to be toxic,8 the chemical basis of its
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union
Medical College, Beijing 100050, People's Republic of China. E-mail: liyong@imm.
ac.cn
Results and discussion
† Electronic supplementary information (ESI) available: 1D and 2D NMR,
HRESIMS, CD, GC analysis, and IR. See DOI: 10.1039/c9ra01736d
‡ Y.-X. Z. and H.-M. Y. contributed equally.
Compound 1 (micranthanoside I) was obtained as white powder
and was found to have a molecular formula of C26H42O8 based
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Fig. 1 Chemical structures of compounds 1–36.
on the (+)-HRESIMS ion at m/z 505.2786 [M + Na]+ (calcd. for correlations from H-1 to C-6 and from H-9 and H2-15 to C-7
26H42O8Na, 505.2772), corresponding to six indices of conrmed the connection of C-5 and C-8 via fragment b. The
hydrogen deciency (IHD). Its IR spectrum suggested the glucose unit was placed at C-16 based on the HMBC correlation
presence of hydroxy (3368 cmꢀ1) and carbonyl (1700 cmꢀ1 from H-10 to C-16. As a result, the planar structure of 1 was fully
functionalities. The 1H NMR (Table 1) data showed resonances established, which is the rst example of a 1,10-secograyanane.
of four methyl groups (dH 1.20, 1.28, 1.64, and 2.17), a glucose The relative conguration of 1 was deduced by the NOESY
C
)
unit (dH 3.93, 3.97, 4.24, 4.25, 4.38, 4.51, and 5.01), and an experiments. Ring A and rings B/C were connected through two
olenic proton at dH 5.27. The 13C NMR data (Table 4), HSQC, methylenes, which should theoretically provide molecular ex-
and DEPT spectra of 1 exhibited 26 carbon resonances, ibility and complicate the stereochemical analysis. The NOESY
including four methyls, eight methylenes, nine methines, and correlations of H3-18/H-6a and H-14a/H-7a (a-side) and H3-19/
ve sp3 carbons. A sugar unit, a carbonyl group, and a double H-6b and H-14b/H-7b (b-side) indicated that bulky substituents
bond account for three of the IHDs, which suggest that the at C-5 and C-8 restrict the rotation of the C-6/C-7 bond and led
remaining part is consisting of three rings.
to a preferential conformation in pyridine-d5 (room tempera-
The COSY (Fig. 2a) and HSQC spectra established four ture) in which C-5 and C-8 were in the anti-position (Fig. 2b).
fragments: CH(OH)–CH2–CH, CH2–CH2, CH–CH2–CH2–CH– The NOESY correlations of H-3/H3-18, H3-18/H-6a, H3-19/H-6b,
CH2, and CH–CH(OH)–CH(OH)–CH(OH)–CH–CH2(OH). Frag- H-6b/H-9, H-9/H-15b, and H3-17/H-12b indicated that H-3 is a-
ment a and the HMBC correlations from two gem-dimethyl oriented and H-9 and CH3-17 are b-oriented. The anomeric
singlets (H3-18 and H3-19) to carbons C-3, C-4, and C-5 allowed proton at H-10 (dH 5.01) showed a large coupling constant (7.8
the ve-membered carbon ring (ring A in Fig. 1) to be dened. Hz), indicating that the glucose unit is in b-conguration. Acid
The HMBC correlations from H-6 to C-1 and from H2-7 to C-5 hydrolysis and GC analysis of the sugar moiety of 1 conrmed
connected C-5 directly to fragment b via C-6. HMBC correla- that the sugar was D-glucose (retention time: 29.35 min). To
tions from H-9 and H-14 (two ends of fragment c) to dC 47.3 (C- determine the absolute conguration of 1, the ECD spectra for
8), from H2-12 to quaternary carbon at dC 88.1 (C-16), as well as 1a (3S,8S,9R,13R,16R,10S,20R,30S,40S,50R) and its enantiomer 1b
correlations from H2-15 to C-8, C-9, C-14, and C-16 revealed were calculated using time-dependent density functional theory
a bicyclo[3.2.1]octane ring system (rings B/C). The HMBC calculations at the B3LYP/6-31+G(d,p) level in methanol.15 The
correlations from H3-20 (dH 2.17) to C-9 (dC 53.6)/C-10 (dC 212.9) measured CD spectrum of 1 agreed well with the calculated ECD
indicated that C-9 was connected to C-10. Finally, key HMBC
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Table 1 1H NMR spectroscopic data for compounds 1–7 in pyridine-d5 (d in ppm, J in Hz)
No 1a
2b
3b
4b
5b
6a
7a
1
2
5.27, brs
2.46, m
5.16, brs
2.55, m
3.39, m
2.33, m
—
3.51, t (9.4)
3.34, d (8.5)
2.19, m
2.41, m
4.38, brs
—
3.29, d (9.3)
2.38, m
2.57, m
4.40, m
—
2.87, dd (16.1, 6.8) 2.41, m
3.34, dd (16.2, 7.3) 2.41, m
4.35, t (7.0)
2.64, dd, (15.3, 6.7) 2.69, dd (14.1, 7.1) 2.63, m
4.26, m
—
3
4.24, t (7.2)
—
4.2, m
—
3.87, m
—
—
4
5
6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2.07, m
2.29, t (13.5)
1.78, m
1.91, m
—
2.00, m
2.15, m
1.65, m
1.88, m
—
1.62, m
1.73, m
1.24, d (11.1)
2.11, m
—
6.07, d (9.8)
—
5.55, d (9.8)
4.25, m
—
2.39, m
2.53, m
—
5.36, brs
—
2.40, m
2.52, m
—
5.51, m
—
2.18, m
2.51, m
—
7
8
—
9
10
11
2.76, brs
—
2.73, d (7.2)
—
2.90, brs
—
4.38, m
—
1.64, m
1.84, m
2.19, t (6.6)
—
—
2.20, m
—
—
2.10, m
2.18, m
—
1.73, m
2.15, m
—
1.57, m
1.59, m
1.55, m
1.92, m
2.53, brs
2.36, m
2.38, m
1.55, m
2.37, m
—
1.59, m
1.66, m
1.58, m
1.91, m
2.19, brs
1.72, m
1.82, m
1.52, m
1.54, m
2.15, m
2.45, dd (15.0, 7.1) 2.61, dd (15.2, 6.5) 1.73, m
1.79, m
1.88, m
2.32, m
12
1.54, m
1.70, m
2.54, brs
1.58, d (10.8)
1.48, m
1.72, m
2.47, brs
1.55, d (11.1) 1.62, d (10.1)
2.23, m 2.14, m
1.74, d (12.8) 1.92, brs
2.26, d (14.2) 1.92, brs
—
13
14
2.30, dd (11.0, 4.7) 1.41, m
2.13, d (11.0)
2.43, d (11.2)
1.67, d (14.0)
2.04, d (13.8)
—
1.58, s
1.21, s
1.98, dd (11.7, 3.3) 2.39, dd (11.0, 4.7) 2.71, m
1.24, d (11.1)
1.79, dd (11.1, 3.3) 2.09, d (12.5)
—
1.43, s
0.99, s
—
15
1.60, d (12.2)
2.41, m
2.90, d (15.2)
—
1.67, s
0.88, s
16
17
18
—
1.39, s
1.34, s
—
1.64, s
1.20, s
—
1.59, s
1.17, s
1.54, s
1.28, s
—
—
19
20
1.28, s
2.17, s
—
5.01, d (7.8)
3.97, m
4.25, m
4.24, m
3.93, brs
4.38, dd (11.6, 5.1)
4.51, dd (11.6, 2.6)
1.27, s
2.15, s
—
4.70, d (7.8)
4.05, m
4.28, m
4.28, m
3.97, brs
1.48, s
5.05, s
5.47, s
4.99, d (7.8)
3.99, m
4.27, m
4.23, m
4.02, m
1.31, s
1.79, s
—
5.03, d (7.8)
4.07, brs
4.28, m
1.32, s
1.84, s
—
5.05, d (7.8)
4.00, m
4.28, m
4.29, m
3.90, m
4.39, m
4.49, d (10.6)
1.22, s
4.96, s
5.09, s
5.01, d (7.8)
4.01 t, (11.1)
4.29, m
4.26, m
3.94, brs
4.36, m
1.28, s
5.00, s
5.20, s
5.04, d (7.8)
4.06, m
4.29, m
4.29, m
4.03, m
4.45, m
10
20
30
40
50
60
4.27, m
4.02, brs
4.42, d (11.8)
4.60, d (11.5)
4.42, dd (11.7, 5.2) 4.42, m
4.58, dd (11.7, 2.4) 4.61, d (11.9)
4.51, d (11.3) 4.62, d (9.6)
a
Recorded at 500 MHz. b Recorded at 600 MHz.
of 1a and is the opposite of that of 1b (Fig. 3). Thus, the absolute
conguration of 1 was established.
Compound 2 (micranthanoside II) was obtained as white seven indices of hydrogen deciency (IHD). The H-NMR data
powder and was found to have a molecular formula of showed resonances of three methyl groups (dH 0.99, 1.43, and
Compound 3 (micranthanoside III) has a molecular formula
of C26H40O8 based on the HRESIMS data, which suggested
1
C
26H42O8. Its NMR data were highly similar to those of 1 except 1.48) and a glucose unit (dH 3.99, 4.02, 4.23, 4.27, 4.42, 4.61, and
13
for the variations in the chemical shis of C-3 (DdC +8.3) and C- 4.99). The C NMR data, HSQC, and DEPT (135ꢁ) spectra of 3
16 (DdC ꢀ8.9), suggesting that the glucose unit was placed at C- exhibited 26 carbon resonances, including three methyls, eight
3. The key HMBC correlation from H-10 to C-3 conrmed the methylenes, ten methines, and ve sp3 carbons. The COSY and
above assignment. The relative conguration of 2 was assigned HSQC spectra established four fragments: CH–CH2–CH, CH2–
the same as 1 by its NOESY correlations of H-3/H3-18 and H-9/H- CH2, CH–CH–CH2–CH–CH2, and CH–CH(OH)–CH(OH)–
15b. The anomeric proton at H-10 (dH 4.70) showed a large CH(OH)–CH–CH2(OH). These structural features are consistent
coupling constant (7.8 Hz), indicating that the glucose unit is in with a grayanane-type diterpenoid glycoside. The overall struc-
b-conguration. Acid hydrolysis and GC analysis of the sugar tural connectivity was established by HSQC and HMBC spec-
moiety of 2 conrmed that the sugar was D-glucose (retention troscopic data. The HMBC correlations from H2-20 (dH 5.05 and
time: 29.32 min). The CD spectrum of 2 was consistent with that 5.47) to C-1 (dC 48.7)/C-9 (dC 54.5)/C-10 (dC 149.7) established an
of 1, showing a negative cotton effect at 303 nm (ESI Fig. S163 exocyclic D10(20) double bond. The HMBC correlations from H3-
and S164†). Thus, the absolute conguration of 2 was assigned 18/H3-19/H2-2 to C-3 (dC 90.1), from H-1/H2-6/H2-7/H3-18/H3-19
as (3S,8S,9R,13R,16R,10R,20R,30S,40S,50R).
to C-5 (dC 83.7), from H-9/H2-12/H-13 to C-11 (dC 84.5), and from
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Fig. 2 (a) Key 1H–1H COSY, HMBC, and NOESY correlations for 1. (b) Conformations of C-6 and C-7, boxes indicate conformations that agree
with the measured data and are energetically favored.
proton at H-10 (dH 4.99) showed a large coupling constant (7.8
Hz), indicating that the glucose unit is found in the b-congu-
ration. Acid hydrolysis and GC analysis of the sugar moiety of 3
conrmed that the sugar was D-glucose (retention time: 29.56
min). The relative conguration of 3 was determined by the
NOESY spectrum. The correlations of H-1/H3-18, H-3/H3-18, and
H-1/H-6a indicated that H-1 and H-3 are cofacial (a-face) and
HO-5 should be b-oriented. The correlations of H-9/H-15b and
H3-17/H-12b indicated that H-9 and CH3-17 are cofacial (b-face).
Thus, the structure of 3 was dened as 11b,16a-epoxy-3b-[(b-D-
glucopyranosyl)oxy]-5b-hydroxygrayanan-10(20)-ene.
Compound 4 (micranthanoside IV) was obtained as white
powder. Its molecular formula was dened as C26H38O7 based
on HRESIMS (m/z 485.2492 [M + Na]+, calcd 485.2510), which
indicated an IHD of eight. Preliminary 1H and 13C analyses of 4
indicated the presence of a common grayanane skeleton. The
Fig. 3 The experimental CD spectrum of 1 (black) and the calculated
ECD spectra of 1a (red) and its enantiomer 1b (blue).
NMR spectroscopic data of 4 were comparable to those of
pierisformoside C.16 The only difference was the location of the
double bond. The HMBC correlations from H3-20/H2-11 to C-9
(dC 142.6)/C-10 (dC 124.3) located the double bond at D9,10
instead of D10,20. The glucose unit was placed at C-3 based on
the HMBC correlation from H-10 to C-3. The anomeric proton at
H-10 (dH 5.03) showed a large coupling constant (7.8 Hz), indi-
cating that the glucose unit is in b-conguration. Acid hydro-
lysis gave a D-glucose, which was identied by GC analysis
(retention time: 29.49 min). In the NOESY spectrum, the
correlations of H-3/H3-18 indicated that H-3 is a-oriented. Thus,
H3-17/H2-14/H2-12/H-13 to C-16 (dC 77.4) indicated the location
of four oxygenated sp3 carbons (C-3, C-5, C-11, and C-16). The
skeleton (ve rings including sugar part) and the exocyclic
double bond accounted for six of the IHDs. The HMBC corre-
lation from H-11 to C-16 suggested that C-11 and C-16 were
connected via oxygen bridge. The glucose unit was placed at C-3
based on the HMBC correlation from H-10 to C-3. The anomeric
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Table 2 1H NMR spectroscopic data for compounds 8–11 in pyridine-d5 (d in ppm, J in Hz)
No
8b
9a
10a
11a
1
2
2.78, dd (11.9, 7.4)
1.54, m
1.65, m
1.47, m
2.39, m
—
3.14, m
2.13, m
2.67, m
4.01, m
—
3.26, t (9.3)
2.16, dd (14.0, 9.3)
2.31, m
4.11, brs
—
3.18, t (9.4)
2.23, m
2.64, m
3.94, d (5.0)
—
—
3
4
5
6
—
—
—
—
—
—
1.66, m
1.72, m
1.83, m
1.89, m
—
1.67, m
1.67, m
1.68, m
1.73, m
—
1.38, m
1.53, m
1.72, m
2.67, m
—
4.48, d (8.6)
—
2.02, d (13.7)
2.76, dd (14.4, 9.8)
—
7
8
9
10
11
2.13, m
—
3.11, m
—
2.29, m
—
2.80, m
—
1.57, m
1.72, m
1.39, m
1.73, m
2.22, brs
1.92, d (11.1)
2.36, dd (11.0, 4.1)
2.11, d (13.7)
2.29, d (14.0)
—
1.55, s
1.34, s
—
1.69, s
1.77, m
1.77, m
1.60, m
2.14, m
2.44, m
1.64, m
2.20, m
1.79, d (13.4)
2.33, d (14.1)
—
1.80, m
1.80, m
1.63, m
2.35, m
2.51, brs
1.50, m
1.70, m
1.88, d (14.0)
2.33, d (14.3)
—
1.63, m
1.82, m
1.59, m
1.81, m
2.62, m
1.88, d (11.1)
2.22, m
1.69, d (13.9)
2.26, d (15.0)
—
12
13
14
15
16
17
18
1.62, s
1.62, s
0.80, s
—
1.29, s
1.60, s
0.80, s
—
1.28, s
3.83, m
3.99, m
1.05, s
19
20
5.22, s
5.10, s
5.17, s
5.19, s
5.31, s
5.14, s
5.19, s
5.22, s
10
20
30
40
50
60
4.99, m
3.97, m
4.27, m
4.25, m
3.91, m
4.36, m
4.50, d (11.5)
5.00, d (7.8)
4.01, m
4.28, m
4.27, m
3.93, brs
4.39, dd (11.6, 5.0)
4.52, dd (11.5, 2.2)
5.00, (8.5)
3.99, brs
4.27, m
4.27, m
3.93, brs
4.40, m
4.54, d (11.3)
4.98, d (7.7)
4.02, t (8.1)
4.17, m
4.18, m
3.87, brs
4.34, dd (11.4, 5.4)
4.54, dd (11.4, 2.7)
a
Recorded at 500 MHz. b Recorded at 600 MHz.
the structure of 4 was dened as 3b-[(b-D-glucopyranosyl)oxy]- and 13C NMR spectroscopic data indicated that 6 has a graya-
16a-hydroxygrayanan-1(5),6(7),9(10)-triene.
nane skeleton. Its 13C NMR spectrum exhibited the typical
HRESIMS analysis of compound 5 (micranthanoside V) resonances of two double bonds and an anomeric carbon at dC
indicated a molecular formula of C26H42O9. Spectroscopic data 107.2, 152.4, 118.3, 156.4, and 99.8. Further HSQC and HMBC
for 5 resembled those of pieroside C, which differs from 5 with experiments allowed full assignment of the 1H and 13C NMR
regard to the position of the glucose unit.17 The HMBC corre- spectra of 6. The location of the two double bonds (D5,6, D10,20
)
lations from H-10 to C-16 placed the glucose unit at C-16 instead was determined via HMBC correlations from H-1 to C-5/C-10,
of C-3 as in pieroside C. A large coupling constant (7.8 Hz) of the from H2-20 to C-1/C-9/C-10, and from H2-7 to C-5/C-6. The
anomeric proton indicated the b-conguration of the glucose. glucose unit was placed at C-16, which was supported by the
In the NOESY spectrum, the correlations of H3-18/H-1, H3-18/H- HMBC correlation from H-10 to C-16. Acid hydrolysis and GC
3, and H3-18/H-6 indicated that H-1, H-3, and H-6 are a- analysis of the sugar moiety of 6 conrmed that the sugar was D-
oriented, and the correlations of H3-17/H-11b indicated that glucose (retention time: 29.56 min). The relative conguration
CH3-17 is b-oriented. Acid hydrolysis and GC analysis of the of 6 was established by the NOESY experiment. The correlations
sugar moiety of 5 conrmed that the sugar was D-glucose of H-1/H3-18, H3-18/H-3, and H-1/H-9 indicated that H-1, H-3,
(retention time: 29.60 min). Thus, the structure of 5 was dened and H-9 are a-oriented. Thus, the structure of 6 was dened
as 16a-[(b-D-glucopyranosyl)oxy]-3b,5b,6b-trihydroxygrayanan- as
9(10)-ene.
Compound 6 (micranthanoside VI) was found by mass
9-epi-16a-[(b-D-glucopyranosyl)oxy]-3b-hydroxygrayanan-
5(6),10(20)-diene.
Compound 7 (micranthanoside VII) was determined to have
spectrometry to have a molecular formula of C26H40O7. The 1H the formula C26H40O7. The planar structure of 7 was found to be
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Table 3 1H NMR spectroscopic data for compounds 12–15 in pyridine-d5 (d in ppm, J in Hz)
No
1
12a
13a
14a
15a
1.72, m
1.97, m
1.42, m
1.72, m
4.68, dd (11.4, 1.9)
—
—
—
4.09, brs
—
1.46, m
2.07, m
1.84, dd (13.9, 2.4)
2.14, m
3.65, d (2.5)
—
—
—
—
—
1.46, m
1.96, m
1.43, m
1.82, m
0.92, d (12.7)
2.68, td (13.3, 4.1)
—
—
—
—
1.66, t (13.7)
2
2.18, m
2.56, m
3.99, m
—
—
—
3
4
5
6
2.53, m
—
7
2.07, m
2.45, t (12.1)
—
2.22, d (11.6)
3.96, d (11.4)
—
2.21, d (11.8)
4.07, d (11.9)
—
1.78, m
1.78, m
—
8
9
10
11
2.09, m
—
2.59, d (8.7)
—
2.57, d (8.5)
—
1.95, m
—
1.42, m
1.60, d (13.1)
1.51, m
1.60, m
2.54, brs
1.96, m
2.29, m
1.60, d (13.1)
2.39, d (14.0)
—
1.46, m
1.64, m
1.53, m
1.57, m
2.51, brs
1.92, d (11.4)
2.30, dd (11.3, 4.0)
1.74, d (14.1)
2.35, d (14.1)
—
1.57, m
1.62, m
1.48, m
1.56, m
2.52, brs
1.91, d (11.4)
2.28, m
1.75, d (14.1)
2.31, d (14.3)
—
1.59, m
1.94, m
1.52, m
1.59, m
2.12, d (2.9)
1.78, t (12.8)
2.02, m
1.86, d (13.9)
2.04, m
—
12
13
14
15
16
17
1.66, s
1.62, s
—
1.61, s
—
1.54, s
—
18
19
20
4.83, s
5.10, s
1.74, s
1.52, s
1.31, s
1.20, s
1.50, s
3.4, d (8.3)
4.56, dd (10.7, 4.7)
1.11, s
1.51, s
1.23, s
1.12, s
4.89, s
—
—
—
5.06, s
10
20
30
40
50
60
4.99, d (7.8)
3.98, brs
4.29, m
4.29, m
3.93, brs
4.40, m
4.52, d (10.1)
4.96, d (7.8)
3.97, m
4.25, m
4.27, m
3.91, brs
4.95, d (8.1)
3.96, brs
4.25, m
4.27, m
3.91, brs
5.02, d (7.7)
4.08, t (8.2)
4.31, m
4.28, m
4.02, m
4.38, dd (11.6, 5.1)
4.51, dd (11.6, 2.3)
4.39, d (12.4)
4.51, d (11.4)
4.41, dd (11.6, 5.4)
4.59, dd (11.6, 2.4)
a
Recorded at 500 MHz.
identical to that of 6 except for the location of the sugar moiety. structure of 8 was dened as 1-epi-16a-[(b-D-glucopyranosyl)
The HMBC correlations from H-10 to C-3 placed the sugar oxy]-5b-hydroxygrayanan-10(20)-ene-18-ol.
moiety at C-3. The correlations of H3-18/H-3, H-3/H-1, and H-1/
Compound 9 (micranthanoside IX) exhibited a molecular
H-9 in the NOESY spectrum indicated that H-1, H-3, and H-9 are formula of C26H42O8 based on the HRESIMS results. The 1H and
a-oriented. Thus, the structure of 7 was dened as 9-epi-3b-[(b-D- 13C NMR spectroscopic data suggested that 9 has a grayanane
glucopyranosyl)oxy]-16a-hydroxygrayanan-5(6),10(20)-diene.
skeleton similar to that of a known analogue, 6-deoxy-
The formula of compound 8 (micranthanoside VIII) was grayanotoxin XVII,18 and an extra sugar moiety was the only
identied as C26H42O8 by HRESIMS, indicating an IHD of six. difference. The glucose unit was placed at C-16 based on the
The 1H (Table 2) and 13C NMR data of 8 were similar to those of HMBC correlation from H-10 to C-16. The NOESY correlations of
micranthanoside E,13 except for the differences associated with H-1/H3-18, H3-17/H-12b, and H-9/H3-17 indicated that H-1 is a-
the locations of the hydroxy groups. In micranthanoside E, the oriented and H-9 and CH3-17 are b-oriented. Acid hydrolysis
hydroxy groups were placed at C-3 and C-6. However, the and GC analysis of the sugar moiety of 9 conrmed that the
hydroxy group was placed at C-18 in 8 indicated by the HMBC sugar was D-glucose (retention time: 29.53 min). Thus, the
correlations from H3-19/H2-3 to C-18. The NOESY correlations structure of 9 was dened as 16a-[(b-D-glucopyranosyl)oxy]-
of H-1/H3-19, H-1/H-9, H-9/H-15b, and H3-17/H-12b suggested 3b,5b-dihydroxygrayanan-9(10)-ene.
that H-1, H-9, CH3-17 and CH3-19 are b-oriented. Thus, the
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Fig. 4 Antinociceptive activities of partial diterpenoids isolated from R. micranthum. Data represent the mean ꢂ SEM. *p < 0.05, **p < 0.01, vs.
vehicle (veh). Control drugs: ibuprofen (ib) and morphine (morph).
The molecular formula of compound 10 (micranthanoside H3-18, H3-17/H-9, and H-9/H-15b indicated that H-1, H-3, and
X) was determined as C26H42O8, implying an IHD of six. The H-6 are a-oriented and H-9 and H3-17 are b-oriented. Thus,
HSQC and HMBC spectra indicated that the planar structure of the structure of 11 was dened as 6b-[(b-D-glucopyranosyl)
10 was the same as that of 9. The NOESY correlations of H-1/H- oxy]-3b,5b,16a-trihydroxygrayanan-9(10)-ene.
9, H-1/HO-5, H3-19/HO-5, and H3-17/H-12b in 10 indicated that
Compound 12 was obtained as white powder and deter-
H-1 is b-oriented, which is the only difference between the two mined to have the molecular formula C26H42O9 by HRESIMS.
1
compounds. Acid hydrolysis and GC analysis of the sugar The H (Table 3) and 13C NMR spectroscopic data suggested
moiety of 10 conrmed that the sugar was D-glucose (retention that 12 has a 4,5-seco-ent-kaurane skeleton similar to known
time: 29.55 min). Thus, the structure of 10 was dened as 1-epi- analogues.14 The 1H, 13C, and HSQC NMR data for 12 indi-
16a-[(b-D-glucopyranosyl)oxy]-3b,5b-dihydroxygrayanan-9(10)-
ene.
cated the presence of a sugar unit (dC 63.4, 72.3, 75.8, 78.3,
79.4, and 99.9) and two olenic carbons (dC 110.5 and 148.0).
The molecular formula of compound 11 (micranthanoside The overall structural connectivity was established by HSQC
XI) was determined as C26H42O9, implying an IHD of six. The and HMBC spectroscopic data. The HMBC correlations from
1H and 13C NMR data of 11 were similar to that of HO-5 to C-5/C-10 and from H-6 to C-7 placed two hydroxy
grayanotoxin-XVIII (17) except for signal of an additional groups at C-5 and C-6. The sugar unit was placed at C-16
sugar moiety.19 The glucose unit was placed at C-6 as sug- suggested by HMBC correlations of H-10 to C-16. An oxygen
gested by the HMBC correlations from H-10 to C-6. The bridge should be present between C-3 and C-5, which was
NOESY correlations of H-3/H3-18, H-1/H-6, H-6/H3-18, H-1/ supported by the HMBC correlation from H-3 to C-5. The
Fig. 5 Inhibitory effects of LPS-induced NO production in BV2 cells.
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Table 4 13C NMR spectroscopic data for compounds 1–16 in pyridine-d5 (d in ppm)
No.
1b
2b
3b
4a
5a
6a
7b
8a
9a
10b
60.6
11a
45.2
12a
32.1
13a
28.2
14b
33.2
15a
42.5
1
118.9
39.0
81.5
49.1
152.1
23.6
38.6
47.3
53.6
212.9
22.9
25.7
46.7
37.9
51.2
88.1
21.4
25.8
20.3
32.6
99.8
75.8
79.3
72.3
78.6
63.5
118.9
37.8
89.8
49.0
151.5
23.0
38.3
47.2
54.2
213.0
22.7
26.0
49.3
37.8
53.3
79.2
25.2
25.9
20.5
32.7
106.5
76.0
79.1
72.2
78.9
63.4
48.7
138.7
41.2
87.8
49.6
144.8
122.7
130.9
50.0
142.6
124.3
24.1
26.4
46.4
39.8
52.5
80.6
29.5
26.5
21.7
18.2
106.5
76.0
79.2
72.3
78.9
63.5
45.1
46.8
46.6
54.8
47.6
2
3
4
5
6
7
8
9
34.7
90.1
51.4
83.7
32.2
34.2
47.7
54.5
149.7
84.5
39.8
45.6
44.1
57.9
86.7
24.0
25.6
18.8
114.6
105.7
76.1
79.2
72.2
79.1
63.4
37.7
81.1
49.5
85.7
68.4
46.8
51.1
136.3
122.1
27.3
29.3
45.8
48.8
55.9
90.1
21.7
24.8
18.3
20.6
99.8
75.8
79.3
72.3
78.5
63.4
37.4
80.6
46.7
156.4
118.3
41.9
46.6
55.8
152.4
26.6
25.5
47.8
35.6
55.4
88.5
21.1
28.1
24.2
107.2
99.8
75.9
79.4
72.4
78.7
63.4
36.2
89.8
46.6
155.7
118.1
42.6
46.7
55.7
151.9
26.3
25.8
50.2
36.0
58.2
79.6
24.8
28.3
24.5
107.5
106.6
76.0
79.2
72.3
78.9
63.4
26.5
33.2
50.5
83.7
26.2
39.3
47.7
54.6
151.0
25.5
33.4
47.0
36.6
56.2
88.9
21.5
68.0
23.5
111.3
99.8
75.7
79.4
72.3
78.6
63.5
39.3
80.7
50.7
84.2
32.2
31.8
46.7
47.5
154.4
25.3
37.8
46.7
24.9
57.8
89.2
21.8
24.3
17.9
111.7
99.8
75.9
79.4
72.4
78.6
63.5
36.7
83.3
50.3
85.6
29.4
36.4
47.2
55.9
155.3
28.1
27.0
47.1
35.6
55.5
88.2
21.6
23.7
19.3
109.8
99.9
75.8
79.3
72.3
78.6
63.5
39.9
82.3
51.7
84.0
79.1
41.8
45.1
53.7
153.0
26.0
24.4
48.4
36.8
63.6
80.0
26.3
23.9
19.9
112.9
103.0
75.5
79.0
72.6
78.7
63.6
26.8
72.9
148.0
99.6
71.1
44.8
45.5
42.6
40.9
18.8
27.2
46.8
38.8
56.3
86.9
21.8
110.5
19.1
23.5
99.9
75.8
79.4
72.3
78.7
63.4
26.3
77.7
40.9
85.3
213.7
51.7
50.0
48.5
48.7
18.5
27.0
46.9
38.7
56.6
86.9
21.6
25.1
24.4
20.9
99.8
75.8
79.3
72.2
78.6
63.4
18.3
32.6
40.3
84.4
216.3
52.5
49.8
47.4
48.5
18.4
27.2
46.8
38.8
56.9
86.9
21.6
20.6
72.0
20.2
99.9
75.8
79.3
72.2
78.6
63.4
30.7
82.3
51.3
213.6
48.3
22.2
46.4
49.9
151.8
36.6
25.3
50.4
39.9
55.9
79.0
25.0
20.6
22.7
106.5
103.2
75.7
79.2
72.4
79.0
63.5
10
11
12
13
14
15
16
17
18
19
20
10
20
30
40
50
60
a
Recorded at 125 MHz. b Recorded at 150 MHz.
NOESY correlations of H-3/H-1b, H-1b/H-9, H-9/H-12b, and indicated that H-9 is b-oriented and CH3-20 is a-oriented.
H3-20/H-6 suggested that H-3 and H-9 are b-oriented and H-6 Acid hydrolysis and GC analysis of 14 conrmed that the
is a-oriented. Thus, the structure of 12 was named as sugar moiety was D-glucose (retention time: 29.50 min). Thus,
micranthanoside XII. Micranthanoside XII (12) represent the the structure of 14 was dened as 16a-[(b-D-glucopyranosyl)
rst example of 3,5-epoxy-4,5-seco-ent-kaurane diterpenoid.
Compound 13 (micranthanoside XIII) has a molecular
oxy]-5b-hydroxy-ent-kaur-6-one-19-ol.
Compound 15 (micranthanoside XV) has a molecular
formula of C26H42O9. Preliminary analyses of the NMR data formula of C26H40O8. Preliminary H and 13C analyses of 15
indicated the presence of an ent-kaurane skeleton. The NMR indicated the presence of a leucothane skeleton. The 13C
spectra indicated the presence of two hydroxy groups, NMR spectrum displayed resonances of a carbonyl group,
a carbonyl group, and a sugar moiety. The two hydroxy groups a sugar moiety and a double bond. The sugar moiety and the
were placed at C-3 and C-5 on the basis of the correlations from carbonyl group were placed at C-3 and C-5 based on the
HO-3 to C-2/C-3/C-4 and from HO-5 to C-4/C-5/C-6, respectively. HMBC correlations from H-10 to C-3 and from H3-18/H3-19/H-
The HMBC correlation from H-10 to C-16 indicated that the 6 to C-5, respectively. The HMBC correlations from H2-20 to
sugar moiety was located at C-16. The HMBC correlations from C-1/C-9/C-10 suggested that the exocyclic double bond is
1
H2-7 to C-6 placed the carbonyl group at C-6. In the NOESY
D
10(20). The NOESY correlations of H-3/H-1, H-3/H3-19, H-6/
spectrum of 13, correlations of H-3/HO-5, H-3/H3-19, H3-18/HO- H3-18, and H-9/H-15b indicated that H-1, H-3, and H-9 are b-
3, H3-20/H-14a, and H-9/H-15b indicated that H-3, HO-5, and H- oriented and H-6 is a-oriented. Acid hydrolysis and GC
9 are b-oriented and CH3-20 is a-oriented. Acid hydrolysis and analysis conrmed that the sugar moiety was D-glucose
GC analysis of 13 conrmed that the sugar moiety was D-glucose (retention time: 29.64 min). Thus, the structure of 15 was
(retention time: 29.54 min). Thus, the structure of 13 was dened as 3a-[(b-D-glucopyranosyl)oxy]-16a-hydroxyleucoth-
dened as 16a-[(b-D-glucopyranosyl)oxy]-3a,5b-dihydroxy-ent- 10(20)-en-5-one.
kaur-6-one.
The known compounds micranthanoside F (16),13 graya-
Compound 14 (micranthanoside XIV) has a molecular notoxin XVIII (17),19 pierisformosin A (18),20 6-deoxy-1-epi-
formula of C26H42O9. The 1H and 13C NMR data of 14 were grayanotoxin XVII (19),18 grayanotoxin IV (20),21 grayanoside
similar to those of 13. The HMBC correlations from H3-18 to C (21),22 grayanotoxin II (22),23 grayanoside B (23),19 micran-
C-19 indicated that the hydroxy group was at C-19 in 14 thanosides C (24) and A (25),13 rhodomicranoside F (26),14
instead of at C-3 as in 13. In the NOESY spectrum, the grayanotoxins I (27),21 III (28),24 VII (29),25 IX (30),26 and VIII
correlations of H3-20/H3-18, H3-20/H-14a, and H-9/H-15b (31),26 19-sophorosyl kaurenoate (32),27 kalmanol (33),28
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rhodomicranoside A (34),14 pieroside B (35),29 and pier-
isformoside F (36)16 were determined by comparison of their
experimental NMR data with those reported in the literature.
The acetic acid-induced writhing test is usually used as
a sensitive model for measuring acute pain.6–9 Compounds 1–
2, 6–11, 13–15, 17–19, 29–33, and 35 were evaluated in the test
for their antinociceptive activity based on the inhibition rates
of writhes. In this test, intraperitoneal (IP) administration
was used for all of the compounds. The obtained data are
summarized in Fig. 4. Compounds 17 and 19 showed higher
inhibitory activities than the other tested compounds,
inhibited 94.3% and 96.4% of the writhes, respectively, when
administered at a dose of 3 mg kgꢀ1. However, they showed
severe toxic reactions when administered at a dose of 10 mg
kgꢀ1. Compounds 2, 6 and 32 showed signicant anti-
nociceptive activity at a dose of 10 mg kgꢀ1. In contrast, in the
same assay, ibuprofen inhibited 58.6% of the writhes when
administered at a dose of 20 mg kgꢀ1 while morphine
inhibited 63.6% of the writhes when administered at a dose
Conclusion
A total of 36 diterpenoids, including een new diterpenoid
glycosides (1–15) and 21 known analogues were obtained from
the leaves and twigs of R. micranthum. In the acetic acid-induced
writhing test, compounds 17 and 19 showed signicant anti-
nociceptive activity at a dose of 3 mg kgꢀ1 and compounds 2, 6
and 32 showed signicant antinociceptive activity at a dose of
10 mg kgꢀ1. Grayananes and kalmanes such as 17, 19, 29–31, 33
are both antinociceptive and toxic components. In addition,
micranthanoside IX (9) and rhodomicranoside F (26) showed
moderate anti-inammatory activity by reducing LPS-induced
NO production in BV2 cells.
Experimental section
General experimental procedures
IR spectra were recorded using a Nicolet 5700 FT-IR spectrom-
eter. Optical rotations were acquired via a Rudolph automatic
polarimeter. HRESIMS analysis was carried out using an Agilent
6520 Accurate-Mass Q-TOF LC/MS spectrometer. NMR spectra
were obtained using INOVA-500, Bruker AV600-III and INOVA
SX-600 spectrometers. A Shimadzu LC-6AD instrument (SPD-
20A and RID-10A detectors) was used for preparative HPLC
separations. Liquid chromatography was conducted using
a YMC ODS column. A D101-type macroporous resin, Baoen
Corporation (Cangzhou, China); Sephadex LH-20, GE Chemical
Corporation (USA); silica gel and GF254 TLC plates, Jiangyou
Corporation (Yantai, China); and ODS (50 mm), Merck (Ger-
many) were used for column chromatography (CC). TLC anal-
yses were carried out on precoated silica gel GF254 plates, and
spots were visualized under UV light (254 and 365 nm) or by
heating aer spraying with a 5% CH3CH2OH–H2SO4 solution.
of 0.1 mg kgꢀ1
.
Compounds 17, 19, 29–31, (grayananes) and 33 (kalmane)
produced antinociceptive effects at lower doses while showed
toxic reactions at higher doses. Mice given higher doses
showed reactions of nausea and convulsion, which is
consistent with the clinical symptoms of “zhaoshanbai”
poisoning.5–7 The results in this test and those reported
previously revealed that among the antinociceptive compo-
nents, grayananes and kalmanes are responsible for both
antinociceptive and toxic effects. Their antinociceptive
activity was positively correlated with the toxicity.8,13,30 Some
kauranes and leucothanes also showed signicant anti-
nociceptive activity at relatively high doses, but no toxic
reaction was observed.10,14 This fact suggests that they may
have different mechanisms of action.
The preliminary analysis of the structures (present and re-
ported) and their bioactivities revealed that the presence of
sugar unit at C-3, C-6, or C-16 decreases the activity as well as
the toxicity (as in 11/17, 10/19, 15/rhododecorumin I, rhodo-
micranoside E/rhodomicranone E).10,14 Compound 2 is more
active than 1, which suggested that sugar unit at C-3 is likely to
hinder activity to a lesser degree. Traditionally, “zhaoshanbai”
was extracted by water decoction. Diterpenoid glycosides rather
than the aglycones were the main components of the extract
because of the polarity, which reduced the toxicity of the extract
Plant material
Twigs and leaves of R. micranthum were collected from Yiyuan,
Shandong Province in August 2014. The plant was authenti-
cated by Prof. Peng Wan (Shandong University of Traditional
Chinese Medicine).
deposited in the herbarium of Institute of Materia Medica,
Chinese Academy of Medical Sciences.
A voucher specimen (ID-s-2586) was
Extraction and isolation
to a certain extent.31,32 However, in order to ensure clinical Twigs and leaves of R. micranthum (107.5 kg) were air-dried and
safety, the content of diterpenoid aglycones in extracts still extracted twice (2 h each time) with EtOH under reux. The
needs to be monitored.
ethanol extracts were evaporated under reduced pressure, and
R. micranthum (zhaoshanbai) has also been widely used as an the residue was suspended in water and then partitioned
anti-inammatory drug to alleviate the symptoms of upper successively with petroleum ether, CH2Cl2, EtOAc, and n-BuOH.
respiratory inammation. Therefore, the anti-inammatory The EtOAc extract was separated using a macroporous resin
activities of the compounds 1–3, 7–11, 13–32, and 34–36 were column eluted sequentially with 70 : 30, 40 : 60, and 5 : 95 (v/v)
evaluated by measuring inhibitory effects of LPS-induced NO H2O–EtOH solutions. Then, silica gel CC was used to separate
production in BV2 cells. At 10 mM, compounds 9 and 26 dis- the 60% EtOH fraction (288.4 g). The column was eluted with
played moderate activity with inhibition rates of 56.31% and a step gradient of CH2Cl2/MeOH (20 : 1, 10 : 1, 5 : 1, and 1 : 1, v/
72.43%, respectively (Fig. 5). At the same concentration, the v). Fractions E60G1–E60G9 were collected based on the TLC
inhibition rate of the positive control (curcumin) is 81.38%.
results. Fraction E60G2 was puried via semipreparative HPLC
(MeCN–H2O, 37 : 63, v/v, 3.5 ml minꢀ1) to afford 30 (5.5 mg, tR ¼
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37.5 min). Fraction E60G3 was separated via a Sephadex LH-20 Fractions G1–G9 were collected based on the results of TLC
column eluted with MeOH–H2O (60 : 40, v/v) and yielded analysis. Fraction E30G5 was further puried via a Sephadex LH-
three fractions (E60G3L1–E60G3L3). E60G3L2 was puried via 20 column to yield 2 fractions, E30G5L1 and E30G5L2. Fraction
semipreparative HPLC (MeCN–H2O, 25 : 75, v/v, 3.5 ml minꢀ1
)
E30G5L1 (37.7 g) was separated via an MCI column and eluted
to afford 17 (72.4 mg, tR ¼ 35.4 min), 29 (20.8 mg, tR ¼ 38.6 with a step gradient of MeOH/H2O (10 : 90, 30 : 70, 40 : 60,
min), 31 (5.5 mg, tR ¼ 40.9 min), 28 (2.1 mg, tR ¼ 15.7 min), 18 50 : 50, 60 : 40, 70 : 30, and 100 : 0, v/v) to yield 5 fractions,
(9.9 mg, tR ¼ 45.6 min), 27 (52.3 mg, tR ¼ 50.3 min). Fraction
E30G5L1M1 to E30G5L1M5. Fraction E30G5L1M3 was separated via
60G6 was further separated using a Sephadex LH-20 column ODS column and eluted with a step gradient of MeOH/H2O
E
eluted with MeOH–H2O (60 : 40, v/v) and yielded 2 fractions (40 : 60, 50 : 50, 70 : 30, 80 : 20 and 100 : 0, v/v) to yield 5 frac-
(E60G6L1–E60G6L2). Fraction E60G6L1 (20.0 g) was separated tions, E30G5L1M3O1 to E30G5L1M3O5. Fraction E30G5L1M3O4 was
using an MCI column and eluted with a step gradient of MeOH/ puried via preparative HPLC to afford eight fractions,
H2O (40 : 60, 50 : 50, 60 : 40, 70 : 30, 80 : 20, and 100 : 0, v/v) to
E30G5L1M3O4-1 to E30G5L1M3O4-5. O4-1 was puried via HPLC
yield 6 fractions, E60G6L1M1 to E60G6L1M6. Fraction E60G6L1M2 (MeCN–H2O, 20 : 80, v/v) to afford 35 (2.1 mg, tR ¼ 48.7 min).
was then separated via preparative HPLC (MeOH–H2O, 50 : 50, O4-2 was puried via HPLC (MeCN–H2O, 25 : 75, v/v) to afford 1
v/v, 5 ml minꢀ1) and yielded nine fractions, E60G6L1M2-1 to (8.4 mg, tR ¼ 25.2 min) and 2 (4.9 mg, tR ¼ 32.9 min). Then,
E
60G6L1M2-9. Fraction M2-5 was puried via semipreparative
E30G5L1M4 was puried via preparative HPLC (MeCN–H2O,
HPLC (MeCN–H2O, 17 : 83, v/v, 3.5 ml minꢀ1) to afford 24 20 : 80, v/v) to afford eight fractions, E30G5L1M4-1 to E30G5L1M4-
(17.5 mg, tR ¼ 22.2 min), 25 (23.2 mg, tR ¼ 24.7 min), and 26 8. M4-3 afforded 20 (13.5 mg, tR ¼ 15.5 min). M4-4 was puried
(8.7 mg, tR ¼ 28.7 min). Fraction M2-6 was puried via semi- via HPLC (MeCN–H2O, 25 : 75, v/v) to afford 15 (7.7 mg, tR
¼
preparative HPLC (MeCN–H2O, 18 : 82, v/v, 3.5 ml minꢀ1) to 23.0 min). M4-5 was puried via HPLC (MeCN–H2O, 25 : 75, v/v)
afford 16 (6.0 mg, tR ¼ 22.7 min). Fraction M2-7 was puried via to afford 22 (8.9 mg, tR ¼ 16.6 min). M4-8 was puried via HPLC
semipreparative HPLC (MeCN–H2O, 16 : 84, v/v, 3.5 ml minꢀ1
)
(MeCN–H2O, 30 : 70, v/v) to afford 36 (49.7 mg, tR ¼ 34.6 min).
to afford 34 (12.0 mg, tR ¼ 36.9 min). Fraction M2-8 was puried Fraction E30G5L1M5 was puried via HPLC (MeCN–H2O, 13 : 87,
via semipreparative HPLC (MeCN–H2O, 25 : 75, v/v, 3.5 v/v) to afford 33 (6.1 mg, tR ¼ 32.1 min).
ml minꢀ1) to afford 9 (23.1 mg, tR ¼ 15.1 min). Fraction M2-9
Micranthanoside I (1). White powder; [a]2D0 ꢀ49.1 (c 0.53,
was puried via semipreparative HPLC (MeCN–H2O, 20 : 80, v/v, MeOH); IR vmax 3368, 2928, 1700, 1460, 1357, 1257, 1159, 1075,
3.5 ml minꢀ1) to afford 5 (1.8 mg, tR ¼ 20.4 min). Fraction 1041, 883, 857, 812, 634 cmꢀ1; 1H and 13C NMR data, see Tables
E
60G6L1M3 was then separated via preparative HPLC (MeOH– 1 and 4; HRESIMS m/z 505.2786 [M + Na]+ (calcd for
H2O, 60 : 40, v/v, 5 ml minꢀ1) and yielded eleven fractions,
60G6L1M3-1 to E60G6L1M3-11. Fraction M3-5 was puried via
semipreparative HPLC (MeCN–H2O, 25 : 75, v/v, 3.5 ml minꢀ1
C
26H42NaO8, 505.2772).
E
Micranthanoside II (2). White powder; [a]2D0 ꢀ83.3 (c 0.48,
)
MeOH); IR vmax 3350, 2928, 1701, 1459, 1357, 1256, 1230, 1201,
to afford 19 (7.0 mg, tR ¼ 25.1 min) and 13 (122.4 mg, tR ¼ 28.7 1163, 1074, 911, 888, 811, 635 cmꢀ1; 1H and 13C NMR data, see
min). Fraction M3-6 was puried via semipreparative HPLC Tables 1 and 4; HRESIMS m/z 505.2781 [M + Na]+ (calcd for
(MeCN–H2O, 24 : 76, v/v, 3.5 ml minꢀ1) to afford 10 (27.3 mg, tR
¼ 29.4 min), 6 (40.1 mg, tR ¼ 29.4 min), and 7 (45.2 mg, tR ¼ 29.4
C
26H42NaO8, 505.2772).
Micranthanoside III (3). White powder; [a]2D0 ꢀ58.1 (c 0.02,
min). Fraction M3-8 was puried via semipreparative HPLC MeOH); IR vmax 3367, 2962, 1627, 1549, 1449, 1378, 1077, 947,
(MeCN–H2O, 27 : 73, v/v, 3.5 ml minꢀ1) to afford 3 (2.2 mg, tR ¼ 903, 830 cmꢀ1
;
1H and 13C NMR data, see Tables 1 and 4;
29.4 min). Fraction M3-9 was puried via semipreparative HPLC HRESIMS m/z 503.2623 [M + Na]+ (calcd for C26H40NaO8,
(MeCN–H2O, 30 : 70, v/v, 3.5 ml minꢀ1) to afford 4 (1.0 mg, tR ¼ 503.2615).
25.4 min). Fraction M3-10 was puried via semipreparative
Micranthanoside IV (4). White powder; [a]2D0 ꢀ50.0 (c 0.01,
HPLC (MeCN–H2O, 30 : 70, v/v, 3.5 ml minꢀ1) to afford 8 MeOH); IR vmax 3382, 2930, 2870, 1630, 1446, 1363, 1314, 1256,
(5.7 mg, tR ¼ 29.1 min) and 14 (12.6 mg, tR ¼ 40.8 min). Fraction 1165, 1078, 1034, 929, 891, 759 cmꢀ1; 1H and 13C NMR data, see
M3-11 afforded 12 (19.5 mg, tR ¼ 58.0 min) without purication. Tables 1 and 4; HRESIMS m/z 485.2492 [M + Na]+ (calcd for
Fraction E60G8 was further separated via a Sephadex LH-20
column eluted with MeOH–H2O (60 : 40, v/v) and yielded four
C
26H38NaO7, 485.251).
Micranthanoside V (5). White powder; [a]2D0 +9.0 (c 0.04,
fractions (E60G8L1–E60G8L4). Fraction E60G8L3 afforded 32 (30.3 MeOH); IR vmax 3389, 2924, 1647, 1598, 1419, 1076, 911, 852,
mg). Fraction E60G8L2 was separated via preparative HPLC 805, 630 cmꢀ1 1H and 13C NMR data, see Tables 1 and 4;
(MeOH–H2O, 50 : 50, v/v, 5 ml minꢀ1) and yielded ve fractions, HRESIMS m/z 521.2722 [M + Na]+ (calcd for C26H42NaO9,
;
E
60G8L2-1 to E60G8L2-5. Fraction E60G8L2-1 afforded 23 (106.6 521.2721).
mg). Fraction E60G8L2-3 was puried via semipreparative HPLC
Micranthanoside VI (6). White powder; [a]2D0 ꢀ58.7 (c 0.06,
(MeCN–H2O, 20 : 80, v/v, 3.5 ml minꢀ1) to afford 21 (9.6 mg, tR ¼ MeOH); IR vmax 3380, 2937, 2869, 1633, 1548, 1447, 1046, 1379,
41.2 min). Fraction E60G8L2-4 was puried via semipreparative 1307, 1266, 1156, 1073, 1034, 915, 890, 828 cmꢀ1; H and 13C
1
HPLC (MeCN–H2O, 20 : 80, v/v, 3.5 ml minꢀ1) to afford 11 NMR data, see Tables 1 and 4; HRESIMS m/z 487.2676 [M + Na]+
(9.8 mg, tR ¼ 35.2 min).
(calcd for C26H40NaO7, 487.2666).
The 30% EtOH fraction of the macroporous resin column
Micranthanoside VII (7). White powder; [a]2D0 ꢀ34.2 (c 0.06,
was also loaded on a silica gel column and eluted with a step MeOH); IR vmax 3379, 2937, 1728, 1677, 1549, 1375, 1306, 1243,
gradient of CH2Cl2/MeOH (20 : 1, 10 : 1, 5 : 1, and 1 : 1, v/v). 1201, 1080, 1048, 885, 643 cmꢀ1 1H and 13C NMR data, see
;
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Tables 1 and 4; HRESIMS m/z 487.2671 [M + Na]+ (calcd for injection temperature, 280 ꢁC; column, HP-5 (60 m ꢃ 0.32 mm
C
26H40NaO7, 487.2666).
ꢃ 0.25 mm); initial column temperature, 200 ꢁC; column
Micranthanoside VIII (8). White powder; [a]D20 ꢀ17.6 (c 0.06, temperature increased to 280 ꢁC (10 ꢁC minꢀ1) and kept at
MeOH); IR vmax 3375, 2938, 2873, 1641, 1549, 1448, 1379, 1306, 280 C for 40 min under N2 carrier gas (1.8 ml minꢀ1).
ꢁ
1158, 1079, 1041, 940, 898, 858, 830, 769, 624, 536 cmꢀ1; 1H and
13C NMR data, see Tables 2 and 4; HRESIMS m/z 505.2788 [M +
Animals
Na]+ (calcd for C26H42NaO8, 505.2772).
Male Kunming mice (16–20 g) were housed for three days prior
Micranthanoside IX (9). White powder; [a]2D0 ꢀ48.6 (c 0.06,
to use. All animal care and experimental procedures were in
MeOH); IR vmax 3407, 2937, 1628, 1448, 1382, 1264, 1158, 1077,
accordance with the guidelines of the National Institutes of
1041, 950, 898, 858, 639 cmꢀ1; 1H and 13C NMR data, see Tables
Health (NIH), and the experimental procedures were approved
2 and 4; HRESIMS m/z 505.2775 [M + Na]+ (calcd for
by the Ethics Committee of Institute of Materia Medica, Chinese
C
26H42NaO8, 505.2772).
Academy of Medical Sciences and Peking Union Medical
College (Beijing, China).
Micranthanoside X (10). White powder; [a]2D0 ꢀ5.0 (c 0.03,
MeOH); IR vmax 3382, 2933, 1637, 1450, 1384, 1222, 1080, 1035,
916, 890, 859, 827, 647 cmꢀ1; 1H and 13C NMR data, see Tables 2
and 4; HRESIMS m/z 505.2766 [M + Na]+ (calcd for C26H42NaO8,
505.2772).
Acetic acid-induced writhing tests
Kunming mice (eight per group) were used in the tests. Control
mice received 0.9% NaCl (10 ml kgꢀ1, ip), and the test mice
received aqueous solutions (10, 3, 1, 0.3, or 0.1 mg kgꢀ1, ip;
injection volume: 10 ml kgꢀ1) with a solution concentration of
1 mg mlꢀ1, 0.3 mg mlꢀ1, 0.1 mg mlꢀ1, 0.03 mg mlꢀ1, or 0.01 mg
mlꢀ1. Compounds 17–19, 29–31, and 33 utilized 1% Tween 80 as
hydrotropic agent. Fieen minutes later, 1% v/v HOAc solution
(0.1 ml/10 g) was administered to the mice by intraperitoneal
injection. The number of writhing events for each mouse was
counted for 15 min.
Micranthanoside XI (11). White powder; [a]2D0 ꢀ27.8 (c 0.2,
MeOH); IR vmax 3368, 2927, 1701, 1460, 1357, 1163, 1074, 1031,
911, 888, 811, 635 cmꢀ1; 1H and 13C NMR data, see Tables 2 and
4 HRESIMS m/z 503.2612 [M + Na]+ (calcd for C26H40NaO8,
503.2615).
Micranthanoside XII (12). White powder; [a]2D0 +66.0 (c 0.05,
MeOH); IR vmax 3393, 2926, 2850, 1645, 1596, 1468, 1453, 1417,
1384, 1261, 1157, 974, 633, 546 cmꢀ1; 1H and 13C NMR data, see
Tables 3 and 4; HRESIMS m/z 521.2708 [M + Na]+ (calcd for
C
26H42NaO9, 521.2721).
Micranthanoside XIII (13). White powder; [a]2D0 ꢀ92.0 (c 0.25,
Anti-inammatory activity assays
MeOH); IR vmax 3557, 3526, 3481, 3330, 2942, 2885, 1716, 1659,
1461, 1434, 1386, 1102, 1076, 1042, 988, 943, 640, 601 cmꢀ1; 1H
and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 521.2719
[M + Na]+ (calcd for C26H42NaO9, 521.2721).
Compounds were tested for their anti-inammatory activity by
measuring inhibitory effects of LPS-induced NO production in
BV2 cells. Curcumin was used as the positive control. The BV2
macrophage cell line was obtained from the Cell Culture Center
at the Institute of Basic Medical Sciences, Peking Union Medical
College. LPS was bought from Sigma-Aldrich company. Aer
preincubation for 24 h in a 96-well plate, the cells were treated
with the test compounds (10ꢀ5 mol Lꢀ1), then stimulated with
LPS for 24 h. The production of NO was determined via
measuring the concentration of nitrite in the culture superna-
tant. NaNO2 was utilized to generate a standard curve. The
absorbances at 550 nm were measured.
Micranthanoside XIV (14). White powder; [a]2D0 ꢀ44.4 (c 0.08,
MeOH); IR vmax 3511, 3321, 2947, 2874, 1698, 1623, 1462, 1442,
1387, 1269, 1104, 1070, 1020, 997, 959, 855, 645, 574, 552 cmꢀ1
;
1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z
521.2711 [M + Na]+ (calcd for C26H42NaO9, 521.2721).
Micranthanoside XV (15). White powder; [a]2D0 ꢀ69.8 (c 0.07,
MeOH); IR vmax 3396, 2935, 2872, 1701, 1643, 1447, 1381, 1299,
1202, 1159, 1077, 1030, 991, 928, 886, 844, 595, 579 cmꢀ1; H
1
and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 503.2615
[M + Na]+ (calcd for C26H0NaO8, 503.2615).
Conflicts of interest
Acid hydrolysis and GC analysis
The authors declare no competing nancial interest.
The conguration of the sugar moiety was established accord-
ing to a published method.33 The compounds were dissolved in
MeOH (2 ml) and then added to 2 N HCl (2 ml). The solution
Acknowledgements
ꢁ
This work was supported by grants from the National Natural
Science Foundation of China (No. 21572274, 21732008, and
81630094) and the CAMS Innovation Fund for Medical Sciences
(No. 2016-I2M-3-012).
was heated at 90 C for 12 h. The reaction mixture was evapo-
rated and partitioned with EtOAc and H2O. The aqueous layer
was concentrated to dryness, dissolved in dry pyridine and
reaꢁcted with L-cysteine methyl ester hydrochloride (2 mg) at
80 C for 1 h. Aer removal of the solvent, N-trimethylsilylimi-
dazole (1 ml) was added, and the mixture was heated at 80 ꢁC for
0.5 h. The residue partitioned into n-hexane and H2O, and the n-
hexane part was analyzed on a GC system equipped with an FID
(detector temperature, 300 ꢁC). Chromatography conditions:
Notes and references
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