Anti-inflammatory 12,20-Epoxypregnane and 11,12-seco-Pregnane Glycosides from the Stems of Hoya kerrii

Article abstract of DOI:10.1021/acs.jnatprod.6b00730

Five 12,20-epoxypregnane glycosides (1-3, 5, and 6) and two 11,12-seco-pregnane glycosides (4 and 7) with spirodilactone motifs, as well as spirodilactone cleavage products 8 and 9, were isolated from the stems of Hoya kerrii. The relative configurations of the three related skeletons were supported by ROESY experiments and X-ray crystallographic analyses. The isolates were evaluated for their anti-inflammatory activity based on the inhibition of NO production in RAW264.7 cells, and some showed IC₅₀ values ranging from 12.6 to 96.5 μM. The most potent compound, 9a, was also examined for its anti-inflammatory mechanism against mRNA expression and was found to down-regulate mRNA expression of iNOS and COX-2 in a dose-dependent manner.

Full text of DOI:10.1021/acs.jnatprod.6b00730

Article
pubs.acs.org/jnp
Anti-inflammatory 12,20-Epoxypregnane and 11,12-seco-Pregnane
Glycosides from the Stems of Hoya kerrii
Chonticha Seeka,† Samran Prabpai,‡ Palangpon Kongsaeree,‡ Supinya Tewtrakul,§
Thitima Lhinhatrakool,⊥ and Somyote Sutthivaiyakit*,†
†Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Hua Mark,
Bangkapi, Bangkok 10240, Thailand
‡Department of Chemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University,
Bangkok 10400, Thailand
§Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkhla University, Hat
Yai, Songkhla 90112, Thailand
⊥College of Oriental Medicine, Rangsit University, Muang Ake, Pathumthani 12000, Thailand
S
* Supporting Information
ABSTRACT: Five 12,20-epoxypregnane glycosides (1−3, 5,
and 6) and two 11,12-seco-pregnane glycosides (4 and 7) with
spirodilactone motifs, as well as spirodilactone cleavage
products 8 and 9, were isolated from the stems of Hoya
kerrii. The relative configurations of the three related skeletons
were supported by ROESY experiments and X-ray crystallo-
graphic analyses. The isolates were evaluated for their anti-
inflammatory activity based on the inhibition of NO
production in RAW264.7 cells, and some showed IC50 values
ranging from 12.6 to 96.5 μM. The most potent compound,
9a, was also examined for its anti-inflammatory mechanism
against mRNA expression and was found to down-regulate
mRNA expression of iNOS and COX-2 in a dose-dependent
manner.
lupeol acetate,9 taraxerol,10 lupeol,10 a mixture of sitosterol and
stigmasterol, p-hydroxybenzaldehyde, scopoletin,11 and laricir-
esinol.12 We report herein the spectroscopic identification of
compounds 1−9 and their anti-inflammatory activity.
Hoya kerrii Craib.1 [syn. H. obovata Decne. var. kerrii (Craib.)
Costantin] of the Apocynaceae family is commonly known in
Thailand as “Dang and Tang”. This plant is among 53 Hoya
species found in Thailand.1 The latex, stems, and leaves of this
plant have been reported to possess wound-healing and anti-
inflammatory properties. A water decoction of the leaves has
been used as an antidiabetic treatment.2 There have been
reports dealing with the chemical constituents of several Hoya
plants, including Hoya australis,3 H. carnosa,4,5 H. lacunose,6
H. naumanii,7 and H. parasitica,8 that led to the isolation of
some 3,4-seco-pentacyclic triterpenoids,3,6−8 pregnanes,4 preg-
nane glycosides,4 androstanes,8 sesquiterpenoids,8 phenolic
compounds,8 and oligosaccharides.5 However, there have
been no reports on any phytochemical investigation of H. kerrii.
The preliminary anti-inflammatory activity assessment of the
inhibition of nitric oxide production in RAW264.7 cells
indicated that the CH2Cl2 extract of the stem was active,
showing an IC50 value of 57.2 μg/mL. An additional in-house
brine shrimp lethality assay indicated that the CH2Cl2 extract of
the stems at a concentration of 250 ppm caused 100% lethality
of Artemia salina nauplii. The use of a combination of
chromatographic techniques led to the isolation of several
new steroids (1−9) in addition to the seven known compounds
RESULTS AND DISCUSSION
■
Compound 1 was obtained as a colorless solid that showed an
HRESIMS sodium adduct molecular ion at m/z 513.2828,
corresponding to a molecular formula of C28H42O7. The FTIR
indicated peaks for the presence of a carbonyl and an olefin
function at νmax 1711 and 1661 cm−1, respectively. The 13C
NMR spectrum revealed the presence of 28 carbon signals
comprising five methyl, seven methylene, 11 methine
(including seven oxymethine and one olefinic), and five
nonprotonated carbons. Among the nonprotonated carbons
was a carbonyl, an oxygenated tertiary carbon, and an olefinic
carbon. The 1H NMR resonance at δH 4.49 (dd, J = 9.8 and 1.9
Hz) showed an HMQC cross-peak with a dioxygenated
methine carbon at δC 98.1, assignable to an anomeric H-1′,
1
together with H−1H COSY cross-peaks between H-2′/H-1′
Received: September 4, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structures of the isolated compounds and their derivatives.
and H-3′, H-4′/H-3′ and H-5′, and H-5′/H3-6′. These
correlations permitted the establishment of the presence of a
2,6-dideoxypyranose unit in 1 (Table 1). The coupling
constants of the oxymethine protons and the HMBC cross-
peak of the OMe singlet at δH 3.37 with C-3′ (δC 78.0)
indicated the presence of a β-diginopyranosyl ring.13 The
remaining 21 carbon resonances of the aglycone with two
methyl groups each attached to tertiary carbons and thus
resonating in the 1H NMR spectrum as two singlets at δH 1.35
and 1.01 showed that the aglycone contained a pregnane
D-diginose, [α]26 +68 (c 0.1, H2O), lit.13,14a +59.6 (c 1.48,
D
H2O). The structure of compound 1 was thus elucidated as
12α,20α-epoxy-14β-hydroxypregn-5-en-11-one 3-O-β-D-digino-
pyranoside. Related rare steroid glycosides having ether
linkages between C-12 and C-20 but with an additional C-15
carbonyl group and no 14-hydroxy function, e.g., 12α,20α-
epoxypregn-5-en-11,15-dione 3-O-β-D-diginopyranoside (digi-
nin), have been isolated from Digitalis ciliate and D. purpurea.14
Compound 2 was obtained as a colorless solid with a
molecular formula of C28H42O8 {HRESIMS [M + Na]+ m/z
529.2759 (calcd for C28H42O8Na, 529.2771)}, thus implying
1
moiety. The olefinic function, as observed by a H NMR
resonance at δH 5.45 d (J = 5.4 Hz) and 13C NMR resonances
at δC 140.9 (C) and 121.7 (CH), was assigned at C-5(6), as
evident from the HMBC cross-peaks between H3-19 (δH 1.01)/
C-1 (δC 37.4), C-5, and C-10 (δC 39.4) as well as H-3/C-1 and
C-5 (Figure S45, Supporting Information). The methyl singlet
at δH 1.35 assigned to H3-18 showed HMBC cross-peaks with
carbon resonances at δC 82.2 (C-14), 91.5 (C-12), and 58.4 (C-
17). The presence of a 1H NMR singlet at δH 3.88, showing an
HMQC cross-peak with C-12, HMBC cross-peaks not only
with C-14, C-17, and C-18 (δC 22.7) but also with the carbonyl
and the oxymethine carbons at δC 211.0 and 82.3 (assigned to
C-20), respectively, required the presence of a carbonyl group
at C-11 and an ether linkage between C-12 and C-20. On the
basis of these data, the aglycone in 1 was defined as 3,14-
dihydroxy-12,20-epoxypregn-5-en-11-one. The connectivity
between the oxygen atom at C-3 and C-1′ of the
diginopyranose unit was evident from the HMBC cross-peaks
between H-3/C-1′ and H-1′/C-3. The O-acetyl derivative 1a
was obtained after reacting 1 with Ac2O−pyridine, and its
ROESY spectrum showed cross-peaks between H3-18/H-8, H-
12, and H-17, while H3-19 showed cross-peaks with H-8 and H-
12. The anomeric H-1′ showed NOE correlations with H-3, H-
3′, and H-5′ (Figure 2a). A single-crystal X-ray crystallographic
analysis of 1 provided the ORTEP plot shown in Figure 2b.
The absolute configuration of the sugar was assigned after acid
hydrolysis of 1 with 0.05 M HCl at 60 °C for 4 h to give the
aglycone 12α,20α-epoxy-14β-hydroxy-pregn-5-en-11-one and
1
that 2 has one more oxygen atom than 1. The H and 13C
NMR resonances of 2 showed close resemblance to those of 1
(Table 1), indicating the presence of diginopyranosyl and
12α,20α-epoxy-14β-hydroxypregn-5-en-11-one moieties, except
that H-12 resonated at δH 4.18, i.e., deshielded relative to 1.
1
Since the H NMR resonance for H-9 (δH 2.20) was a singlet,
instead of a doublet as in 1, and both H-9 and H-6 (δH 5.38)
showed HMBC cross-peaks with the same oxygenated tertiary
carbon at δC 76.7, the presence of the hydroxy group at C-8 was
indicated. On the basis of the acid hydrolysis of 1, which
provided D-diginose, the structure of compound 2 was therefore
elucidated as 12α,20α-epoxy-8β,14β-dihydroxypregn-5-en-11-
one 3-O-β-D-diginopyranoside. The 4′-O-acetyl derivative 2a
was obtained after reacting 2 with Ac2O in pyridine (Table S1,
Supporting Information).
Compound 3, a colorless solid, had the same molecular mass
1
as 2 based on HRESIMS. The H and 13C NMR spectra also
exhibited similar patterns of resonances to those observed in 2,
1
except that the H NMR singlet at ca. δH 4.18 and the 13C
NMR resonance at δC 90.6, assigned to H-12 and C-12 in 2,
were absent (Table 1). The important HMBC cross-peaks of
H-9 (δH 2.28) and H-17 (δH 2.07) with a dioxygenated carbon
at δC 102.7 led to the placement of a hydroxy group at C-12.
Acid hydrolysis of 1 afforded D-diginose; thus the structure of
compound 3 was assigned as 12α,20α-epoxy-12β,14β-dihy-
droxypregn-5-en-11-one 3-O-β-D-diginopyranoside.
B
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diginopyranosyl moiety [δH 4.48 d (J = 9.7 Hz, H-1′), 1.31 (d, J
= 6.3 Hz, H3-6′)] were also apparent (Table 1). The other
methyl doublet at δH 1.40 (J = 6.2 Hz), assigned to H3-21,
showed a 1H−1H COSY cross-peak with a deshielded quintet at
δH 4.25 (H-20). This deshielded signal compared with those of
C-20 in 1−3 suggested the attachment of an O-acyl group to C-
20. The HMBC cross-peaks between H3-18/C-17, the less-
shielded C-14 (δC 93.9), and a carbonyl carbon (δC 179.1,
assigned to C-12), as well as an H-20/C-12 cross-peak (Figure
S45, Supporting Information), required a lactone linkage
between C-20 and C-13. The other important HMBC cross-
peaks of both H-8 (δH 2.55) and H-9 (δH 2.54) with C-14 and
a carbonyl carbon at δC 175.1 required another lactone moiety
between C-14 and C-9. Compound 4 could therefore be
identified as an 11,12-seco-pregnane derivative possessing a
spirodilactone motif (Scheme 1, postulated transformation).
Acetylation of 4 using Ac2O−pyridine gave 4a. The ROESY
spectrum of 4a showed cross-peaks between H-8/H3-19, H-17/
H3-18 and H3-21, and H3-18/H3-21, as well as cross-peaks
between H-1′/H-3, H-3′, and H-5′. The X-ray crystallographic
structure of 4a, as illustrated in Figure 3a and b, defined the
relative configurations of the stereogenic centers in 4. On the
basis of the acid hydrolysis of 1, which gave D-diginose, the
structure of compound 4 was proposed as kerriipregnane A 3-
O-β-D-diginopyranoside.
Figure 2. (a) Key NOE correlations of 1a and (b) an ORTEP plot of
1.
Compound 4 was isolated as a colorless solid with the
molecular formula of C28H40O8 based on its HRESIMS
spectrum, showing an [M + Na]+ ion at m/z 527.2625 (calcd
for C28H40O8Na, 527.2615). The 13C NMR spectrum showed
28 carbons comprising five methyl, seven methylene, 10
methine, and six nonprotonated carbons including two
deshielded carbonyl carbons (δC 175.1 and 179.1), one olefinic
carbon (δC 141.0), one oxygenated tertiary carbon (δC 93.9),
Compound 5 was obtained as a colorless solid. The
HRESIMS revealed a molecular formula of C34H52O12. The
1H and 13C NMR spectra established resonances of the
diginopyranosyl and the 12α,20α-epoxy-14β-hydroxypregn-5-
1
and two quaternary carbons. The H NMR resonances of a
Scheme 1. Postulated Biosynthesis Transformations of 1−3, 4, and 8/9
D
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methyl doublets [δH 1.18 (d, J = 7.6 Hz), 1.42 (d, J = 6.1 Hz,
H3-21), and 1.34 (d, J = 6.5 Hz, H3-6′)], instead of two as
encountered in 4, together with two methoxy singlets at δH 3.65
1
and 3.39 (OCH3-3′) (Table 3). The H,1H−COSY spectrum
exhibited connectivities between the less-shielded H-20/H3-21
and H-17 (δH 1.65), H-17/H-13 (δH 2.28) and H-16, and H-
13/H3-18 (δH 1.28). The key HMBC cross-peaks between H-
17/C-12 (δC 178.4), C-13, C-18, C-20, and C-21 revealed a
lactone linkage between C-20 and C-13 (Supporting
Information). The placements of a carbonyl group at C-14
and an ester carbonyl at C-11 were based on the HMBC cross-
peaks between H-8/C-7, C-9, C-10, and C-14 and H-9/C-7, C-
10, C-11 (δC 174.3), and C-14, respectively. The methoxy
singlet at δH 3.65 showing an HMBC cross-peak with C-11 was
therefore assigned to OCH3-11. Owing to the overlap of
1
relevant H NMR resonances, important NOE correlations
could not be unambiguously specified from the ROESY
spectrum of either 8 or its acetate 8a. However, the relative
configurations of the stereogenic centers in 8 were obtained
from the X-ray structure of 8a, as shown in Figure 4. On the
basis of the acid hydrolysis of 1, affording D-diginose, the
structure of compound 8 was defined as kerriipregnane B 3-O-
β-D-diginopyranoside.
Figure 3. (a) Key NOE correlations and (b) an ORTEP plot of 4a.
en-11-one skeletons as found in 1 (Table 2). The presence of a
1
glucopyranosyl ring was indicated by the H and 13C NMR
resonances at δH 5.18 d (J = 7.6 Hz, H-1″), δC 104.6 (CH, C-
1″), δH 4.57 dd (J = 11.4 and 1.3 Hz, H-6″a), 4.33 dd (J = 11.4
and 5.9 Hz, H-6″b), and δC 63.3 (CH2, C-6″). The HMBC
cross-peaks between H-4′/C-1″ and H-1″/C-4′ indicated the
attachment of C-1″ of the glucopyranosyl ring to the oxygen
atom at C-4′. Upon acid hydrolysis of 5 with 0.05 M HCl at 60
°C for 6 h and subsequent chromatographic purification, the
same aglycone as that of 1, together with D-diginose, β-D-
glucopyranosyl-(1→4)-D-diginopyranose, and D-glucose, was
obtained. Compound 5 was therefore identified as 12α,20α-
epoxy-14β-hydroxypregn-5-en-11-one 3-O-β-D-glucopyranosyl-
(1→4)-O-β-D-diginopyranoside.
Compound 9 was obtained as a sticky liquid, separable from
8 by HPLC purification and having the same molecular formula
as that of 8 (HRESIMS [M + Na]+ m/z 559.2881, calcd for
1
C29H44O9Na, 559.2871). The H and 13C NMR resonances
were similar to those of 8 except that the H-20 resonance was
deshielded [δH 4.27 quint (J = 6.3 Hz)] and the H3-18
resonance was shielded [δH 1.18 d (J = 7.6 Hz)] (Table 3). On
the basis of the postulated transformation route (Scheme 1)
and the ROESY spectra of 9 and its O-acetyl derivative 9a,
which exhibited NOE cross-peaks between H-8/H3-19, H-17/
H-13 and H3-21, H-13/H3-21, and H-1′/H-3, H-3′, and H-5′
(Figure 5), compound 9 was thus concluded to be the C-13
epimer of 8 and named 13-epi-kerriipregnane B 3-O-β-D-
diginopyranoside.
We propose that compounds 1−3 are inter-related by an
oxygenation reaction (Scheme 1). The formation of 4 was
proposed from 3 by a nucleophilic attack of the OH-14 group
at the C-11 carbonyl function, leading to intermediate A. After
oxidative rearrangement, resulting in cleavage of the bond
between C-11 and C-12, and some biological oxidant
(presumably NADP+) accepting a hydride ion, a 11,12-seco-
pregnane derivative with a spirodilactone motif, 4, was
established. Hydrolysis of 4 followed by a retro-aldol reaction,
which could be by either a one- or two-step reaction, could lead
to the formation of either 8A or 9A depending on which face
the protonation occurred. Subsequent methylation, most
probably with S-adenosylmethionine, provided 8 and 9. It is,
however, noteworthy that compound 4 remained intact after
stirring 4 (3 mg) with silica gel (50 mg) in MeOH (5 mL) at
room temperature for 5 days.
Compound 6 was isolated as a colorless solid with the
1
molecular formula of C34H52O13 based on HRESIMS. The H
and 13C NMR spectra (Table 2) showed sets of resonances of
the diginopyranosyl and the 12α,20α-epoxy-8β,14β-dihydrox-
1
ypregn-5-en-11-one units as observed in 2, but with extra H
and 13C NMR resonances of a glucopyranosyl unit. The HMBC
cross-peak between H-1″/C-4′ confirmed the connectivity of
C-4′-O to C-1″. On the basis of the acid hydrolysis of 5, which
provided D-diginose and D-glucose, the structure of compound
6 was thus assigned as 12α,20α-epoxy-8β,14β-dihydroxypregn-
5-en-11-one 3-O-β-D-glucopyranosyl-(1→4)-O-β-D-diginopyra-
noside.
Compound 7 was isolated as a colorless solid with the
molecular formula C34H50O13 based on HRESIMS. The 1H and
13C NMR spectra showed similar sets of resonances to those
found in 4 (Table 2) but with the presence of additional signals
of a glucopyranosyl moiety. The HMBC cross-peak between H-
1″ and C-4′ indicated connectivity between C-1″ and the
oxygen atom at C-4′ and led to the structural assignment of
compound 7 as kerriipregnane A 3-O-β-D-glucopyranosyl-(1→
4)-O-β-D-diginopyranoside.
Compound 8 was obtained as a colorless sticky liquid with a
molecular formula of C29H44O9 based on HRESIMS. The 13C
NMR spectrum showed 29 carbon resonances comprising six
methyl, seven methylene, 11 methine, and five nonprotonated
carbons, among which there were three carbonyl (δC 212.4,
178.4, and 174.3), one olefinic (δC 140.3), and one quaternary
carbon. The 1H NMR spectrum showed resonances for a
diginopyranosyl moiety, H-6 (δH 5.40) and H-20 (δH 4.09, dq, J
= 9.1 and 6.1 Hz), similar to those found in 4 but with three
Table 4 shows the anti-inflammatory activities of selected
compounds based on their inhibition of nitric oxide (NO)
production in RAW264.7 cells.16 Among the test compounds,
compound 9a showed the most potent inhibitory activity, with
an IC50 value of 12.6 μM, which is comparable to that of the
positive control, caffeic acid phenethyl ester, CAPE (IC50 = 9.3
μM), while compound 8a exhibited an IC50 value of 31.6 μM,
which is more potent than aspirin, ibuprofen, indomethacin,
and L-nitroarginine (L-NA). Compound 9a was also examined
for its anti-inflammatory mechanism against mRNA expression.
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sugar units were proven by acid hydrolysis of some selected
glycosides. Some compounds showed potent to moderate anti-
inflammatory activity.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured using an Electrothermal melting point apparatus and are
uncorrected. Optical rotations were recorded on a JASCO DIP 1020
polarimeter. The IR spectra were obtained on a PerkinElmer 1760x
FT-IR spectrophotometer. The 1H and 13C NMR spectra were
recorded with a Bruker AVANCE 400 MHz spectrometer. Chemical
shifts are referenced to the residual solvent signals (CDCl3: δH 7.24
and δC 77.0). HRESIMS was recorded on a Bruker Daltonics
microTOF mass spectrometer. HPLC separation was performed using
a Merck LiChrospher 100 RP-18 (5 μm, 250 × 4.0 mm) column with
a TSP SpectraSYSTEM P2000 pump and a TSP SpectraSYSTEM
UV2000 detector.
Figure 4. ORTEP plot of 8a.
Plant Material. The plant, Hoya kerrii (Apocynaceae), was
collected from Padaeng Subdistrict, Chattrakan District, Pitsanuloke
Province, on May 12, 2009. The plant was identified by Associate
Professor Dr. Nijsiri Ruangrangsi, Faculty of Pharmacy, Rangsit
University, Pathumthani. A voucher specimen (SSHK-2009) is
maintained at the Chemistry Department, Ramkhamhaeng University.
Extraction and Isolation. Fresh stem (5.2 kg) of H. kerrii was cut
into small pieces, ground, and soaked in MeOH (24 L) at room
temperature for 30 d. The concentrated extract, obtained as a greenish,
waxy residue after removal of solvent under reduced pressure, was
partitioned successively with hexanes (28.4 L), CH2Cl2 (30 L), and
EtOAc (24 L) to yield dark green hexanes (83.16 g), dark green
CH2Cl2 (48.02 g), reddish-brown EtOAc (80.69 g), and reddish-
brown aqueous (MeOH) (312.46 g) extracts. The stem CH2Cl2
extract (48.02 g) was fractionated by column chromatography (silica
gel, hexanes−CH2Cl2, 70:30−30:70) to obtain 15 fractions. Fraction 2
(269.9 mg) was purified by column chromatography (CC, silica gel,
hexanes−EtOAc, 99.7:0.3) to give three subfractions; the third
subfraction (61 mg) was chromatographed (silica gel, hexanes−
EtOAc, 99.7:0.3) to give 63.9 mg of a colorless solid of lupeol acetate.
Fraction 4 (465.9 mg) was chromatographed (silica gel, hexanes−
EtOAc, 98:2 to 95:5) to give three subfractions (4.1−4.3); subfraction
4.2 (242.2 mg) was recrystallized from hexanes−EtOAc (8:2) to give
taraxerol (33.2 mg). Fraction 5 (938.6 mg) was recrystallized from
hexanes−EtOAc (8:2) to give an additional quantity of taraxerol (67.7
mg), and the concentrated mother liquor was recrystallized from
CH2Cl2−MeOH (4:6) to give lupeol (77.1 mg). Fraction 6 (939.5
mg) was purified by CC (silica gel, hexanes−EtOAc, 95:5 to 50:50) to
give three subfractions (6.1−6.3); subfraction 6.2 (356.1 mg) after CC
(silica gel, hexanes−CH2Cl2, 50:50, to CH2Cl2−MeOH, 99.9:0.5) gave
a mixture of sitosterol and stigmasterol (142.8 mg). Fraction 8 (323.4
mg) was purified by CC (silica gel, hexanes−EtOAc, 80:20−50:50) to
give three subfractions (8.1−8.3); subfraction 8.2 (44.2 mg) gave p-
hydroxybenzaldehyde (9.3 mg). Subfraction 8.3 (14.0 mg) was
separated by chromatography (RP-18, MeOH−H2O, 70:30−100:0)
to give scopoletin (5.9 mg). Fraction 10 (11.40 g) was fractionated
(CC, silica gel, hexanes−EtOAc, 70:30, CH2Cl2−MeOH, 99.5:0.5) to
give six subfractions (10.1−10.6); subfraction 10.2 (1.63 g) after CC
(silica gel, CH2Cl2−MeOH, 99.7:0.3−96:4) gave five subfractions
(10.2.1−10.2.5). Subfraction 10.2.2 (996.7 mg) after CC (RP-18,
MeOH−H2O, 65:35) and then silica gel (hexanes−EtOAc, 50:50)
gave colorless compound 4 (58.8 mg); an aliquot of 4 after acetylation
(using Ac2O, pyridine) and purification (CC, silica gel, hexanes
−EtOAc, 50:5) gave a colorless solid of 4a. Subfraction 10.2.3 (76.7
mg) was chromatographed (silica gel, hexanes−EtOAc, 50:50) to give
four subfractions; the third subfraction was purified by CC (silica gel,
hexanes−EtOAc, 50:50) to give four subfractions, the second
subfraction of which (31.5 mg) was purified by HPLC, RP-18,
MeOH−H2O, 60:40 to 100:0, TSP 2000, flow rate 1 mL/min,
detected at 220 nm, to give 9, tR = 12.8 min (3.4 mg), and 8, tR = 13.7
min (11.2 mg). This subfraction containing a mixture of 8 and 9 (62.3
mg) was acetylated using Ac2O−pyridine, the crude product (53.6
Figure 5. Key NOE correlations of 9.
Table 4. Anti-inflammatory Activity Based on the Inhibition
of Nitric Oxide Production in RAW264.7 Cells
a
compound
IC50 (μM)
compound
1a
IC50 (μM)
b
1
96.5
91.2
87.8
2
62.5
84.0
2a
3
4
93.8
4a
6
60.4
5
>100
c
>100
31.6
8
87.3
8a
9a
9
66.1
43.2
46.5
37.7
b
d
12.6
aspirin
d
d
ibuprofen
54.5
9.3
indomethacin
d
d
CAPE
L-NA
a
b
c
Each value represents mean
SEM of four determinations.
Cytotoxic effect was observed at ≥100 μM (by the MTT assay).
d
Cytotoxic effect was observed at ≥10 μM. Positive control
compounds, caffeic acid phenethyl ester (CAPE), L-nitroarginine (L-
NA).
The compound was found to down-regulate mRNA expression
of iNOS and COX-2 in a dose-dependent manner (Figure 6).
Figure 6. Effect of 9a at various concentrations (3, 10, 30, 100 μM) on
the mRNA expression of iNOS and COX-2 using RAW264.7 cells [N
= LPS (−), sample (−); C = LPS (+), sample (−); 3−100 μM = LPS
(+), sample (+)].
In summary, five rare 12,20-epoxypregnane glycosides and
the first two 11,12-seco-pregnane glycosides with a spirodi-
lactone motif, as well as the spirodilactone cleavage products,
were isolated from the stems of H. kerrii. Their structures were
elucidated on the basis of extensive spectroscopic and X-ray
crystallographic analyses. The absolute configurations of the
H
DOI: 10.1021/acs.jnatprod.6b00730
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Journal of Natural Products
Article
mg) was fractionated using Sephadex LH-20 and MeOH, and the
major fraction was further purified using HPLC, MeOH−H2O, 60:40
to 100:0, to give 9a, tR = 19.7 min (4.7 mg), and 8a, tR = 20.8 min (8.4
mg). Subfraction 10.2.3.4 (79.3 mg) was fractionated (Sephadex LH-
20, MeOH, and then RP-18, MeOH−H2O, 50:50) to give lariciresinol
(9.0 mg). Fraction 10.2.4 (202.8 mg) was purified by CC (Sephadex
LH-20, MeOH) to obtain three subfractions (10.2.4.1−10.2.4.3);
subfraction 10.2.4.2 (45.6 mg), after CC (RP-18, MeOH−H2O, 60:40
to 70:30), gave a colorless solid (18.8 mg) of 3. Subfraction 10.3 (1.01
g) was recrystallized (hexanes−CH2Cl2, 6:4) to give colorless needles
(114.6 mg) of 1. An aliquot of 1 (30 mg) was acetylated using Ac2O−
pyridine to obtain 1a after purification (silica gel, hexanes−EtOAc,
50:50) (28.9 mg). Subfraction 10.4 (2.24 g) was chromatographed
(silica gel, CH2Cl2−MeOH, 99.5:0.5−93:7) to give six subfractions
(10.4.1−10.4.6). Subfraction 10.4.2 (282.5 mg) was recrystallized from
hexanes−CH2Cl2 6:4 to give fine needles (47.1 mg) of 1. Subfraction
10.4.4 (324.0 mg) was purified by CC (RP-18, MeOH−H2O, 50:50)
and then silica gel (hexanes−EtOAc, 45:65) to give five subfractions
(10.4.4.1−10.4.4.5); subfraction 10.4.4.2 gave an additional quantity of
1 (20.7 mg), and subfraction 10.4.4.4 gave 2 (17.4 mg). Subfraction
10.5 (1.495 g) was subjected to CC (silica gel, hexanes−EtOAc, 20:80,
to CH2Cl2−MeOH, 98:2) to give four subfractions (10.5.1−10.5.4).
Subfraction 10.5.2 (1.17 g) was separated by chromatography (RP-18,
MeOH−H2O, 50:50−100:0) to obtain two subfractions (10.5.2.1−
10.5.2.2); subfraction 10.5.2.1 (365.9 mg) was fractionated (CC,
Sephadex LH-20, MeOH) to give three subfractions. Subfraction
10.5.2.1.2 (193.8 mg) after CC (RP-18, MeOH−H2O, 60:40−100:0,
and then Sephadex LH-20, MeOH) gave an additional quantity of a
colorless solid (36.2 mg) of 2. The fraction containing 2 as a major
compound was acetylated using Ac2O−pyridine to obtain 2a as a
colorless solid after CC (silica gel, hexanes−EtOAc, 50:50). Fraction
14 (11.35 g) was fractionated (CC, Sephadex LH-20, MeOH) to
obtain three subfractions (14.1−14.3); subfraction 14.2 (8.0 g) was
column chromatographed (silica gel, CH2Cl2−MeOH, 93:7−80:20) to
give four subfractions (14.2.1−14.2.4). Subfraction 14.2.2 (1.73 g)
after further CC [(RP-18 MeOH−H2O, 60:40 to 100:0, then
Sephadex LH-20 (MeOH)] and finally CC [(silica gel, CH2Cl2−
MeOH, 93:7)] gave compound 7 (12.3 mg) and compound 5 (9.4
mg). Subfraction 14.2.3 (3.42 g) was subjected to CC (silica gel,
CH2Cl2−MeOH, 95:5−90:10) to give four subfractions (14.2.3.1−
14.2.3.4); subfraction 14.2.3.2 (351.2 mg) after CC (RP-18, MeOH−
H2O, 50:50, then Sephadex LH-20, MeOH) gave an additional
quantity of 5 (29.5 mg). Subfraction 14.2.3.4 (763.4 mg) was
subjected to CC (RP-18, MeOH−H2O, 50:50) to obtain compound 6
(21.1 mg).
1), 1.317 (d, J = 6.0 Hz, H-21), 1.27 (H-16), 1.03 s (H-18); 13C NMR
(CDCl3, 100 MHz) δC 211.2 (C, C-11), 140.8 (C, C-5), 121.7 (CH,
C-6), 91.5 (CH, C-12), 82.3 (CH, C-20), 82.2 (C, C-14), 71.6 (CH,
C-3), 63.2 (C, C-13), 59.6 (CH, C-9), 58.4 (CH, C-17), 41.4 (CH2,
C-4), 39.2 (C, C-10), 37.3 (CH2, C-1), 36.5 (CH, C-8), 32.4 (CH2, C-
15), 31.2 (CH2, C-2), 28.1 (CH2, C-7), 22.7 (CH3, C-18), 20.2 (CH3,
C-21), 20.0 (CH3, C-19); HRESIMS m/z 369.2026 [M + Na]+ (calcd
for C21H30O4Na, 369.2034). The aqueous phase was neutralized with
saturated NaHCO3 and evaporated to give an aqueous extract (8.1
mg). Column chromatography (silica gel, CH2Cl2−MeOH, 92:8 to
75:25) of the crude extract gave D-diginose (0.9 mg, Rf 0.57), β-D-
glucopyranosyl-(1→4)-D-diginopyranose (1.1 mg, Rf 0.15), and D-
glucose (5.4 mg, Rf 0.03) [silica gel TLC, thickness 0.2 mm, CH2Cl2−
MeOH (92:8)]. The optical rotation values were measured after 24 h
of dissolution in H2O: D-diginose [α]26 +59 (c 0.1, H2O) (lit.13,14a
D
+59.6 (c 1.48, H2O); β-D-glucopyranosyl-(1→4)-D-diginopyranose,
[α]26 +62 (c 0.1, H2O) (lit.15 +50.8 (c 1.3, MeOH); and D-glucose,
D
[α]26 +59 (c 0.1, H2O) (lit.14a +53.2 (c 0.1, H2O).
D
3-β-O-12α,20α-Epoxy-14β-hydroxypregn-5-en-11-one β-D-digi-
nopyranoside (1): colorless solid, mp 192−194 °C; [α]D −182 (c
0.5, CHCl3); FT-IR (ATR) νmax 3463, 2936, 2890, 1711, 1661, 1445,
1367, 1322, 1269, 1194, 1167, 1129, 1102, 1060, 1031, 974, 893, 813,
784, 725, 658, 580 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR
(CDCl3, 100 MHz) data, see Table 1; HRESIMS m/z 513.2828 [M +
Na]+ (calcd for C28H42O7Na, 513.2816).
4′-O-Acetyl-3-β-O-12α,20α-epoxy-14β-hydroxypregn-5-en-11-
one β-D-diginopyranoside (1a): [α]D −132 (c 0.4, CHCl3); FT-IR
(ATR) νmax 3445, 2967, 2956, 2930, 2889, 2841, 1735, 1711, 1439,
1379, 1374, 1360, 1235, 1172, 1140, 1112, 1059, 1034, 1019, 973, 951,
1
884, 863, 847, 814, 661 cm−1; H NMR (CDCl3, 400 MHz) and 13C
NMR (CDCl3, 100 MHz) data, see Table S1; HRESIMS m/z
555.2956 [M + Na]+ (calcd for C30H44O8Na, 555.2928).
12α,20α-Epoxy-8α,14β-dihydroxypregn-5-en-11-one 3-O-β-D-
diginopyranoside (2): colorless solid, mp 146−148 °C; [α]D −57 (c
0.7, CHCl3); FT-IR (ATR) νmax 3436, 2925, 2856, 1736, 1710, 1460,
1445, 1371, 1260, 1191, 1371, 1260, 1191, 1166, 1098, 1059, 1047,
1024, 975, 886, 864, 809, 727 cm−1; 1H NMR (CDCl3, 400 MHz) and
13C NMR (CDCl3, 100 MHz) data, see Table 1; HRESIMS m/z
529.2759 [M + Na]+ (calcd for C28H42O8Na 529.2766).
4′-O-Acetyl-12α,20α-epoxy-8α,14β-dihydroxypregn-5-en-11-one
3-O-β-D-diginopyranoside (2a): colorless solid, mp 204−206 °C;
[α]D −84 (c 0.6, CHCl3): FT-IR (ATR) νmax 3518, 3426, 2965, 2928,
2872, 1747, 1698, 1457, 1439, 1378, 1367, 1342, 1169, 1227, 1132,
1
1103, 1063, 1022, 967, 979, 946, 915, 888, 860, 809, 728 cm−1; H
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Table S1; HRESIMS m/z 571.2898 [M + Na]+ (calcd for
C30H44O9Na, 571.2877).
Acid Hydrolysis of 1. A mixture of compound 1 (6.5 mg) and
0.05 M HCl (1.5 mL) was refluxed at 60 °C for 4 h. After cooling, the
reaction mixture was extracted with EtOAc (3 × 10 mL). The
combined EtOAc extract was washed with water and concentrated to
give the EtOAc extract (5.1 mg). Reversed-phase column chromatog-
raphy (MeOH−H2O, 40:60) of the extract gave the agylcone 12α,20α-
epoxy-14β-hydroxypregn-5-en-11-one (3.4 mg, Rf 0.35) and D-diginose
(1.8 mg, Rf 0.52) [silica gel TLC, thickness 0.2 mm, CH2Cl2−MeOH
(94:6)]. The aqueous extract after neutralization and concentration
gave an additional quantity of D-diginose (0.9 mg). The optical
rotation value was measured after 24 h of dissolution in H2O: D-
12α,20α-Epoxy-12β,14β-dihydroxypregn-5-en-11-one 3-O-β-D-
diginopyranoside (3): [α]D −49 (c 0.5, CHCl3); FT-IR (ATR) νmax
3468, 2965, 2933, 2867, 1717, 1458, 1442, 1375, 1268, 1189, 1166,
1118, 1098, 1062, 1030, 1017, 972, 941, 913, 886, 843, 816, 723 cm−1;
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data,
see Table 1; HRESIMS m/z 529.2779 [M + Na]+ (calcd for
C28H42O8Na, 529.2766).
Kerriipregnane A 3-O-β-D-diginopyranoside (4): colorless solid,
mp 158−162 °C; [α]D −78 (c 0.5, CHCl3); FT-IR (ATR) νmax 3511,
2969, 2937, 2874, 1756, 1447, 1376, 1367, 1192, 1167, 1098, 1060,
1028, 979, 966, 937, 894, 851, 812, 719 cm−1; 1H NMR (CDCl3, 400
MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; HRESIMS
m/z 527.2625 [M + Na]+ (calcd for C28H40O8Na, 527.2615).
4′-O-Acetylkerriipregnane A 3-O-β-D-diginopyranoside (4a):
colorless solid, mp 172−173 °C; [α]D −25 (c 0.8, CHCl3); FT-IR
(ATR) νmax 2978, 2946, 2883, 1757, 1715, 1456, 1439, 1380, 1361,
1240, 1253, 1192, 1168, 1103, 1062, 1029, 961, 950, 918, 726 cm−1;
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data,
diginose [α]26 +68 (c 0.1, H2O) (lit.13,14a +59.6 (c 1.48, H2O)).
D
Acid Hydrolysis of 5. A mixture of compound 5 (13.8 mg) and
0.05 M HCl (3.0 mL) was refluxed at 60 °C for 6 h. After cooling, the
reaction mixture was extracted with EtOAc (3 × 10 mL). The
combined EtOAc extract after usual workup (5.1 mg) and column
chromatography (silica gel, CH2Cl2−MeOH, 98:2) gave 12α,20α-
epoxy-14β-hydroxypregn-5-en-11-one (4.1 mg), Rf 0.35 (CH2Cl2−
MeOH, 96:4): [α]26 −200 (c 0.4, CHCl3); FT-IR (ATR) νmax 3442,
D
2928, 2876, 1712, 1458, 1378, 1270, 1144, 1113, 1067, 1047, 1033,
1020, 973 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.47 (d, J = 5.5 Hz,
H-6), 3.90 (s, H-12), 3.53 (H-3), 3.46 (H-20), 2.36 (dt, J = 11.9, and
4.4 Hz, H-8); 2.35 (H-4), 2.27 (H-7), 2.14 (brt, J = 11.0 Hz, H-4),
2.02 (H-1), 2.02 (d, J = 12.3 Hz, H-9), 1.95 (H-16), 1.86 (H-2), 1.83
(H-17), 1.75 (H-7), 1.58 (H-15), 1.45(H-2), 1.39 s (H-19), 1.35 (H-
see Table S1; HRESIMS m/z 569.2725 [M + Na]+ (calcd for
C30H42O9Na, 569.2715).
12α,20α-Epoxy-14β-hydroxypregn-5-en-11-one 3-O-β-D-gluco-
pyranosyl-(1→4)-O-β-D-diginopyranoside (5): [α]D −79 (c 0.7,
CHCl3); FT-IR (ATR) νmax 3373, 2926, 2870, 2855, 1741, 1711,
I
DOI: 10.1021/acs.jnatprod.6b00730
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1655, 1596, 1451, 1369, 1322, 1271, 1220, 1195, 1166, 1099, 1057,
1026, 1017, 972, 889, 863, 811 cm−1; 1H NMR (pyridine-d5, 400
MHz) and 13C NMR (pyridine-d5, 100 MHz) data, see Table 2;
HRESIMS m/z 675.3356 [M + Na]+ (calcd for C34H52O12Na,
675.3342).
30, 100 μM) using the RNeasy mini kit (Qiagen Operon Co. Ltd.,
USA). The total RNA from each sample was used for cDNA synthesis
using a first-strand cDNA synthesis kit (Rever Tra Ace-α, TOYOBO
Co., Ltd., Japan), followed by RT-PCR (Rever Tra Dash, TOYOBO
Co., Ltd., Japan). The primers for iNOS and COX-2 were used
(forward primer for iNOS: 5′-ATCTGGATCAGGAACCTGAA-3′
and its reverse primer: 5′-CCTTTTTTGCCCCATAGGAA-3′;
forward primer for COX-2:5′-GGAGAGACTATCAAGATA-
GTGATC-3′ and its reverse primer: 5′-ATGGTCAGTAGACTTT-
TACAGCTC-3′; forward primer for β-actin (an internal standard): 5′-
TGTGATGGTGGGAATGGGTCAG-3′ and reverse primer: 5′-
TTTGATGTCACGCACGATTTCC-3′). The solution for cDNA
synthesis consisted of 11 μL of RNA solution, 4 μL of 5× RT buffer, 2
μL of dNTP mixture (10 mM), 1 μL of RNase inhibitor (10 U/μL), 1
μL of Oligo(dT)20, and 1 μL of Rever Tra Ace (reverese transcriptase
enzyme) for a 20 μL reaction. The conditions for cDNA synthesis
were as follows: 42 °C for 20 min, 99 °C for 5 min, and 4 °C for 5
min. After that, 1/10 the volume (2 μL) of the cDNA product was
further used for PCR. The PCR mixture consisted of 2 μL of RT
reaction mixture (cDNA product): 85 μL of sterilized water, 10 μL of
10× PCR buffer, 1 μL of forward primer (10 pmol/ μL), 1 μL of
reverse primer (10 pmol/ μL), and 1 μL of KOD Dash (polymerase
enzyme) for a final volume of 100 μL. The conditions for PCR were as
follows: denaturation at 94 °C for 1 min, 98 °C for 30 s, 55 °C for 30
s, and 74 °C for 1 min (30 cycles). The PCR products were analyzed
in 1.2% agarose gel electrophoresis and visualized by SYBR safe
staining and UV irradiation under a wavelength of 312 nm.
12α,20α-Epoxy-8α,14β-dihydroxypregn-5-en-11-one 3-O-β-D-
glucopyranosyl-(1→4)-O-β-D-diginopyranoside (6). colorless solid,
mp 190−194 °C; [α]D −90 (c 0.5, CHCl3); FT-IR (ATR) νmax3406,
2973, 2935, 2871, 1700, 1647, 1596, 1456, 1445, 1370, 1320, 1276,
1223, 1199, 1167, 1153, 1100, 1071, 1051, 1022, 994, 977, 954, 944,
1
916, 892, 863 cm−1; H NMR (pyridine-d5, 400 MHz) and 13C NMR
(pyridine-d5, 100 MHz) data, see Table 2; HRESIMS m/z 691.3310
[M + Na]+ (calcd for C34H52O13Na 691.3300).
Kerriipregnane A 3-O-β-D-glucopyranosyl-(1→4)-O-β-D-digino-
pyranoside (7): colorless solid, mp 158−162 °C; FT-IR (ATR) νmax
3384, 2970, 2934, 2872,1761, 1732, 1456, 1442, 1369, 1170, 1099,
1
1056, 1020 cm−1; H NMR (pyridine-d5, 400 MHz) and 13C NMR
(pyridine-d5, 100 MHz) data, see Table 2; HRESIMS m/z 689.3204
[M + Na]+ (calcd for C34H50O13Na, 689.3134).
Kerriipregnane B 3-O-β-D-diginopyranoside (8): colorless solid,
1
mp 132−134 °C; [α]D −48 (c 1.3, CHCl3); H NMR (CDCl3, 400
MHz) and 13C NMR (CDCl3, 100 MHz) data see Table 3; HRESIMS
m/z 559.2881 [M + Na]+ (calcd for C29H44O9Na, 559.2871).
1
4′-O-Acetylkerriipregnane B 3-O-β-D-diginopyranoside (8a): H
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Table 3; HRESIMS m/z 601.3006 [M + Na]+ (calcd for C31H46O10Na,
601.2983).
13-epi-Kerriipregnane B 3-O-β-D-diginopyranoside (9): [α]D
−46.46 (c 0.24, CHCl3); 1H NMR (CDCl3, 400 MHz) and 13C
NMR (CDCl3, 100 MHz) data, see Table 3; HRESIMS m/z [M + Na]
559.2888 (calcd for C29H44O9Na 559.2871).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00730.
■
S
4′-O-Acetyl-13-epi-kerriipregnane B 3-O-β-D-diginopyranoside
(9a): FT-IR (ATR) νmax 3445, 2966, 2929, 2891, 2842, 1734, 1711,
1438, 1380, 1235, 1170, 1138, 1111, 1059, 1034, 1019, 973, 862 cm−1;
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data,
HMBC correlations of compounds 1−4 and 8 (PDF)
X-ray data of 1 (CIF)
X-ray data of 4a (CIF)
see Table 3; HRESIMS m/z 601.3023 [M + Na]+ (calcd for
C31H46O10Na, 601.2983).
X-ray Crystallographic Analysis of Compounds 1, 4a, and
8a. X-ray analysis was carried out on a Bruker-Nonius kappaCCD
diffractometer using a graphite monochromator with Mo Kα radiation
(λ = 0.710 73 Å) at 298(2) K. The structures were solved by direct
methods using SIR9717 and refined with full-matrix least-squares
calculation on F2 using SHELXL-97.18 The crystallographic data have
been deposited at the Cambridge Crystallographic Data Centre under
the reference numbers CCDC 1484810−1484812.
Crystal data for 1: 2(C28H42O7) 3(H2O), MW = 1035.3, 0.15 ×
0.15 × 0.20 mm3, monoclinic, space group P21 (No. 4), a = 7.2520(1)
Å, b = 35.8010(5) Å, c = 10.7280(2) Å, β = 90.0590(5)°, V =
2785.30(8) Å3, Z = 2, T = 298(2) K, μ(Mo Kα) = 0.090 mm−1, Dx =
1.235 g/cm3, reflections measured/unique: 16 175/3306, observed [I
> 2σ(I)]: 3154, R1 = 0.0464, wR2 = 0.1250 (all data).
Crystal data for 4a: C30H42O9, MW = 546.64, 0.25 × 0.25 × 0.30
mm3, monoclinic, space group P21 (No. 4) a = 12.0520(6), Å, b =
8.5300(4) Å, c = 14.5650(7) Å, β = 95.408(3)°, V = 1490.67(12) Å3, Z
= 2, T = 298(2) K, μ(Mo Kα) = 0.090 mm−1, Dx = 1.218 g/cm3,
reflections measured/unique: 6495/1868, observed [I > 2σ(I)]: 1439,
R1 = 0.0577, wR2 = 0.1710 (all data).
Crystal data for 8a: 2(C30H46O710), MW = 578.68, 0.15 × 0.15 ×
0.30 mm3, orthorhombic, space group P21212 (No. 18), a =
27.6220(4) Å, b = 19.96800(10) Å, c = 5.6990(5) Å, V = 3143.3(3)
Å3, Z = 4, T = 298(2) K, μ(Mo Kα) = 0.090 mm−1, Dx = 1.223 g/cm3,
reflections measured/unique: 5050/4650, observed [I > 2σ(I)]: 4200,
R1 = 0.0520, wR2 = 0.1346 (all data).
X-ray data of 8a (CIF)
1H and 13C NMR spectroscopic data of 1a, 2a, and 4a in
CDCl3 (PDF)
1H and 13C NMR spectra of compounds 1−9; 2D NMR
spectra of 1, 4, and 8 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: (662) 319-0931. Fax: (662) 319-1900. E-mail: somyote_
s@yahoo.com; s_somyote@ru.ac.th.
ORCID
Somyote Sutthivaiyakit: 0000-0002-3249-1380
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
■
We are grateful to the Thailand Research Fund (BRG5580012)
and Ramkhamhaeng University for financial support. C.S.
acknowledges the Royal Golden Jubilee Ph.D. Program,
Thailand Research Fund, and the Center of Excellence for
Innovation in Chemistry (PERCH−CIC), the Commission on
Higher Education, the Ministry of Education, for a scholarship.
P.K. gratefully acknowledges the support from Mahidol
University and the Office of the Higher Education Commission
under the National Research Universities Initiative. We
acknowledge Mr. M. Taegyong, Mr. S. Singha, Miss S.
Kruakaew, and the late Mr. W. Komkhunthot for technical
assistance.
Bioassays. The assay for anti-inflammatory activity based on the
inhibition of NO production in RAW264.7 cells was conducted using a
previously published protocol.15
To acquire the mechanism of action of the cytokine release of 9a,
assays for the mRNA expression of iNOS and COX-2 were performed.
The total RNA was isolated from RAW264.7 cells and was harvested
after 20 h of incubation with samples at various concentrations (3, 10,
J
DOI: 10.1021/acs.jnatprod.6b00730
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Journal of Natural Products
Article
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DOI: 10.1021/acs.jnatprod.6b00730
J. Nat. Prod. XXXX, XXX, XXX−XXX