Enantiomeric neolignans and sesquineolignans from Jatropha integerrima and their absolute configurations

Article abstract of DOI:10.1039/c4ra15966g

Two pairs of new sesquineolignan enantiomers, (±)-jatrointelignans A and B (1a/1b and 2a/2b), one pair of new neolignan enantiomers, (±)-jatrointelignan D (4a/4b), and two new neolignans, (+)-jatrointelignan C (3a), and (+)-schisphenlignan I (5a) together

Full text of DOI:10.1039/c4ra15966g

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Enantiomeric neolignans and sesquineolignans
from Jatropha integerrima and their absolute
configurations†
Cite this: RSC Adv., 2015, 5, 12202
Jian-Yong Zhu,‡a Bao Cheng,‡b Yin-Jia Zheng,c Zhen Dong,a Shu-Ling Lin,a
a
a
a
*
Gui-Hua Tang, Qiong Gu and Sheng Yin
Two pairs of new sesquineolignan enantiomers, (ꢀ)-jatrointelignans A and B (1a/1b and 2a/2b), one pair of
new neolignan enantiomers, (ꢀ)-jatrointelignan D (4a/4b), and two new neolignans, (+)-jatrointelignan C
(3a), and (+)-schisphenlignan I (5a) together with seven known analogues (3b, 5b, 6a, 6b, 7a, 7b, and 8)
were isolated from the trunks of Jatropha integerrima. The structures were determined by combined
spectroscopic and chemical methods, and their absolute configurations were elucidated by the circular
dichroism (CD) method, in particular the configurations of the aryl glycerol-700,800-yloxy moiety in 1 and 2
were determined via the Rh2(OCOCF3)4-induced CD analysis. All compounds were examined for their
inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in BV-2 microglial
cells, and compounds 5a, 6a, and 4b exhibited pronounced inhibition on NO production with IC50 values
in the range of 5.9–8.9 mM, being more active than the positive control, quercetin (IC50 ¼ 17.0 mM).
Received 8th December 2014
Accepted 13th January 2015
DOI: 10.1039/c4ra15966g
www.rsc.org/advances
microglial cells might be an important and attractive thera-
peutic target for the treatment of neurodegenerative diseases.
Introduction
Jatropha integerrima Jacq (Euphorbiaceae), is a common
ornamental shrub widely distributed in subtropical and tropical
areas of the world.7 Its leaves have been reported to possess a
strong purgative effect.8 Previous chemical investigation of this
plant led to the isolation of macrocyclic diterpenes,8 sesquiter-
penes,9 sesquiterpene-coumarin,9 and cyclic peptides,10 some of
which showed antimicrobial,11 antiplasmodial,9 and antituber-
culosis activities.9 In our screening program aimed at the
discovery of novel NO inhibitors from natural resource,12 the
EtOAc fraction of the ethanolic extract of J. integerrima showed a
certain inhibitory activity against the lipopolysaccharide (LPS)-
induced NO production in BV-2 microglial cells. Subsequent
chemical investigation led to the isolation of seven pairs of
lignan enantiomers and a racemic mixture, including eight new
compounds. Bioassay veried that 13 compounds were
responsible for the NO inhibitory activities of the EtOAc frac-
tion, with IC50 values ranging from 5.9 to 38.0 mM. Herein,
details of the isolation, structural elucidation, and NO inhibi-
tory activities of these compounds were described.
Neuroinammation has been considered as one of the patho-
logical factors in neurodegenerative diseases including Alz-
heimer's disease (AD), Parkinson's disease (PD), stroke,
dementia, and amyotrophic lateral sclerosis (ALS).1,2 Activation
of brain microglial cells and consequent overexpression of
proinammatory mediators such as nitric oxide (NO) are
involved in the neuroinammatory process. The production of
NO and superoxide also lead to the formation of peroxynitrite,
which results in lots of oxidation and potential destruction of
host cellular constituents causing dysfunctional critical cellular
processes, cell signaling pathway disruption, and brain cell
death via cell apoptosis and necrosis.3,4 There is numerous
evidence suggesting that suppression of proinammatory
mediators (such as NO) and further inhibition of the neuro-
inammatory responses in microglia could attenuate the
severity or delay the progress of these neurodegenerative
disorders.5,6 Therefore, suppression of NO production in
aSchool of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong
510006, P. R. China. E-mail: yinsh2@mail.sysu.edu.cn; Fax: +86-20-39943090; Tel:
+86-20-39943090
bInstitute of Chinese Medical Sciences, Guangdong Pharmaceutical University,
Results and discussion
Guangzhou, Guangdong 510006, P. R. China
cSchool of Health Management, Guangzhou Medical University, Guangzhou,
Guangdong 510182, P. R. China
The air-dried powder of the trunks of J. integerrima was extrac-
ted with 95% EtOH at room temperature (rt) to give a crude
extract, which was suspended in H2O and successively parti-
tioned with petroleum ether, EtOAc, and n-BuOH. Various
column chromatographic separations of the EtOAc extract
† Electronic supplementary information (ESI) available: IR, HRESIMS, CD, 1D and
2D NMR spectra of 1–5, 1H and 13C NMR spectra of known compounds (6–8). See
DOI: 10.1039/c4ra15966g
‡ These authors have contributed equally to this work.
12202 | RSC Adv., 2015, 5, 12202–12208
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
afforded seven pairs of enantiomers 1a/1b–7a/7b and a racemic J ¼ 8.0, 2.0, 2.0 Hz, H-600), 6.75 (1H, d, J ¼ 8.0 Hz, H-500), and 6.99
mixture (8).
(1H, dd, J ¼ 2.0, 2.0 Hz, H-200)]. The 13C NMR spectrum, in
Compound 1 (1a/1b), colorless oil, exhibited a molecular combination with DEPT experiments, resolved 31 carbon reso-
formula of C31H36O11 as determined by the 13C NMR data and a nances attributable to three benzene rings, one double bond,
HRESIMS ion at m/z 607.2121 [M + Na]+ (calcd for C31H36O11Na, three oxygenated methylenes, four methines (three oxygenated
607.2129). The IR spectrum exhibited absorption bands for OH methines), and four methoxy groups. Above-mentioned data
(3460 cmꢁ1) and aromatic (1595, 1502, and 1464 cmꢁ1) func- implied that 1 possessed a sesquineolignan feature13,14 and
tionalities. The 1H NMR spectrum revealed the presence of four resembled that of buddlenol B isolated from Buddleja davidii.15
methoxy groups [dH 3.89 (3H, s), 3.82 (3H ꢂ 2, s), and 3.81 (3H, The gross structure of 1 was further established by analysis of its
s)], three oxygenated methylenes [dH 4.19 (2H, dd, J ¼ 6.0, 1.2 2D NMR data. The 1H–1H COSY spectrum established three C3
Hz, H-90), 3.86 (1H, m, H-9a), 3.80 (1H, m, H-9b), 3.73 (1H, m, H- spin systems from C-7 to C-9, C-70 to C-90, and C-700 to C-900. The
900a), and 3.34 (1H, m, H-900b)], three oxygenated methines [dH HMBC correlations (Fig. 1) from H-7 to C-1, C-2, and C-6, from
5.58 (1H, d, J ¼ 6.0 Hz, H-7), 4.98 (1H, d, J ¼ 6.9 Hz, H-700), and H-70 to C-10, C-20, and C-60, and from H-700 to C-100, C-200, and C-600
4.08 (1H, ddd, J ¼ 6.9, 5.0, 3.5 Hz, H-800)], one disubstituted E connected the three C3 units to three benzene rings, respec-
double bond [dH 6.54 (1H, d, J ¼ 15.8 Hz, H-70) and 6.23 (1H, dt, tively, thus indicating the presence of three phenylpropanoid
J ¼ 15.8, 6.0 Hz, H-80)], a symmetrically 1,2,3,5-tetrasubstituted units. The connectivities of the three phenylpropanoid units
benzene ring (ring A) [dH 6.72 (2H, s, H-2/6)], an 1,2,3,5-tetra- were established by HMBC correlations from both H-8, H-7 to
substituted benzene ring (ring B) [dH 6.95 (2H, brs, H-20/60)], and C-30 and C-40 and from H-800 to C-4, which necessitated the
an 1,2,4-trisubstituted benzene ring (ring C) [dH 6.86 (1H, ddd, presence of a 2,3-dihydrofuran ring. The locations of the
Fig. 1 Selected 1H–1H COSY (—) and HMBC (/) correlations of 1 and 3.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 12202–12208 | 12203

View Article Online
RSC Advances
Paper
methoxyl groups were further dened by detailed HMBC anal- that 2 shared the same planar structure as that of 1, indicating
yses (Fig. 1). The relative conguration of 1 was established on that 2 was a stereoisomer of 1 with variation at conguration at
the basis of the NOESY experiment and interpretation of 1H–1H C-700 or C-800. An erythro conguration for the aryl glycerol-800-
coupling constants. A coupling constant of 15.8 Hz between H- yloxy moiety in 2 was further determined by the coupling
70
80 and H-70 and strong NOE interaction of H-90/H-70 assigned D
constants between H-700 and H-800(J7 ,8
¼
5.1 Hz).13,14,17
00 00
as E conguration. A coupling constant of 6.0 Hz between H-7 Compound 2 also had negligible optical activity and CD spec-
and H-8 implied the trans-relationship between these two trum. Subsequent chiral resolution of 2 afforded the enantio-
protons,16 which was further supported by NOESY correlations mers 2a and 2b. Using the same methods as described for 1a
between H-7 and H2-9 (ESI S48†), while the coupling constant and 1b, the 7S,700S,8R,800R conguration of 2a and the
between H-700 and H-800 (6.9 Hz) implied a threo conguration of 7R,700R,8S,800S conguration of 2b were elucidated (ESI, S15 and
these two protons.17,18
S16†). Thus, 2a was assigned as (+)-(7S,700S,8R,800R,70E)-400-
Compound 1 was primarily obtained with the specic rota- hydroxy-3,300,5,50-tetramethoxy-40,7-epoxy-8,30-sesquineolign-70-
tion being almost zero and no Cotton effect in its electronic en-700,9,90,900-tetraol, and was given a trivial name (+)-jatrointe-
circular dichroism (ECD), indicating a near racemic nature. lignan B. Consequently, its enantiomer 2b was named as
Subsequent chiral resolution of 1 afforded the anticipated (ꢁ)-jatrointelignan B.
enantiomers 1a and 1b, which showed mirror image-like ECD
Compound 3 (3a/3b) was obtained as a pale gum. The spec-
curves (Fig. 2A) and opposite specic rotation ([a]2D0 +15 in 1a, troscopic data of 3 were identical to those of (ꢁ)-(70R,80S)-50-
[a]2D0 ꢁ13 in 1b). The absolute congurations (ACs) of 1a and 1b methoxyl-(dimeric coniferyl acetate) (3b),25 except for a negligible
were determined by CD analysis. According to the reversed specic rotation and CD spectrum in 3, indicating 3 was a racemic
helicity rule for the 7-methoxy-2,3-dihydrobenzo[b]furan chro- mixture. Subsequent chiral resolution of 3 afforded the antici-
mophore, P/M helicity of the heterocyclic ring leads to positive/ pated enantiomers 3a and 3b, which were opposite in terms of
negative 1Lb band CD (at 260–310 nm).19,20 In the CD spectrum ECD curves and optical rotation, respectively. The ACs of 3a and
of 1a, a positive Cotton effect at 273 nm (D3 +2.77) indicated a 3b were determined by using the same CD methods as described
P-helicity and thus assigned the ACs of C-7 and C-8 as 7S and for 7-methoxy-2,3-dihydrobenzo[b]furan chromophore in 1a and
8R.21,22 The absolute conguration of C-700 was assigned by 1b. In the CD spectrum of 3a, (Fig. 2A) a positive Cotton effect at
Rh2(OCOCF3)4-induced CD analysis. On the basis of the bulki- 273 nm (D3 +3.58) indicated a 7S,8R conguration,21 which was
ness rule for secondary alcohols, a positive Cotton effect at supported by its optical rotation ([a]2D0 +102), as neolignans con-
around 350 nm (the E band) in the Rh2(OCOCF3)4-induced CD taining a dihydrobenzo[b]furan skeleton with 7S,8R conguration
spectrum indicated a S-conguration, while negative Cotton exhibited a positive optical rotation.22 Thus, 3a was dened as
effect implied a R-conguration.23,24 Thus, a negative Cotton (+)-(7S,8R,70E)-4-hydroxy-3,5,50,-trimethoxy-40,7-epoxy-8,30-neolign-
effect at 353 nm (the E band) in the Rh2(OCOCF3)4-induced CD 70-en-9,90-diyil diacetate and was given a trivial name (+)-jatroin-
spectrum of 1a assigned the 700R conguration.13 (Fig. 2B) telignan C. Consequently, 3b was identied as the known
Consequently, the 800R conguration was dened by the 700,800- compound (ꢁ)-(70R,80S)-50-methoxyl-(dimeric coniferyl acetate).
threo relationship in 1a. Thus, 1a was identied as
Compound 4 (4a/4b), obtained as a pale gum, gave a [M +
(+)-(7S,700R,8R,800R,70E)-400-hydroxy-3,300,5,50-tetramethoxy-40,7- Na]+ ion at m/z 395.1458 in the HRESIMS corresponding to a
epoxy-8,30-sesquineolign-70-en-700,9,90,900-tetraol and was given a molecular formula of C21H24O6. The spectroscopic data showed
trivial name (+)-jatrointelignan A. Consequently, its enantiomer similarity to that of co-isolated known compound 6b.26 In
1b was named as (ꢁ)-jatrointelignan A.
comparison with 6b, the major differences of 4 were due to 90-
Compound 2 (2a/2b), colorless oil, had the same molecular OCH3 and 9-OH functionalities. This was supported by the
formula as that of 1. The NMR data of 2 was very similar to that HMBC correlation from the methoxy protons (dH 3.39, s) to C-90
of 1, except for the slight discrepancy arising from the oxygen- (dC 73.2) and the upeld-shied H2-9 signals in 4 with respect to
ated methines (C-700 and C-800). Detailed 2D analysis revealed those of 6b [dH 3.97 (H-9a), 3.92 (H-9b) in 4; dH 4.42 (H-9a), 4.28
Fig. 2 The CD spectra of 1a, 1b, 3a, and 3b in MeCN (A) and the Rh2(OCOCF3)4 induced CD spectra of 1a and 1b in CH2Cl2 (B).
12204 | RSC Adv., 2015, 5, 12202–12208
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
(H-9b) in 6b]. The relative conguration of 4 was assigned to be specic rotation and CD spectrum. Chiral analysis of 8 in HPLC
the same as that of 3 based on a coupling constant of 7.5 Hz revealed two blunt peaks with poor resolution. Attempts to
between H-7 and H-8 and NOE correlations between H-7 and H2- separate each peak failed in various conditions probably due to
9. Compound 4 also had negligible optical activity and CD its low polarity. To increase the polarity of 8, a reduction reaction
spectra. Subsequent chiral resolution of 4 afforded the enan- was carried out under NaBH4 to transform the formyl group to
tiomers 4a and 4b. The ACs of the 4a and 4b were determined to alcohol which subsequently generated 2. Compound 2 was
be 7S,8R and 7R,8S, respectively, by using the same methods as further puried by chiral separation to give 2a and 2b. Therefore
described for those of 3a and 3b (ESI, S34†). Thus, 4a was the buddlenol A (8) was conrmed as a racemic mixture.
elucidated the same ACs as 3a, and was dened as
The known compounds (ꢁ)-(70R,80S)-50-methoxyl-(dimeric
(+)-(7S,8R,70E)-4-hydroxy-3,50,90-trimethoxy-40,7-epoxy-8,30-neo- coniferyl acetate) (3b),25 schisphenlignan I (5b),27,28 dimeric
lign-70-en-9-ol and was given a trivial name (+)-jatrointelignan coniferyl acetate (6a),29 (ꢁ)-(70E)-(7R,8S,700E)-4-hydroxy-3,50-
D. Consequently, its enantiomer 4b was named as (ꢁ)-jatroin- dimethoxy-40,7-epoxy-8,30-neolig-70-en-9,90-diyil
diacetate
telignan D.
(6b),26 (+)-balanophonin (7a),22,30 (ꢁ)-balanophonin (7b),31 and
Compound 5 (5a/5b), obtained as a yellow oil, gave a [M + Na]+ buddlenol A (8)15 were identied by comparison of their NMR
ion at m/z 423.1404 in the HRESIMS appropriate for the molec- data, optical rotations, and CD spectra with those in the
ular formula of C22H24O7. The NMR data of 5 were identical to literature.
those of schisphenlignan I, which was isolated from Clematis
All compounds were evaluated for their inhibitory effects
armandii27 and Schisandra sphenanthera.28 Chiral resolution of 5 on the NO production in LPS-induced BV-2 microglial cells
afforded the enantiomers 5a and 5b. The specic rotation and using the Griess assay. Compounds 1a and 1b, were inactive
CD data of 5b were almost identical to those of schisphenlignan (<50% inhibition at 50 mM), while compounds 2a, 2b, 3a, 3b,
I, indicating 5b possessed the same 7R,8S conguration (ESI, 4a, 5b, 6b, 7a, 7b, and 8 showed moderate inhibitory activities
S41†). Thus, compound 5a was elucidated as (+)-(7S,8R,70E)-4- with IC50 values ranging from 12.5 to 38.0 mM. Compounds
hydroxy-3,50-dimethoxy-40,7-epoxy-8,30-neoligna-70-en-9-acetyl-90- 4b, 6a, and 5a showed remarkable inhibitory activities with
ol and was given a trivial name (+)-schisphenlignan I.
IC50 values of 8.9, 7.9, and 5.9 mM, respectively, more active
Compound 8, a colorless oil, had a molecular formula than the positive control quercetin (IC50 ¼ 17.0 mM), a well-
C31H34O11 as determined by a HRESIMS ion at m/z 605.1967 [M + known NO inhibitor (Table 3). The inhibitory curves of 4b,
Na]+ (calcd for C31H34O11Na, 605.1969). The spectroscopic data 6a, 5a, and quercetin were represented in Fig. 3. To investigate
of 8 were identical to those of buddlenol A15 with a negligible whether the inhibitory activities of the active compounds
Table 1 1H NMR data of compounds 1–5a
Position
1b
2b
3c
4c
5c
2
6.72, s
6.70, s
6.62, s
6.91, s
6.89, s
5
6.88, s
6.87, s
6
6.72, s
6.70, s
6.62, s
6.89, s
6.87, s
7
8
9a
5.58, d (6.0)
3.47, ddd (6.5, 6.0, 5.7)
3.86, m
3.80, m
6.95, brs
5.57, d (6.0)
3.48, ddd (6.5, 6.0, 5.7)
3.90, m
3.87, m
6.95, brs
5.45, d (7.5)
5.58, d (7.5)
5.46, d (7.5)
3.76, ddd (7.5, 7.3, 5.5)
4.43, dd (11.2, 5.5)
4.30, m
6.87, s
6.87, s
6.55, d (15.8)
6.24, dt (15.8, 6.0)
4.30, (2H, m)
3.78, ddd (7.5, 7.3, 5.5)
4.45, dd (11.2, 5.5)
4.32, dd (11.2, 7.3)
6.89, s
6.89, s
6.60, d (15.8)
3.62, ddd (7.5, 6.0, 5.0)
3.97, dd (11.2, 6.0)
3.92, dd (11.2, 5.0)
6.89, s
9b
20
60
6.95, brs
6.95, brs
6.89, s
70
6.54, d (15.8)
6.23, dt (15.8, 6.0)
4.19, dd (2H, 6.0, 1.2)
6.99, dd (2.0, 2.0)
6.75, d (8.0)
6.86, ddd (8.0, 2.0, 2.0)
4.98, d (6.9)
4.08, ddd (6.9, 5.0, 3.5)
3.73, m
3.34, m
3.82, s
3.82, s
3.89, s
6.54, d (15.8)
6.23, dt (15.8, 6.0)
4.19, dd (2H, 6.0, 1.2)
6.96, brs
6.74, d (8.0)
6.79, dd (8.0, 1.6)
4.90, d (5.1)
4.25, ddd (5.1, 4.7, 3.5)
3.90, m
6.55, d (15.8)
6.15, dt (15.8, 6.0)
4.07, dd (2H, 6.0, 1.2)
800
6.16, dt (15.8, 6.0)
4.72, dd (2H, 6.0, 1.2)
900
200
500
6
700
800
900a
900b
3.58, dd (12.0, 3.5)
3.78, s
3.78, s
3-OCH3
5-OCH3
50-OCH3
90-OCH3
300-OCH3
9-OAc
90-OAc
3.87, s
3.87, s
3.91, s
3.86, s
3.85, s
3.90, s
3.89, s
3.90, s
3.39, s
3.81, s
3.80, s
2.04, s
2.10, s
2.02, s
a
b
Data were recorded at 400 MHz, chemical shis are in ppm, coupling constant J is in Hz. In CD3OD. c In CDCl3.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 12202–12208 | 12205

View Article Online
RSC Advances
Paper
Table 2 13C NMR (100 MHz) data (d) of compounds 1–5
Position 1a
2a
3b
4b
5b
1
139.5, C
139.4, C
134.9, C
132.9, C
132.3, C
2
103.9, CH 104.0, CH 103.1, CH 108.8, CH 108.6, CH
3
4
5
154.4, C
136.9, C
154.4, C
154.6, C
136.4, C
154.6, C
147.2, C
148.2, C
147.2, C
146.7, C
145.7, C
114.3, CH 114.3, CH
146.7, C
145.8, C
6
103.9, CH 103.9, CH 103.1, CH 119.4, CH 119.9, CH
7
8
88.9, CH
55.4, CH
88.9, CH
55.4, CH
89.0, CH
50.4, CH
88.2, CH
53.5, CH
88.7, CH
50.3, CH
9
64.9, CH2 64.9, CH2 65.3, CH2 64.0, CH2 65.3, CH2
132.8, C 132.8, C 130.6, C 130.9, C 131.0, C
116.5, CH 116.5, CH 115.3, CH 114.8, CH 115.0, CH
10
20
30
130.0, C
149.1, C
145.5, C
130.0, C
149.1, C
145.5, C
127.7, C
148.2, C
144.4, C
128.0, C
148.3, C
144.4, C
127.7, C
147.9, C
144.4, C
40
Fig. 3 The inhibitory curves of compounds 4b, 5a, 6a, and quercetin
(positive control) on LPS-induced NO production in BV-2 cells.
50
60
112.2, CH 112.2, CH 110.6, CH 110.5, CH 110.5, CH
131.9, CH 131.9, CH 134.3, CH 132.5, CH 131.3, CH
127.8, CH 127.7, CH 121.2, CH 123.9, CH 126.5, CH
63.8, CH2 63.8, CH2 65.2, CH2 73.2, CH2 63.7, CH2
70
800
Conclusions
900
100
133.4, C
133.8, C
In summary, four new sesquineolignans, four new neolignans,
and seven known analogues were isolated from the trunks of J.
integerrima. The structures were determined by combined
spectroscopic and chemical methods, and their ACs were
elucidated by circular dichroism (CD) method, particularly the
congurations of the aryl glycerol-700,800-yloxy moiety in 1 and 2
were determined via the Rh2(OCOCF3)4-induced CD analysis. All
compounds were examined for their inhibitory effects on the
nitric oxide (NO) production induced by lipopolysaccharide
(LPS) in BV-2 microglial cells, and compounds 5a, 6a, and 4b
exhibited pronounced inhibition on NO production with IC50
values in the range of 5.9–8.9 mM, being more active than the
positive control, quercetin (IC50 ¼ 17.0 mM). J. integerrima is
known as a rich source of macrocyclic diterpenoids. The current
study not only enriched the chemodiversity of this spices by the
isolation of a series of bioactive lignans, but also dig out the
potential usage of this plant as anti-neuroinammatory
therapy. However, the mechanism of inhibition against NO
production of these compounds requires further investigation.
200
111.7, CH 111.7, CH
300
148.7, C
147.1, C
148.6, C
146.8, C
400
5
115.8, CH 115.7, CH
120.9, CH 120.7, CH
600
700
74.5, CH
88.9, CH
74.0, CH
87.3, CH
800
900
61.8, CH2 61.6, CH2
3-OCH3
5-OCH3
56.7, CH3 56.6, CH3 56.4, CH3 56.0, CH3 56.0, CH3
56.7, CH3 56.6, CH3 56.4, CH3
50-OCH3 56.8, CH3 56.8, CH3 56.0, CH3 56.0, CH3 55.9, CH3
300-OCH3 56.4, CH3 56.3, CH3
90-OCH3
9-OAc
57.9, CH3
170.7, C
21.0, CH3
170.9, C
20.8, CH3
170.8, C
20.7, CH3
90-OAc
In CD3OD. b In CDCl3.
a
Table
3 IC50 values of active compounds on LPS-induced NO
production in BV-2 cells
a
IC50a (mM)
Experimental section
Compound
IC50 (mM)
Compound
General experimental procedures
2a
2b
3a
3b
4a
4b
5a
12.5 ꢀ 1.2
38.0 ꢀ 2.1
32.7 ꢀ 2.3
25.9 ꢀ 1.7
26.4 ꢀ 2.0
8.9 ꢀ 0.9
5.9 ꢀ 0.8
5b
6a
6b
7a
7b
8
14.7 ꢀ 1.2
7.9 ꢀ 2.5
Optical rotations were measured on a Rudolph Autopol I auto-
matic polarimeter. CD spectra were obtained on an Applied
Photophysics Chirascan spectrometer. UV spectra were recor-
ded on a Shimadzu UV-2450 spectrophotometer. IR spectra
were determined on a Bruker Tensor 37 infrared spectropho-
tometer. NMR spectra were measured on a Bruker AM-400
spectrometer at 25 ꢃC. HRESIMS was performed on a Waters
Micromass Q-TOF spectrometer. A Shimadzu LC-20 AT equip-
ped with a SPD-M20A PDA detector was used for HPLC. A chiral
column (Phenomenex Lux, cellulose-2, 250 ꢂ 10 mm, 5 mm) was
used for semipreparative HPLC separation. Semipreparative
HPLC was performed on a YMC-pack ODS-A (250 ꢂ 10 mm, S-
5 mm, 12 nm). Silica gel (300–400 mesh, Qingdao Haiyang
Chemical Co., Ltd.), and MCI gel (CHP20P, 75–150 mm, Mitsu-
bishi Chemical Industries Ltd.) were used for column
35.5 ꢀ 3.1
24.9 ꢀ 2.1
29.8 ꢀ 1.3
12.8 ꢀ 1.3
17.0 ꢀ 2.2
Quercetinb
a
Values are represented as means ꢀ SD based on three independent
experiments. b Positive control.
were generated from their cytotoxicity, the effects of
compounds 2a, 3b, 4a, 4b, 5a, 6a, 6b, and 8 on LPS-induced
BV-2 microglial cell viability were measured using the MTT
method. These eight compounds (up to 80 mM) did not show
any signicant cytotoxicity with LPS treatment for 24 h
(ESI, S49†).
12206 | RSC Adv., 2015, 5, 12202–12208
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
chromatography (CC). All solvents were of analytical grade 1125 cmꢁ1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/
(Guangzhou Chemical Reagents Company, Ltd.).
z 607.2121 [M + Na]+ (calcd for C31H36O11Na, 607.2129).
(ꢁ)-Jatrointelignan A (1b). Colorless oil; [a]2D0 ꢁ13 (c 0.19,
CHCl3); CD (c 3.25 ꢂ 10ꢁ4 M, CH3CN) lmax (D3) 208 (+6.21), 222
(ꢁ2.02), 240 (+2.78), 273 (ꢁ3.28) nm; UV, IR, NMR, and HRE-
SIMS were the same with those of 1a.
Plant material
Trunks of J. integerrima were collected in the East campus of Sun
Yat-sen University in the Guangzhou city, P. R. China, in
November 2013, and were authenticated by Associate Professor
Lin Jiang of Sun Yat-sen University. A voucher specimen
(accession number: YJIZ-201311) has been deposited at the
School of Pharmaceutical Sciences, Sun Yat-sen University.
(+)-Jatrointelignan B (2a). Colorless oil; [a]2D0 +38 (c 0.15,
CHCl3); UV (MeOH) lmax (log 3) 276 (4.14), 225 (4.61) nm; CD (c
2.57 ꢂ 10ꢁ4 M, CH3CN) lmax (D3) 208 (ꢁ6.53), 222 (+0.55), 240
(ꢁ4.35), 268 (+2.73) nm; IR (KBr) nmax 3446, 2918, 1595, 1502,
1464 cmꢁ1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS
m/z 607.2134 [M + Na]+ (calcd for C31H36O11Na, 607.2140).
(ꢁ)-Jatrointelignan B (2b). Colorless oil; [a]2D0 ꢁ42 (c 0.12,
Extraction and isolation
The air-dried powder of the trunks of J. integerrima (6 kg) was CHCl3); CD (c 2.05 ꢂ 10ꢁ4 M, CH3CN) lmax (D3) 208 (+9.82), 222
extracted with 95% EtOH (3 ꢂ 10 L) at rt to give 462 g of crude (ꢁ1.67), 240 (+4.38), 268 (ꢁ5.18) nm; UV, IR, NMR, and HRE-
extract. The extract was suspended in H2O (2 L) and successively SIMS were the same with those of 2a.
partitioned with petroleum ether (PE, 3 ꢂ 2 L), EtOAc (3 ꢂ 2 L),
(+)-Jatrointelignan C (3a). Pale gum; [a]2D0 +102 (c 0.10,
and n-BuOH (3 ꢂ 2 L) to yield three corresponding portions. The CHCl3); UV (MeOH) lmax (log 3) 275 (4.15), 213 (4.46) nm; CD (c
EtOAc extract (34 g) was subjected to MCI gel CC eluted with a 2.12 ꢂ 10ꢁ4 M, CH3CN) lmax (D3) 204 (ꢁ8.24), 220 (+1.43), 242
MeOH/H2O gradient (2 : 8 / 10 : 0) to afford ve fractions (I–V). (ꢁ2.15), 273 (+3.58) nm; IR (KBr) nmax 3410, 2928, 2214, 1735,
Fr. I (17 g) was subjected to silica gel chromatography using 1605, 1501, 1454, 1236, 1113 cmꢁ1; H and 13C NMR data, see
1
hexane/EtOAc mixtures (v/v 1 : 0 / 0 : 1) to afford six fractions Tables 1 and 2; HREIMS m/z 495.1613 [M + Na]+ (calcd for
Ia–If. Fr. Ib (3.4 g) was subjected to silica gel chromatography
using PE–acetone mixtures (v/v 1 : 0, 15 : 1, 10 : 1, 1 : 1) to yield
C
25H28O9Na, 495.1616).
(+)-Jatrointelignan D (4a). Pale gum; [a]2D0 +74 (c 0.14, CHCl3);
four fractions, Fr. Ib (1–4). Fr. Ib3 was puried using semi- UV (MeOH) lmax (log 3) 276 (3.76), 215 (4.14) nm; CD (c 3.49 ꢂ
preparative HPLC with a YMC-pack ODS-A column (MeOH/H2O, 10ꢁ4 M, CH3CN) lmax (D3) 208 (ꢁ6.43), 221 (+0.56), 239 (ꢁ3.25),
7 : 3, 3 mL minꢁ1), followed by semipreparative chiral HPLC 273 (+2.42) nm; IR (KBr) nmax 3473, 2923, 1662, 1516, 1094 cmꢁ1
;
(CH3OH/H2O, 9 : 1, 3 mL minꢁ1) to give 4a (3 mg, tR 11 min) and 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z
4b (2.5 mg, tR 12 min). Fr. Ic (1.5 g) was loaded on a Sephadex LH- 395.1458 [M + Na]+ (calcd for C21H24O6Na 395.1465).
20 column and eluted with CH2Cl2–MeOH (1 : 1) to yield four
(ꢁ)-Jatrointelignan D (4b). Pale gum; [a]2D0 ꢁ63 (c 0.15,
fractions, Fr. Ic (1–4). Fr. Ic2 was further puried by semi- CHCl3); CD (c 3.32 ꢂ 10ꢁ4 M, CH3CN) lmax (D3) 204 (+6.61), 220
preparative chiral HPLC (MeOH/H2O, 8 : 2, 3 mL minꢁ1) to give (ꢁ2.35), 242 (+2.90), 273 (ꢁ3.34) nm; UV, IR, NMR, and HRE-
5a (4 mg, tR 15 min) and 5b (5 mg, tR 17 min). Fr. Ic3 was followed SIMS were the same with those of 4a.
by semipreparative chiral HPLC (CH3CN/H2O, 4.5 : 5.5,
(+)-Schisphenlignan I (5a). Yellow oil; [a]2D0 +72 (c 0.17,
3 mL minꢁ1) to give 7a (9 mg, tR 10 min) and 7b (11 mg, tR 12 CHCl3); UV (MeOH) lmax (log 3) 276 (3.73), 215 (3.94) nm; CD
min). Fr. Ie (1.2 g) was loaded on a Sephadex LH-20 column and (c 2.69 ꢂ 10ꢁ4 M, CH3CN) lmax (D3) 215 (+0.49), 236 (ꢁ1.76), 268
eluted with CH2Cl2–MeOH (1 : 1) to yield four fractions, Fr. Ie (1– (+2.19), 285 (+2.65) nm; IR (KBr) nmax 3442, 2931, 1734, 1603, 1506,
4). Fr. Ie3 was puried using semipreparative HPLC with a YMC- 1234 cmꢁ1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS
pack ODS-A column (MeOH/H2O, 6 : 4, 3 mL minꢁ1) to give 8 m/z 423.1404 [M + Na]+ (calcd for C22H24O7Na, 423.1414).
(17 mg). Fr. If (962 mg) was separated by a Sephadex LH-20
column (CHCl3/MeOH, 1 : 1), followed by semipreparative
HPLC with a YMC-pack ODS-A column (MeOH/H2O, 5.5 : 4.5, 3
mL minꢁ1) to give Fr. If1 and Fr. If2, which were further puried
Determination of the absolute conguration of the secondary
alcohol units in 1 (1a/1b) and 2 (2a/2b)
Following the reported procedure,23,24 a 1 : 2 mixture of
secondary alcohol–Rh2(OCOCF3)4 for 1a was subjected to CD
measurements at a concentration of 0.1 mg mLꢁ1 in anhydrous
CH2Cl2. The rst CD spectrum was recorded immediately aer
mixing, and its time evolution was monitored until stationary
(about 10 min aer mixing). The inherent CD was subtracted.
The observed sign of the band at around 350 nm in the induced
CD spectrum was correlated to the absolute conguration of the
secondary alcohol. Compounds 1b, 2a, and 2b were measured
by the same methods as 1a.
by semipreparative chiral HPLC (CH3CN/H2O, 4 : 6, 3 mL minꢁ1
)
to give 2b (7 mg, tR 22 min) and 2a (8 mg, tR 23 min), 1b (8 mg, tR
23 min) and 1a (10 mg, tR 25 min), respectively. Fr. III (4 g) was
subjected to silica gel eluted with hexane/EtOAc mixtures (v/v
1 : 0, 10 : 1, 5 : 1, 0 : 1) to afford six fractions, Fr. III (a–f). Fr.
IIIe (900 mg) was loaded on a Sephadex LH-20 column and eluted
with CH2Cl2–MeOH (1 : 1) to yield four fractions, Fr. IIIe (1–4). Fr.
IIIe2 and Fr. IIIe3 were further puried using semipreparative
chiral HPLC (CH3OH/H2O, 9 : 1, 3 mL minꢁ1) to give 3a (1.5 mg)
and 3b (1.6 mg), 6a (10 mg) and 6b (11 mg), respectively.
(+)-Jatrointelignan A (1a). Colorless oil; [a]2D0 +15 (c 0.16,
CHCl3); UV (MeOH) lmax (log 3) 276 (4.00), 225 (4.46) nm; CD (c
Chemical transformation of 8 to 2
2.74 ꢂ 10ꢁ4 M, CH3CN) lmax (D3) 208 (ꢁ9.54), 222 (+0.80), 240 NaBH4 (2 mg) was added to a stirred solution of 8 (4 mg) in
(ꢁ4.65), 273 (+2.77) nm; IR (KBr) nmax 3460, 2918, 1595, 1502, 1464, MeOH (2 mL), and the reaction was stirred for 15 min at rt. The
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 12202–12208 | 12207

View Article Online
RSC Advances
Paper
mixture was then puried by HPLC on a semi-preparative YMC-
pack ODS-A column (MeOH/H2O, 5.5 : 4.5, 3 mL minꢁ1) to
afford 2. Compound 2 was further puried by semipreparative
chiral HPLC (CH3CN/H2O, 4 : 6, 3 mL minꢁ1) to give 2a and 2b.
Those compounds were identied by their 1H NMR spectra, MS,
and CD data.
6 B. Liu and J. S. Hong, J. Pharmacol. Exp. Ther., 2003, 304, 1–7.
7 M. Sujatha, N. Sivaraj and M. S. Prasad, Biol. Plant., 2000, 43,
167–171.
8 S. Sutthivaiyakit, W. Mongkolvisut, P. Ponsitipiboon,
S. Prabpai, P. Kongsaeree, S. Ruchirawat and C. Mahidol,
Tetrahedron Lett., 2003, 44, 3637–3640.
9 S. Sutthivaiyakit, W. Mongkolvisut, S. Prabpai and
P. Kongsaeree, J. Nat. Prod., 2009, 72, 2024–2027.
10 A. Wele, C. Baragueye, W. Ndiaye, D. Fall, I. Ndoye, Y. Diop,
L. Dubosq and B. Bodo, Dakar Med., 2007, 52, 209–215.
11 A. O. Eshilokun, A. A. Kasali, I. A. Ogunwande, T. M. Walker
and W. N. Setzer, Nat. Prod. Commun., 2007, 2, 853–855.
12 Z. Dong, Q. Gu, B. Cheng, Z. B. Cheng, G. H. Tang, Z. H. Sun,
J. S. Zhang, J. M. Bao and S. Yin, RSC Adv., 2014, 4, 55036–
55043.
13 L. Xiong, C. Zhu, Y. Li, Y. Tian, S. Lin, S. Yuan, J. Hu, Q. Hou,
N. Chen, Y. Yang and J. Shi, J. Nat. Prod., 2011, 74, 1188–
1200.
14 F. Cutillo, B. D'Abrosca, M. DellaGreca, A. Fiorentino and
A. Zarrelli, J. Agric. Food Chem., 2003, 51, 6165–6172.
15 P. J. Houghton, Phytochemistry, 1985, 24, 819–826.
16 K. H. Kim, E. Moon, S. Y. Kim and K. R. Lee, J. Agric. Food
Chem., 2010, 58, 4779–4785.
Cell culture and viability assay
BV-2 microglial cells were obtained from Southern Medical
University (SMU) Cell Bank (Guangzhou, People's Republic of
China). Cells were plated into a 96-well plate (2 ꢂ 104 cells per
well). Aer 24 h, they were pretreated with samples for 30 min
and stimulated with 1 mg mLꢁ1 LPS for another 24 h. The
cell viability of the cultured cells was assessed by MTT assay.
Briey, BV-2 cells were incubated with 200 mL MTT solution
(0.5 mg mLꢁ1 in medium) for 4 h at 37 ꢃC, and then the
supernatants were removed and residues were dissolved in
200 mL DMSO. The absorbance was detected at 570 nm using a
microplate reader (Molecular Devices, USA) and analyzed using
a SoMax Pro 5 soware (Molecular Devices, USA).
Measurement of NO production
The NO concentration was measured by the Griess reaction.
Briey, BV-2 cells were treated with LPS (1.0 mg mLꢁ1) and
compounds for 24 h. Aer that, 100 mL of culture supernatant
was allowed to react with 100 mL of Griess reagent (1% sulfa-
nilamide, 0.1% N-1-naphthylethylenediamine dihydrochloride
in 5% phosphoric acid) for 10 min at rt in the dark. Then, the
optical density (100 mL per well) was measured at 540 nm using a
microplate reader (Molecular Devices, USA). Sodium nitrite was
used as a standard to calculate the nitrite concentration. Inhi-
bition (%) ¼ (1 ꢁ (ALPS+sample ꢁ Auntreated)/(ALPS ꢁ Auntreated)) ꢂ
100. The experiments were performed in triplicates, and the data
were expressed as the mean ꢀ standard deviation (SD) values.
Quercetin was used as a positive control.
17 S. Besombes, D. Robert, J. P. Utille, F. R. Taravel and
K. Mazeau, J. Agric. Food Chem., 2002, 51, 34–42.
18 K. Kijima, H. Otsuka, T. Ide, C. Ogimi, E. Hirata, A. Takushi
and Y. Takeda, Phytochemistry, 1998, 48, 669–676.
´
´
19 L. Kiss, T. Kurtan, S. Antus and A. Benyei, Chirality, 2003, 15,
558–563.
20 A. Goel, A. Kumar and A. Raghuvanshi, Chem. Rev., 2013,
113, 1614–1640.
´
´
21 S. Antus, T. Kurtan, L. Juhasz, L. Kiss, M. Hollosi and
Z. Majer, Chirality, 2001, 13, 493–506.
22 M. S. M. Yuen, F. Xue, T. C. W. Mak and H. N. C. Wong,
Tetrahedron, 1998, 54, 12429–12444.
23 J. Frelek and W. J. Szczepek, Tetrahedron: Asymmetry, 1999,
10, 1507–1520.
Acknowledgements
24 M. Gerards and G. Snatzke, Tetrahedron: Asymmetry, 1990, 1,
221–236.
The authors thank the National Natural Science Foundation of
China (no. 81102339) and the Opening Project of Guangdong
Provincial Key Laboratory of New Drug Design and Evaluation
(no. 2011A060901014) for providing nancial support to this
work.
25 Q. Guo, Y. Li, Y. B. Liu, H. S. Gu, Y. D. Wang, Q. Hou and
S. S. Yu, J. Asian Nat. Prod. Res., 2014, 16, 1–10.
26 K. H. Kim, S. K. Ha, S. Y. Kim, H. J. Youn and K. R. Lee, J.
Enzyme Inhib. Med. Chem., 2010, 25, 887–892.
27 J. Xiong, V. B. Bui, X. H. Liu, Z. L. Hong, G. X. Yang and
J. F. Hu, J. Ethnopharmacol., 2014, 153, 737–743.
28 C. Q. Liang, J. Hu, R. H. Luo, Y. M. Shi, S. Z. Shang, Z. H. Gao,
R. R. Wang, Y. T. Zheng, W. Y. Xiong, H. B. Zhang, W. L. Xiao
and H. D. Sun, Fitoterapia, 2013, 86, 171–177.
29 A. H. Banskota, Y. Tezuka, J. K. Prasain, K. Matsushige,
I. Saiki and S. Kadota, J. Nat. Prod., 1998, 61, 896–900.
30 S. Falah, T. Katayama and T. Suzuki, J. Wood Sci., 2008, 54,
483–489.
Notes and references
1 D. Luo, T. C. T. Or, C. L. H. Yang and A. S. Y. Lau, ACS Chem.
Neurosci., 2014, 5, 855–866.
2 S. D. Skaper, P. Giusti and L. Facci, FASEB J., 2012, 26, 3103–
3117.
3 P. Wang and J. L. Zweier, J. Biol. Chem., 1996, 271, 29223–
29230.
4 R. Radi, A. Cassina and R. Hodara, Biol. Chem., 2002, 383,
401–409.
31 M. Haruna, T. Koube, K. Ito and H. Murata, Chem. Pharm.
Bull., 1982, 30, 1525–1527.
5 J. Li, K. W. Zeng, S. P. Shi, Y. Jiang and P. F. Tu, Fitoterapia,
2012, 83, 896–900.
12208 | RSC Adv., 2015, 5, 12202–12208
This journal is © The Royal Society of Chemistry 2015