1,5-Diphenylpent-3-en-1-ynes and methyl naphthalene carboxylates from Lawsonia
inermis and their anti-inflammatory activity
Jing-Ru Liou a,1, Mohamed El-Shazly a,b,1, Ying-Chi Du a, Chao-Neng Tseng a, Tsong-Long Hwang c,
Yueh-Lin Chuang aa,e,f,
, Yu-Ming
Hsu a, Pei-Wen
Hsieh c, Chin-Chun Wu a, Shu-Li Chen a, Ming-Feng Hou
⇑
a,g,h,i,⇑
Fang-Rong Chang
, Yang-Chang Wu
d,e
,
a
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street 11566, Abassia, Cairo, Egypt
c
Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
d
Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
e
Cancer Center, Kaohsiung Medical University Hospital, No. 100 Tz-You First Road, Kaohsiung 807, Taiwan
f
Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
g
Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan
h
Natural Medicinal Products Research Center, China Medical University Hospital, Taichung 404, Taiwan
i
School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan
b
a b s t r a c t
Keywords:
Henna
Lawsonia inermis
Lythraceae
1,5-Diphenylpent-3-en-1-yne
Methyl naphthalene carboxylates
Anti-inflammatory
Superoxide anion
Elastase
Lawsonia inermis (Lythraceae) known as henna is one of the most popular and ancient plants used in cosmetics and hair dying. It is cultivated for its leaves but other parts such as seeds, flowers, stem bark and
roots are also used in traditional medicine for millennia. Henna tattoo paste also proved to be beneficial
for wound healing and in several skin diseases suggesting potent anti-inflammatory activity. To evaluate
henna anti-inflammatory activity, 31 compounds, including three 1,5-diphenylpent-3-en-1-yne derivatives, lawsochylin A-C and three methyl naphthalene carboxylates, lawsonaphthoate A-C, were isolated
from the stems and leaves of henna utilizing a bioassay-guided fractionation. The structures of the compounds were elucidated by spectroscopic data. Two compounds, lawsochylin A and lawsonaphthoate A
showed potent anti-inflammatory activity by inhibition of superoxide anion generation (IC50 = 1.80
and 1.90 lg/ml) and elastase release (IC50 = 1.58 and 3.17 lg/ml) of human neutrophils in response to
fMLP or cytochalasin B. Moreover, the known compounds, luteolin, apigenin, 4S-4-hydroxy-a-tetralone,
and 2-butoxysuccinic acid, also showed potent inhibition of superoxide anion generation (IC50 = 0.75–
1.78 lg/ml) and elastase release (IC50 = 1.62–3.61 lg/ml).
。
1. Introduction
Lawsonia is a monotypic genus represented by henna (Lawsonia
inermis Linn.), a spinescent shrub or small tree with grayish brown
bark. Henna leaves produce an orange-red dye and its paste or
powder is traditionally used for dyeing hair, decorating hands,
nails and feet (henna tattoo). Ancient literature indicated that
henna leaves and bark decoction were prescribed for alleviating
jaundice, skin diseases, venereal diseases, smallpox and spermatorrhoea (Chaudhary et al., 2010). Historically, henna origin is
traced back to ancient Egypt from which it was introduced to India
followed by China, Japan, Korea and Taiwan. Despite the wide use
⇑
Corresponding authors. Tel.: +886 7 3121101x2162; fax: +886 7 3114773 (F.-R.
Chang), tel.: +886 4 22057153; fax: +886 4 22060248 (Y.-C. Wu).
E-mail addresses: aaronfrc@kmu.edu.tw (F.-R.
edu.tw (Y.-C. Wu).
1
These authors contributed equally to this work.
Chang), yachwu@mail.cmu.
of henna in India and elsewhere, its use was restricted in China and
South East Asia up to the early years of twentieth century. Foreign
cosmetics were considered barbaric and horrid resulting in the
banning of henna tattoo. However, the rising popularity of henna
tattoo among young Europeans and Americans in recent years
has led to the reintroduction of henna culture to China and South
East Asia. Other than decoration, traditional healers in China and
South East Asia started prescribing henna tattoo paste for various
inflammatory disorders such as hemorrhoids (Miczak, 2001). The
increasing attention to the therapeutic potential of henna has
encouraged the study herein of the phytochemical constituents
of henna grown in South East Asia and to evaluate their biological
activity.
The phytochemical investigation of henna over the last decades
led to the isolation of several classes of secondary metabolites such
as: b-sitosterolglucosides, flavonoids, quinoids, naphthalene derivatives, triterpenoids, coumarins, xanthones and phenolic
68
glycosides (Ahmed et al., 2000). The isolated compounds and different henna extracts were evaluated for their antibacterial, antifungal, antimycotic and antiparasitic activities (Babu
and
Subhasree, 2009). Despite the historical use of henna paste to treat
different skin diseases and wounds, few studies have reported its
anti-inflammatory effect (Alia et al., 1995; Nayak et al., 2007; Yogisha et al., 2002). In these previous reports, it was found that the
ethanolic extract of henna along with its CHCl3 and n-BuOH fractions possessed potent anti-inflammatory activity. However, the
major lead anti-inflammatory compounds in the extract were not
fully investigated (Alia et al., 1995).
In our continuing efforts to discover new anti-inflammatory
agents from natural sources, the CHCl3 and n-BuOH fractions from
L. inermis stems showed significant activity in inhibiting superoxide anion generation (IC50 = 2.80 ± 0.16 and 3.53 ± 0.23 lg/ml)
and elastase release (IC50 = 1.31 ± 0.23 and 3.72 ± 1.20 lg/ml),
respectively. Bioassay-guided fractionation of the extracts led to
the isolation of three 1,5-diphenylpent-3-en-1-ynes (1–3), and
three methyl naphthalene carboxylates (4–6) (Fig. 1), along with
17 known compounds, including eight flavonoids: luteolin (7) (Li
et al., 2008), luteolin 7-O-b-D-glucopyranoside (8) (Li et al., 2008),
luteolin 40 -O-b-D-glucopyranoside (9) (Yoshizaki et al., 1987),
apigenin (10) (Mikhaeil et al., 2004), apigenin 7-O-b-D-glucopyranoside (11) (Oyama and Kondo, 2004), apigenin 40 -O-b-D-glucopyranoside (12) (Oyama and Kondo, 2004), luteolin 7-O-rutinoside
(13) (Siciliano et al., 2004), and diosmetin 7-O-rutinoside (14)
(Siciliano et al., 2004); one naphthoquinone: 3-amino-2-methoxycarbonyl-1,4-naphthoquinone (15) (Jacobs et al., 2009); two a-tetralones: (4S)-4-hydroxy-a-tetralone (16) (Liu et al., 2004), and
3S,4R-dihydro-3,4-dihydroxynaphthalen-1(2H)-one (17) (Mukherjee et al., 2010); three C13 nor-isoprenoids: 9-hydroxy-4-megastigmen-3-one (18) (D’Abrosca et al., 2004), (6R,7E)-9-hydroxy-4,7megastigmadien-3-one (19) (D’Abrosca et al., 2004), and (6S)dehydrovomifoliol (20) (Knapp et al., 1997); one neolignan:
(+)-dihydrodehydrodiconiferyl alcohol (21) (Nabeta et al., 1994);
two benzenoids: 4-hydroxybenzaldehyde (22) (Shin and Yoon,
2009), and 4,6-dihydroxy-2-O-(b-D-glucopyranosyl)acetophenone
(23) (Suksamrarn et al., 1997) and a mixture of b-sitosterol-D-glucoside and b-stigmasterol-D-glucoside (24).
Additionally, the 75% aq. MeOH and n-BuOH fractions from the
leaves were evaluated for their anti-inflammatory activity at a concentration of 10 lg/ml resulting in the inhibition of superoxide anion generation (inhibition% = 75.33 ± 7.69% and 78.79 ± 5.51%) and
elastase release (55.91 ± 5.50% and 62.82 ± 7.12%), respectively.
Utilizing bioassay-guided fractionation, compounds 7, 9, 10, 23,
4''
R4
3
R1
R2
R3
4'
1
1
2
3
3''
1
3'
R2
OH
OH
OH
R
H
OH
H
R3
H
H
OCH3
R1
R4
OH
OH
OH
O
1
9
OCH 3
2
6
HO
5
10
R2
4
5
6
R1
OH
OCH3
OH
4
OCH 3
R2
H
H
OCH3
Fig. 1. Chemical structures of six compounds isolated from L. inermis.
24, one
naphthalene: 1,2,4-trihydroxynaphthalene-1-O-b-Dglycopyranoside (25) (Hsouna et al., 2011); three carbohydrates:
O-n-butyl-b-D-glucopyranoside (26) (Yu et al., 2007), D-glucopyranose (27) (Roslund et al., 2008), and D-mannitol (28) (Rodrigues
et al., 2010); one chlorophyll: methyl pheophorbide b (29) (Closs
et al., 1963); two fatty acids: oleamide (30) (Cravatt et al., 1995)
and 2-butoxysuccinic acid (31) (Lin et al., 2011) were isolated.
The structures of the isolates were established by spectroscopic
analysis and comparison with reported physical data. Some of
the isolated compounds were evaluated for their anti-inflammatory activity.
2. Results and discussion
Compound 1 was obtained as a pale brown powder. Its molecular formula was calculated as C17H14O2 from the analysis of its
HRESIMS data, corresponding to 11 degrees of unsaturation. The
UV spectrum exhibited absorption maxima at 283 and 298 nm.
Its IR spectrum showed absorption bands for hydroxyl
(3380 cm-1), internal alkyne (weak band at 2189 cm-1), and aromatic (1604 and 1509 cm-1) functionalities. Two 1,4-disubstituted
benzene rings were suggested from the 1H NMR signals at dH 7.26
(2H, d, J = 8.8 Hz, H-20 , H-60 ) and dH 6.75 (2H, d, J = 8.8 Hz, H-30 , H50 ), along with dH 7.05 (2H, d, J = 8.8 Hz, H-200 , H-600 ) and dH 6.71
(2H, d, J = 8.8 Hz, H-300 , H-500 ). The proton resonances of two olefinic
methine [dH 6.00 (1H, dt, J = 10.4, 7.6 Hz, H-4) and dH 5.71 (1H, d,
J = 10.4 Hz, H-3)] and one methylene [dH 3.58 (2H, d, J = 7.6 Hz,
H-5)] indicated a partial structure of cis-double bond with an adjacent methylene group. The presence of a pent-3-en-1-yne moiety
in 1 was predicted from the COSY correlations of H-3/H-4 (dH
5.71/dH 6.00) and H-4/H2-5 (dH 6.00/dH 3.58), as well as the HMBC
correlations of H-3/C-1 (dH 5.71/dC 94.8), H-3/C-5 (dH 5.71/dC 36.6),
H2-5/C-3 (dH 3.58/dC 110.3), and H2-5/C-4 (dH 3.58/dC 142.4). The
connection of the two phenol groups (1,4-disubstituted benzene
rings) to the (Z)-pent-3-en-1-yne moiety was determined by the
HMBC correlations of H-20 , H-60 /C-1 (dH 7.26/dC 94.8), H2-5/C-100
(dH 3.58/dC 132.0), H2-5/C-200 , C-600 (dH 3.58/dC 130.4), and H-200 ,
H-600 /C-5 (dH 7.05/dC 36.6). Thus, lawsochylin A (1) was assigned
as (Z)-40 ,400 -dihydroxy-1,5-diphenylpent-3-en-1-yne.
Compound 2 was obtained as a pale brown powder. The molecular formula was determined as C17H14O3 based on HRESIMS. The
IR spectrum suggested hydroxyl (3358 cm-1), internal alkyne
(2194 cm-1), and aromatic (1597 and 1512 cm-1) functionalities.
The 1D and 2D NMR spectroscopic data of 2 closely resembled
those of 1, except for the presence of an additional hydroxyl group
on one of the aromatic rings [dH 6.84 (1H, d, J = 2.0 Hz, H-20 ), dH
6.79 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), and dH 6.71 (1H, d, J = 8.0 Hz,
H-50 )]. The assignments of the hydroxyl groups were achieved by
HMBC correlations (Fig. 2). Accordingly, the structure of 2 (lawsochylin B) was determined as (Z)-30 ,40 ,400 -trihydroxy-1,5-diphenylpent-3-en-1-yne.
Compound 3 was isolated as a pale brown powder, and was assigned the molecular formula of C18H16O3 based on HRESIMS. The
UV, IR, 1H NMR, HSQC, and 1H–1H COSY spectroscopic data of 3
were similar to 1 except for an extra methoxy group [dH 3.81
(3H, s, 300 -OMe), dC 56.3]. It was suggested that the methoxy group
is connected at C-300 based on the HMBC data showing correlations
between H-200 , H-500 , H-600 , 300 -OMe and C-300 (dH 6.83, 6.71, 6,67,
3.81/dC 149.0) and also the NOESY correlation between H-200 and
300 -OMe (dH 6.83/dH 3.81). Consequently, lawsochylin C (3) was elucidated as (Z)-40 ,400 -dihydroxy-300 -methoxy-1,5-diphenylpent-3en-1-yne.
The enyne moiety is present in some biologically active compounds such as the cytotoxic alkaloid hachijodine G (Tsukamoto
et al., 2000), the antifungal drug terbinafine (Nussbaumer et al.,
69
OH
OH
OH
O
HO
HO
1
HO
2
CH3
3
OH
OCH3
OH O
O
OH O
OCH3
OCH3
HO
HO
HO
OCH3
OCH3
4
OCH3
OCH3OCH3
6
5
HMBC
COSY
Fig. 2. Key COSY and HMBC correlations for 1–6.
1995), and the antibiotic neocarzinostatin (Lhermitte and Grierson,
1996). Also compounds with enyne moiety have found several
applications in synthetic chemistry as important intermediates
for the synthesis of conjugated dienes (Kumar et al., 2005), polyenes (Crousse et al., 1999), and polysubstituted benzenes (Saito
and Yamamoto, 2000). However, compounds with 1,5-diphenylpent-3-en-1-yne skeleton have never been isolated from natural
sources and few reports have described their synthesis (Masuyama
et al., 1993; Yoshimatsu et al., 1994). The biosynthesis of these
enynes (1–3) is suggested to proceed through phenylpropanoid
pathway (see supporting information S45).
Compound 4 was obtained as a white powder. Its molecular formula (C13H12O5) was determined by HRESIMS corresponding to
eight degrees of unsaturation. The UV spectrum exhibited absorption maxima at 271 and 352 nm. Its IR spectrum showed absorption bands for hydroxyl (3477 cm-1), carbonyl (1633 cm-1), and
aromatic (1604 and 1520 cm-1) functionalities. The 13C NMR and
HSQC data suggested the presence of a naphthalene ring (ten carbon signals in the range dC 101.8–160.0), one carbonyl ester (dC
172.8), and two methoxy groups (dC 56.0 and 52.6), indicating that
4 is a methyl naphthalene carboxylate derivative. HMBC correlations supported the structural assignment of 4 (Fig. 2). Additionally, the 1H NMR pattern of one singlet peak at dH 6.99 (1H, s, H3) and the typical ABX type aromatic signals [dH 8.19 (1H, d,
J = 9.2 Hz, H-8), dH 7.41 (1H, d, J = 2.4 Hz, H-5), and dH 7.09 (1H,
dd, J = 9.2, 2.4 Hz, H-7)] indicated the presence of mono-substitution on one ring and tri-substitutions on the other ring of naphthalene core. Intramolecular hydrogen bonding between the hydroxyl
and carbonyl functionalities was suggested on the basis of the lowfield shifted resonances at dH 11.64 and 12.20 in CDCl3 and C5D5N,
respectively, indicating the presence of a hydroxyl group ortho to
the ester group. The HMBC correlation of the methoxy protons
(dH 3.97) and the carbonyl carbon (dc 172.8) (Fig. 2), as well as
the NOESY cross-peak between H-3 (dH 6.99) and 4-OMe (dH
3.92), suggested that the methoxy group is connected to C-4 and
the ester group is connected to C-2. Thus, lawsonaphthoate A (4)
was elucidated as methyl 1,6 dihydroxy-4-methoxynaphthalene2-carboxylate.
Compound 5, a white powder, has the molecular formula
C14H14O5, as established by HRESIMS corresponding also to eight
degrees of unsaturation. The UV spectrum exhibited absorption
maxima at 261 and 319 nm. Its IR spectrum showed absorption
bands for hydroxyl (3347 cm-1), carbonyl (1688 cm-1), and aromatic (1610 and 1521 cm-1) functionalities. Although the 1H and
13
C NMR spectra of 4 and 5 are similar, the NMR spectra of 5
showed an additional methoxy signal (dH 4.08/dC 63.3) without
the intramolecular hydrogen bonding signal present in 4. The
assignments of 1-OMe, 2-COOMe, 4-OMe, and 6-OH were achieved
by NOESY and HMBC correlations. Finally, lawsonaphthoate B (5)
was identified as methyl 1,4-dimethoxy-6-hydroxynaphthalene2-carboxylate.
Compound 6 was isolated as a white powder and the molecular
formula was calculated as C14H14O6 by the analysis of its HRESIMS
data, corresponding to eight degrees of unsaturation. The UV, IR
and NMR spectroscopic data of 6 closely resembled those of 4
and 5. However, the 1H NMR spectrum of 6 in comparison to 4
showed two doublets and one singlet signals in the aromatic region and one singlet resonance corresponding to a methoxy group
(dH 3.89, 3H, s). Also 13C NMR and DEPT experiments showed primary carbon signal (dC 62.8). Both the HMBC correlations and the
NOE enhancement indicated that the methoxy group (dH 3.89,
3H, s) is linked to C-5 (Fig. 2 and supporting information S42).
Compound 6 (lawsonaphthoate C) was established as methyl 1,6dihydroxy-4,5-dimethoxy naphthalene-2-carboxylate. Compounds
4–6 are small phenolic derivatives, which are believed to be originated in henna through shikimic acid pathway (see supporting
information S46).
The limited quantities of some isolates, allowed only compounds 1, 4–6 and the known compounds, 7–18, and 21–31 to
be evaluated for their anti-inflammatory activity. Inhibition of
superoxide anion generation and elastase release by human
neutrophils in response to fMLP were utilized to measure the
anti-inflammatory activity of the isolated compounds (Table 3).
Genistein was used as the positive control in superoxide anion generation assay (IC50 = 0.54 lg/ml) and in the elastase release assay
(IC50 = 6.99 lg/ml) (Chung et al., 2011).
Compound 1 with the characteristic enyne moiety inhibited
superoxide anion generation (IC50 = 1.80 lg/ml) and elastase release (IC50 = 1.58 lg/ml). Compounds with the enyne moiety have
been previously utilized for the synthesis of anti-inflammatory
agents with Z,E,E-triene unit such as protectin D1 (Ogawa and
Kobayashi, 2011). However, the anti-inflammatory activity of compounds with an enyne moiety has not been thoroughly investigated. The activity of 1 offers the prospect of the utilization of
enyne containing compounds not only as synthetic intermediates
but also as potential anti-inflammatory agents.
The other class of isolates, methyl naphthalene-2-carboxylates
(4–6) showed significant activity. Compound 4 exhibited the highest activity against superoxide anion generation (IC50 = 1.90 lg/ml)
and elastase release (IC50 = 3.17 lg/ml). The structural resemblance of methyl naphthalene-2-carboxylates to salicylates (commercial anti-inflammatory agents) opens an interesting field of
research in finding new therapeutic agents based on 2-naphthoate
nucleus.
70
Additionally, the known compounds, 7, 10, 15, 16, 25, 29, and
31 showed inhibitory activity against superoxide anion generation
and elastase release with 4S-4-hydroxy-a-tetralone (16) showing
the highest activity. Compounds 22 and 26 showed selective inhibitory effects on superoxide anion generation.
Interestingly, the aglycones luteolin (7) and apigenin (10) inhibited superoxide anion generation with IC50 values of 0.75 and
1.12 lg/ml, respectively, but their glycosides, with sugar moiety
at C-7 or C-40 (8, 9, 11, 12, and 13) were inactive. This finding is
supported by previous anti-inflammatory studies on flavonoids
and their glycosidic derivatives showing that the aglycones exhibit
more potent anti-inflammatory activity compared to their corresponding glycosides (Hostetler et al., 2012).
3. Conclusion
Three 1,5-diphenylpent-3-en-1-ynes (1–3), three methyl naphthalene carboxylates (4–6), together with 25 known compounds
were isolated from the stems and the leaves of L. inermis. The structures of these compounds were identified by various spectroscopic
techniques, and their anti-inflammatory activities was evaluated
by superoxide anion generation and elastase release. Among the
six isolated compounds, 1 showed the highest anti-inflammatory
activity against superoxide anion generation (IC50 = 1.80 lg/ml)
and elastase release (IC50 = 1.58 lg/ml). The anti-inflammatory
activity of the isolates sheds light on the scientific basis of henna
utilization in skin diseases and wound healing.
4. Experimental procedures
4.1. General experimental procedures
Silica gel (Kieselgel 60, 70–230, and 230–400 mesh, Merck
KGaA, Darmstadt, Germany), Diaion HP-20 (Sigma–Aldrich Corp.
St. Louis, MO), and Sephadex LH-20 (Pharmacia Fine Chemicals
AB, Uppsala, Sweden) were used for column chromatography
(CC), while TLC analysis was carried out on silica gel (Kieselgel
60, F254, Merck KGaA) and RP-18 (F254s, Merck KGaA) pre-coated
plates. TLC spots were detected under UV light at 254 nm and
365 nm and also by spraying with 50% H2O–H2SO4 (1:1) followed
by heating on a hot plate. HPLC was performed with JASCO PU980 pumps in conjunction with JASCO UV-970 UV/VIS detector
(JASCO Inc., Tokyo, Japan). Reversed phase columns (Discovery
HS C18, 5 lm, 250 x 10 mm; Ascentis C18, 5 lm, 250 x 10 and
250 x 21.2 mm; Atlantis T3, 5 lm, 150 x 4.6 mm, Thermo Fisher
Scientific Inc., Rockford, IL) were applied for HPLC separation. 1H
and 13C NMR spectra were recorded on Varian Unity Inova-600,
Varian Unity Plus-400, or Varian Gemini-200 NMR spectrometers
(Varian Inc., Palo Alto, CA). Chemical shifts are reported in parts
per million (d), and coupling constants (J) are expressed in hertz.
UV spectra were obtained using a JASCO V-530 UV/vis spectrophotometer. IR spectra were recorded on a Mattson Genesis II FT-IR
spectrophotometer (Mattson Instruments, Madison, WI). LRESIMS
were measured on a Finnigan PolarisQ mass spectrometer (Thermo
Finnigan, Austin, TX). HRESIMS were measured on a Bruker Daltonics APEX II mass spectrometer (Bruker Instruments, Billerica, MA).
Optical rotations were measured with a JASCO-P-1020 polarimeter
(JASCO Inc.).
4.2. Plant material
Stems and leaves of L. inermis were donated by Mr. Yun-Chao
Chen (collected from Xinshi Township, Tainan County, Taiwan) in
October, 2006 and identified by the botanist Dr. Ming-Hong Yen.
Voucher specimens [Lawsonia-01 (leaves) and Lawsonia-02
(stems)] were deposited at the Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
4.3. Extraction and isolation of compounds from Lawsonia inermis
stems
Air-dried stems of L. inermis (3.2 kg) were extracted with MeOH
(4 x 20 l) at room temperature and concentrated under reduced
pressure. The MeOH extract (280.0 g) was partitioned between
MeOH–H2O (1:1, 4 x 1 l) and CHCl3 (4 x 1 l) to yield an aq. MeOH
layer and a CHCl3 layer. The CHCl3 layer (concentrated to give
37.0 g) was then partitioned with n-hexane (4 x 1 l) and MeOH–
H2O (19:2, 4 x 1 l). The resulting aq. MeOH layer was further partitioned with CHCl3 (4 x 1 l) and H2O (4 x 1 l) to obtain a further
CHCl3 layer. The obtained CHCl3 layer (27.9 g) was subjected to silica gel CC using mixtures of n-hexane p-CHCl3-MeOH with increasing polarity as eluants to afford 29 fractions. Fraction 4 (614.4 mg)
was separated on Sephadex LH-20 with MeOH to give six subfractions. Fraction 4–4 (44.2 mg) was further purified using Sephadex
LH-20 CC, eluted with a mixture of CHCl3–MeOH (1:1), to give
six subfractions. Fraction 4–4–4 (21.9 mg) was purified over a
RP-HPLC column (Ascentis C18, 250 x 21.2 mm, monitored at
254 nm) using MeOH–H2O (70:30) as eluent to give 6 (1.7 mg, tR
60.4 min, flow rate 4 ml/min). Fraction 5 (148.1 mg) was separated
on Sephadex LH-20 with CHCl3–MeOH (1:1) to give five subfractions. Fraction 5–4 (26.1 mg) was purified on RP-HPLC (254 nm;
Discovery HS C18, 250 x 10 mm) eluted with MeOH–H2O (75:25)
to give 4 (9.1 mg, tR 18.3 min, flow rate 2 ml/min). Fraction 7
(99.9 mg) was separated on Sephadex LH-20 with CHCl3–MeOH
(1:1) as eluent to give 11 subfractions. Fraction 8 (120.3 mg) was
purified on Sephadex LH-20 with CHCl3–MeOH (1:1) as eluent to
give 18 subfractions. According to TLC behaviour, fractions 7–8
(18.8 mg) and 8–9 (9.5 mg) were combined and then subjected
to RP-HPLC (254 nm; Discovery HS C18, 250 x 10 mm) using
MeOH–H2O (70:30) as the mobile phase to give 5 (2.2 mg, tR
12.8 min, flow rate 2 ml/min). Fraction 9 (161.2 mg) was separated
on Sephadex LH-20 with CHCl3–MeOH (1:1) as eluent to give six
subfractions. Fraction 9–5 (21.1 mg) was purified on RP-HPLC
(254 nm; Discovery HS C18, 250 x 10 mm) using MeOH-H2O
(70:30) as eluent to give 3 (0.5 mg, tR 22.1 min, flow rate 2 ml/
min). Fraction 17 (1.1 g) was separated on Sephadex LH-20 with
CHCl3–MeOH (1:1) as eluent to give five subfractions. Fraction
17–3 (318.9 mg) was subjected to C18 silica gel CC with MeOH
and then purified with Sephadex LH-20 (CHCl3–MeOH, 1:1) to
yield 1 (15.6 mg). Fraction 20 (446.5 mg) was separated on Sephadex LH-20 with CHCl3–MeOH (1:1) to give six subfractions. Fraction 20–5 (16.7 mg) was purified using RP-HPLC (254 nm;
Ascentis C18, 250 x 21.2 mm) with MeOH-H2O (65:35) as eluent
to give 2 (1.8 mg, tR 46.3 min, flow rate 4 ml/min).
4.4. Spectroscopic data
4.4.1. Lawsochylin A (1)
Pale brown, amorphous powder; UV (MeOH) kmax (log e) nm:
283 (4.32), 298 (4.24); IR (neat) mmax cm-1: 3380, 2923, 2189,
1649, 1604, 1509, 1441; for 1H (400 MHz, CD3OD) and 13C NMR
data (100 MHz, CD3OD) see Table 1; ESIMS m/z 251 [M+H]+, 273
[M+Na]+; HRESIMS m/z 273.0894 (calcd for C17H14O2Na, 273.0891).
4.4.2. Lawsochylin B (2)
Pale brown, amorphous powder; UV (MeOH) kmax (log e) nm:
285 (3.92), 302 (3.82); IR (neat) mmax cm-1: 3358, 2919, 2194,
1648, 1597, 1512, 1240; for 1H (400 MHz, CD3OD) and 13C NMR
data (100 MHz, CD3OD) see Table 1; ESIMS m/z 267 [M+H]+, 289
[M+Na]+; HRESIMS m/z 289.0839 (calcd for C17H14O3Na, 289.0841).
71
Table 1
1
H and 13C NMR spectroscopic data for compounds 1–3.a
Position
Lawsochylin A (1)
dC
1
2
3
4
5
10
20
30
40
50
60
100
200
300
400
500
600
Lawsochylin B (2)
dH (mult., J in Hz)
94.8
85.2
110.3
142.4
36.6
115.6
133.9
116.5
159.0
116.5
133.9
132.0
130.4
116.3
156.7
116.3
130.4
5.71 (d, 1H, 10.4)
6.00 (dt, 1H, 10.4, 7.6)
3.58 (d, 2H, 7.6)
7.26 (d, 1H, 8.8)
6.75 (d, 1H, 8.8)
6.75 (d, 1H, 8.8)
7.26 (d, 1H, 8.8)
7.05 (d, 1H, 8.8)
6.71 (d, 1H, 8.8)
6.71 (d, 1H, 8.8)
7.05 (d, 1H, 8.8)
dC
Lawsochylin C (3)
dH (mult., J in Hz)
93.9
83.5
109.1
141.2
35.4
114.7
118.0
145.0
146.2
115.2
123.6
130.8
129.2
115.1
155.6
115.1
129.2
dC
5.70 (d, 1H, 10.4)
6.00 (dt, 1H, 10.4, 8.0)
3.58 (d, 2H, 8.0)
6.84 (d, 1H, 2.0)
6.71 (d, 1H, 8.0)
6.79 (dd, 1H, 8.0, 2.0)
7.05 (d, 1H, 8.8)
6.71 (d, 1H, 8.8)
6.71 (d, 1H, 8.8)
7.05 (d, 1H, 8.8)
C300 -OMe
a
dH (mult., J in Hz)
94.9
85.2
110.4
142.4
37.0
115.6
133.9
116.5
159.0
116.5
133.9
132.8
113.1
149.0
145.9
116.2
121.9
5.73 (d, 1H, 10.8)
6.05 (dt, 1H, 10.8, 7.2)
3.60 (d, 2H,7.2)
7.27 (d, 1H, 9.0)
6.75 (d, 1H, 9.0)
6.75 (d, 1H, 9.0)
7.27 (d, 1H, 9.0)
6.83 (d, 1H, 1.8)
6.71 (d, 1H, 7.8)
6.67 (dd, 1H, 7.8, 1.8)
56.3
3.81 (s, 3H)
NMR data (d) were measured in CD3OD at 400 MHz for 1 and 2, and in CD3OD at 600 MHz for 3.
Table 2
1
H and 13C NMR spectroscopic data for compounds 4–6.a
Position
Lawsonaphthoate A (4)
Lawsonaphthoate B (5)
Lawsonaphthoate C (6)
dC
dC
dC
dH (mult., J in Hz)
CD3OD
1
2
3
4
5
6
7
8
9
10
C1-OMe
C2-COOMe
C2-COOMe
C4-OMe
C5-OMe
C1-OH
a
157.0
102.8
101.8
148.0
105.4
160.0
118.8
126.7
120.6
133.4
172.8
52.6
56.0
C 5D 5N
CDCl3
C5D5N
6.99 (s, 1H)
7.15 (s, 1H)
7.00 (s, 1H)
7.41 (d, 1H, 2.4)
8.03 (d, 1H, 2.4)
7.48 (d, 1H, 2.4)
7.09 (dd, 1H, 9.2, 2.4)
8.19 (d, 1H, 9.2)
7.55 (dd, 1H, 8.8, 2.4)
8.60 (dd, 1H, 8.8, 0.4)
7.13 (dd, 1H, 9.2, 2.4)
8.30 (d, 1H, 9.2)
3.97 (s, 3H)
3.92 (s, 3H)
dH (mult., J in Hz)
3.85 (s, 3H)
3.81 (s, 3H)
3.98 (s, 3H)
3.95 (s, 3H)
12.20 (s, 1H)
11.64 (s, 1H)
153.4
116.0
104.7
150.4
105.3
159.4
120.3
126.3
123.8
131.5
63.3
166.8
51.9
55.6
7.38 (s, 1H)
8.05 (d, 1H, 2.4)
7.58 (dd, 1H, 9.6, 2.4)
8.33 (d, 1H, 9.6)
dH (mult., J in Hz)
CDCl3
156.0
102.5
103.4
146.6
140.1
150.3
116.6
121.9
121.6
124.5
7.07 (s, 1H)
7.27 (d, 1H, 8.4)
8.18 (d, 1H, 8.4)
4.08 (s, 3H)
3.91 (s, 3H)
3.82 (s, 3H)
171.1
52.2
56.5
62.8
3.99 (s, 3H)
3.96 (s, 3H)
3.89 (s, 3H)
11.62 (s, 1H)
NMR data (d) were measured in CD3OD, C5D5N and CDCl3 at 400 MHz for 4, and in C5D5N at 600 MHz for 5, and at CDCl3 at 600 MHz for 6.
4.4.3. Lawsochylin C (3)
Pale brown, amorphous powder; UV (MeOH) kmax (log e) nm:
283 (3.67), 300 (3.59); IR (neat) mmax cm-1: 3285, 2920, 2188,
1693, 1511, 1204; for 1H (600 MHz, CD3OD) and 13C NMR data
(150 MHz, CD3OD) see Table 1; ESIMS m/z 281 [M+H]+, 303
[M+Na]+; HRESIMS m/z 303.0995 (calcd for C18H16O3Na, 303.0997).
4.4.6. Lawsonaphthoate C (6)
White, amorphous powder; UV (EtOH) kmax (log e) nm: 262
(3.88), 358 (3.26); IR (neat) mmax cm-1: 3396, 2925, 1658, 1620,
1603, 1512, 1256; for 1H (600 MHz, CDCl3) and 13C NMR data
(150 MHz, CDCl3) see Table 2; ESIMS m/z 279 [M+H]+, 301
[M+Na]+; HRESIMS m/z 301.0690 (calcd for C14H14O6Na, 301.0688).
4.4.4. Lawsonaphthoate A (4)
White, amorphous powder; UV (EtOH) kmax (log e) nm: 271
(4.29), 352 (3.23); IR (neat) mmax cm-1: 3477, 2921, 1633, 1604,
1520, 1381; for 1H (400 MHz) and 13C NMR data (100 MHz) see
Table 2; ESIMS m/z 249 [M+H]+, 271 [M+Na]+; HRESIMS m/z
271.0584 (calcd for C13H12O5Na, 271.0582).
4.5. Biological assays
4.4.5. Lawsonaphthoate B (5)
White, amorphous powder; UV (EtOH) kmax (log e) nm: 261
(4.03), 319 (3.15); IR (neat) mmax cm-1: 3347, 2922, 1688, 1610,
1521, 1234; for 1H (600 MHz, C5D5N) and 13C NMR data
(150 MHz, C5D5N) see Table 2; ESIMS m/z 263 [M+H]+, 285
[M+Na]+; HRESIMS m/z 285.0737 (calcd for C14H14O5Na, 285.0739).
4.5.1. Preparation of human neutrophils
Blood was taken from healthy human donors (20–35 years old)
by venipuncture using a protocol approved by the institutional review board at Chang Gung Memorial Hospital. Neutrophils were
isolated using a standard method as previously described (Chung
et al., 2011).
4.5.2. Measurement of superoxide generation
SOD inhibition was measured by the reduction of ferricytochrome c as described previously (Chung et al., 2011). Neutrophils
in 0.5 mg/ml ferricytochrome c and 1 mM Ca2+ were equilibrated at
72
Table 3
Inhibitory effects of compounds from L. inermis on superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB.
IC50 (lg/ml)a
Compound
Lawsochylin A (1)
Lawsonaphthoate A (4)
Lawsonaphthoate B (5)
Lawsonaphthoate C (6)
Luteolin (7)
Luteolin 7-O-b-D-glucopyranoside (8)
Luteolin 40 -O-b-D-glucopyranoside (9)
Apigenin (10)
Apigenin 7-O-b-D-glucopyranoside (11)
Apigenin 40 -O-b-D-glucopyranoside (12)
Luteolin 7-O-rutinoside (13)
Diosmetin 7-O-rutinoside (14)
3-Amino-2-methoxycarbonyl-1,4-naphthoquinone (15)
(4S)-4-Hydroxy-a-tetralone (16)
3a,4a-Dihydroxy-a-tetralone (17)
9-Hydroxy-4-megastigmen-3-one (18)
(+)-Dihydrodehydrodiconiferyl alcohol (21)
4-Hydroxybenzaldehyde (22)
1,2,4-Trihydroxynaphthalene-1-O-b-D-glycopyranoside (25)
O-n-Butyl b-D-glucopyranoside (26)
D-Glucopyranose
(27)
D-Mannitol
(28)
Methyl pheophorbide b (29)
Oleamide (30)
2-Butoxysuccinic acid (31)
Genistein b
Superoxide anion
Elastase
1.80 ±
1.90 ±
2.07 ±
nd
0.75 ±
>10
>10
1.12 ±
>10
>10
>10
>10
7.13 ±
1.61 ±
>10
>10
>10
5.85 ±
2.86 ±
3.21 ±
>10
1.58 ±
3.17 ±
5.07 ±
6.18 ±
2.19 ±
nd
6.15 ±
3.61 ±
nd
>10
nd
>10
5.18 ±
1.62 ±
>10
>10
>10
>10
2.72 ±
>10
>10
0.22
0.56
0.37
0.25
0.19
0.42
0.44
0.13
0.45
0.34
0.43
0.83
0.49
0.34
0.22
2.36
0.10
0.23
0.65
0.11
>10
>10
6.28 ± 0.47
>10
1.78 ± 0.59
0.54 ± 0.11
8.22 ± 8.22
>10
2.36 ± 0.51
6.99 ± 1.62
nd: not determined (compound reacts with cytochrome c, and OD value is too high, so that no value can be detected).
a
Concentration necessary for 50% inhibition (IC50). Results are presented as mean ± SEM (n = 3–4).
b
Genistein was used as the positive control.
37 0C for 2 min and then incubated with the test compounds for
5 min. Cells were activated with formyl-methionyl-leucyl-phenylalanie (fMLP, 100 nM)/cytochalasin B (CB, 1 lg/ml) for 10 min.
The absorbance was continuously monitored at 550 nm in a double-beam, six-cell position spectrophotometer Hitachi U-3010
with constant stirring (Hitachi Inc., Tokyo, Japan). Calculations
were based on the differences in absorbance with and without
SOD (100 U/ml) divided by the extinction coefficient for the reduction of ferricytochrome c (e = 21.1/mM/10 mm).
4.5.3. Measurement of elastase release
Elastase release was measured by degranulation of azurophilic
granules (Chung et al., 2011). Neutrophils were equilibrated in
MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 lM), an elastase
substrate, at 37 0C for 2 min and then incubated with test compounds for 5 min. Cells were activated by 100 nM fMLP and
0.5 lg/ml CB, and changes in the absorbance at 405 nm were continuously monitored to monitor elastase release. The results are
expressed as the percent of the initial rate of elastase release in
the fMLP/CB-activated, drug-free control system.
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgments
This work was supported by grants from the Department of
Health, Executive Yuan, Taiwan (DOH100-TD-C-111-002) and National Science Council, Taiwan awarded to Dr. Y.C. Wu and Dr.
F.R. Chang.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
11.010.
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