Industrial Crops & Products 125 (2018) 454–461

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Industrial Crops & Products

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Anti-inflammatory diarylheptanoids and phenolics from the rhizomes of T

kencur (Kaempferia galanga L.)
⁎ ⁎
Fazhuang Yaoa,b, Yuying Huanga,b, Yihai Wanga,b, , Xiangjiu Hea,b,
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
b
Guangdong Engineering Research Center for Lead Compounds & Drug Discovery, Guangzhou 510006, China

A R T I C LE I N FO A B S T R A C T

Keywords: Kencur (Kaempferia galanga L.) is an aromatic plant of the Zingiberaceae family. It is widely cultured as a crops
Kencur and its rhizome has been widely used as a flavor spice existing in various cooking. In this study, three new
Kaempferia galanga L. diarylheptanoids (1–3), one new phenolic glycoside (4), together with thirteen known phenolics were isolated
Diarylheptanoids from the rhizomes of kencur. Their chemical structures were elucidated based on IR, 1D and 2D NMR, HR-ESI-
Anti-inflammatory
MS spectroscopic analysis and electronic circular dichroism (ECD) methods. Diarylheptanoids were evaluated for
their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in a macrophage RAW
264.7 cell line. Except for compound 7, most of diarylheptanoids exhibited pronounced inhibitory activities
compared with indomethacin. The results demonstrated that the diarylheptanoids may be partially responsible
for the anti-inflammatory activity of kencur, which suggested that it could be served as an anti-inflammatory
agent for healthy or medicinal products.

1. Introduction
Kencur, also called as aromatic ginger, sand ginger, is an aromatic
plant of Kaempferia galanga L. (Zingiberaceae family). It’s mainly dis-
tributed in the tropical and subtropical Asia including China, Myanmar,
Indonesia, Malaysia, and Thailand, and known as a popular condiment
in cuisine, but rarely known in the Western countries (Wang and Tang,
1980). It is widely cultivated in Southern China, as an industrial crop
sold in the market, and its rhizome has been used as a flavor spice
existing in various cooking. In traditional Chinese medicine, the rhi-
zome of kencur is prescribed for relieving pain and promoting digestion
(Chinese Pharmacopeia Commission, 2015). In folk medicine, it has
been extensively used for the treatment of hypertension, inflammations,
and tumors for a long history (He et al., 2012).
Over the past two decades, according to modern pharmacological
research, the rhizome of K. galanga has been reported possessed a lot of
biological activities, such as antiangiogenic effect (He et al., 2012),
anti-inflammation (Jagadish et al., 2016; Ridtitid et al., 2009; Umar
et al., 2012), anti-cholangiocarcinoma (Amuamuta et al., 2017), hypo-
pigmentary activity (Ko et al., 2014), and sedative activity (Huang
et al., 2008). Phytochemical studies revealed that it manly contained
volatile oil (about 2.5–4.0%), which included ethyl trans-p-methox-
ycinnamate, borneol, ethyl cinnamate, pentadecane, and some other
constituents of 1,8-cineole, 3-carene, (E)-cinnamaldehyde, eucalyptol,
kaempferol, methyl p-coumaric acid ethyl ester (Fan et al., 2005; Wong
et al., 1992). Ethyl p-methoxycinnamate isolated from this plant have
been considered to be responsible for most of the biological activities,
and its derivatives could be widely used in food, cosmetics, and medi-
cines for its sedative, insecticidal, and antibacterial activities (Pandji
et al., 1993; Umar et al., 2014). However, there were very few non-
volatile components reported from this medicinal spicy food.
Inflammation is a normal biological response to injury and infec-
tion, and involves the recruitment of the immune system to neutralize
invading pathogens, repair injured tissues, and promote wound healing.
Nitric oxide (NO) is a well-known endogenous cellular signaling mo-
lecule and served as an important mediator in many physiological
processes. However, pharmacological studies have demonstrated that
inflammation is related to overproduction of NO. During chronic or

Abbreviations: COSY, correlation spectroscopy; DEPT, distortionless enhancement by polarization transfer; EtOAc, ethyl acetate; EtOH, ethanol; GC, gas chroma-
tography; HPLC, high-performance liquid chromatography; HR-ESI-MS, high-resolution electrospray mass spectrum; HSQC, heteronuclear single quantum coherence;
LDH, lactate dehydrogenase; LPS, lipopolysaccharides; MTT, 3-45-dimethylthiazol-2-yl) -25-diphenyltetrazolium bromide; NMR, nuclear magnetic resonance; NO,
nitric oxide; NOESY, nuclear Overhauser enhancement spectroscopy; n-BuOH, n-butyl alcohol; ODS, octadecyl silane; Q-TOF-MS, quadrupole ion trap time of flight
mass; TFA, trifluoroacetic acid
⁎
Corresponding authors at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China.
E-mail addresses: wangyih88@163.com (Y. Wang), hexiangjiu@163.com (X. He).

https://doi.org/10.1016/j.indcrop.2018.09.026
Received 26 December 2017; Received in revised form 8 September 2018; Accepted 11 September 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
F. Yao et al.
excessive activation of the immune system, NO and pro-inflammatory
cytokines such as interleukin 6 and interleukin 1β are released, which
are implicated as promoter of the severity of a series of diseases such as
cancer, diabetes, and stroke. Therefore, the level of NO has been used as
an important marker of inflammation and diseases pathogenesis
(Baumann and Gauldie, 1994; Miranda et al., 2001).
As part of our ongoing program to screen bioactive components
from medicinal plants, fruits, vegetables, and foods, phytochemical
investigation on the rhizomes of K. galanga was carried out in this
study. As a result, 3 new diarylheptanoids, 1 new phenolic glycoside,
along with 13 known phenolics were isolated and characterization from
the rhizomes of K. galanga. Their inhibitory effects on NO production in
macrophage RAW 264.7 cells were also evaluated.
2. Materials and methods
2.1. General experimental procedures
Optical rotations were measured using a Jasco P-2000 polarimeter
(Jasco Inc., Tokyo, Japan). IR spectra were obtained on a Perkin Elmer
100 spectrometer (Perkin Elmer Inc., Waltham, MA, USA) with KBr
pellets. UV spectra were acquired on a Shimadzu UV-2550 spectro-
photometer (Shimadzu Inc., Kyoto, Japan). The ECD spectra were re-
corded on a Chirascan spectropolarimeter (Applied Photophysics Inc.,
Leatherhead, Surrey, UK). 1D, 2D NMR were recorded on Bruker
Avance III-500 NMR and Bruker Avance III-400 NMR spectrometer
(Bruker Inc., Falanden, Switzerland). HR-ESI-MS was recorded on a
Waters UPLC-Q-TOF-MS Ultima mass spectrometer (Waters Inc.,
Milford, MA, USA). Semi-preparative HPLC was applied with a
Shimadzu HPLC system, including a pump model LC-20AR and a UV
detector model SPD-20 A (Shimadzu Inc., Kyoto, Japan). Two Cosmosil
HPLC columns (5C18-MS-II, 10ID × 250 mm; 5C18-AR-II,
10ID × 250 mm; Nacalai Tesque, Kyoto, Japan) were applied in the
purification. Column chromatography (CC) was employed on silica gel
(200–300 mesh; Anhui Liangchen Silicon Material Co. Ltd., Lu’an,
China), ODS (40–60 μm, Merck, Darmstadt, Germany), and Sephadex
LH-20 (Pharmacia, Pittsburgh, PA, USA), respectively. Sugar reagents
(Sigma, St. Louis, MO, USA) were used for GC analysis. Gas chroma-
tography for sugar analysis was utilized on a Thermo Trace-1300 GC
(Thermo Inc., Waltham, MA, USA).
2.2. Plant material
The rhizomes of kencur were purchased from Qingping herbal
medicine market, Guangzhou on Oct. 2015 and were identified as
Kaempferia galanga L. (Zingiberaceae) by Prof. X. J. He of Guangdong
Pharmaceutical University. A voucher specimen (No.GDPU-NPR-
201510) was deposited in the Lead Compounds Laboratory, School of
Pharmacy, Guangdong Pharmaceutical University, China.
2.3. Extraction, isolation and purification
Dried rhizomes of kencur (20.0 kg) were refluxed with 75% EtOH
(3 × 55 L) for 2 h and filtered. Then, the ethanol solution was evapo-
rated under vacuum at 50 °C to get 15 L ethanol-free extract, and the
extract was suspended in 15 L distilled water and successively extracted
with cyclohexane, chloroform, ethyl acetate, and n-butanol to yield a
CHCl3-soluble fraction (172.5 g), an EtOAc-soluble fraction (44.6 g),
and an n-BuOH-soluble fraction (301.9 g), respectively. Compound 8
(12.9 g) was recrystallized in the CHCl3-soluble fraction (172.5 g), and
the rest of CHCl3 extract was fractioned by silica gel column chroma-
tography (CC, 200–300 mesh, 2.0 kg, 80 × 820 mm) and eluted with
CHCl3/MeOH (100:1 to 0:1, v/v) to yield 17 fractions (b1 to b17) on the
basis of TLC analysis. Fraction b8 (2.5 g) was further subjected to a
silica CC, Sephadex LH-20 (CHCl3/MeOH, 2:1, v/v) column chroma-
tography, and finally purified by the semi-preparative HPLC (C18
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Industrial Crops & Products 125 (2018) 454–461
column, 10 × 250 mm) using MeOH/H2O/TFA (30:70:0.1, v/v/v) to
get compounds 17 (9.4 mg, tR = 12.0 min) and 9 (12.9 mg,
tR = 21.5 min). The EtOAc extract (44.6 g) was chromatographed by a
silica gel column (200–300 mesh, 1.3 kg, 65 × 960 mm) eluted with a
gradient CHCl3/MeOH (100:1 to 0:1, v/v) to afford 11 fractions (c1 to
c11). Fraction c4 (4.8 g) was further subjected to an ODS MPLC and
eluted with MeOH/H2O (10:90 to 100:0, v/v) to obtain ten fractions
(c4-1 to c4-10). Fr. c4-3 (187.0 mg) was divided by the semi-pre-
parative HPLC (C18 column, 10 × 250 mm) with MeOH/H2O (26:74, v/
v) to yield compound 16 (45.4 mg, tR = 22.7 min) and Fr. c4-3-1
(50.8 mg), which was further purified using preparative TLC with
CHCl3/acetone (5:1, v/v) to yield compound 12 (23.6 mg). Fr. c4-5
(133.2 mg) was separated by the semi-preparative HPLC (C18 column,
10 × 250 mm) with MeOH/H2O (30:70, v/v) to yield compound 13
(10.7 mg, tR = 26.4 min). Compound 11 (27.1 mg) was recrystallized
from the fraction c4-7 (254.8 mg). Fr. c4-6 (648.4 mg) was isolated with
Sephadex LH-20 (CHCl3/MeOH, 2:1, v/v) column chromatography and
further purified by the semi-preparative HPLC (C18 column,
10 × 250 mm) with MeOH/H2O (35:65, v/v) to get compound 15
(5.2 mg, tR = 15.9 min). And compound 14 (40.0 mg) was also re-
crystallized from the fraction c5 (1.03 g). The mother solution was
further purified by the semi-preparative HPLC (C18 column,
10 × 250 mm) with MeOH/H2O/TFA (26:74:0.1, v/v/v) to afford
compound 10 (8.5 mg, tR = 31.0 min). Fraction c6 (5.1 g) was con-
secutively subjected to an ODS MPLC eluted with gradient MeOH/H2O
(10:90 to 100:0, v/v), using a Sephadex LH-20 (CHCl3/MeOH, 2:1, v/v)
column chromatography and was purified using a semi-preparative
HPLC (C18 column, 10 × 250 mm) with MeOH/H2O/TFA (35:65:0.1, v/
v/v) as the mobile phase to obtain compounds 1 (6.0 mg,
tR = 22.9 min), 2 (22.0 mg, tR = 16.4 min), 4 (19.7 mg, tR = 26.9 min),
5 (16.9 mg, tR = 23.7 min), 6 (8.5 mg, tR = 23.1 min), and 7 (3.7 mg,
tR = 15.4 min). Fraction c8 (2.8 g) was divided by an ODS MPLC
(MeOH/H2O, 10:90 to 70:30, v/v) to give eight subfractions. The
second subfraction (102.9 mg) was purified by the semi-preparative
HPLC (C18 column, 10 × 250 mm) with MeOH/H2O/TFA (35:65:0.1, v/
v/v) to yield compound 3 (11.6 mg, tR = 19.1 min).
2.3.1. (1R,3R,5R)-1,5-epoxy-3-hydroxy-1-(3,4-dihydroxyphenyl)-7-(3,4-
dihydroxyphenyl) heptane (1)
Yellow oil (MeOH); [α]29D +26.0 (c 0.5, MeOH); IR νmax (KBr)
cm−1: 3200 (OH), 2925 (CH2), 1717, 1521, 1231, 995, 495; UV
(MeOH) λmax 284 nm; 1H and 13C NMR data see Table 1; HR-ESI-Q-
TOF-MS m/z [M – H]– 345.1341 (calcd for C19H21O6 [M – H]–,
345.1338).
2.3.2. 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)heptane-
1,2,3,5,6-pentaol (2)
Yellow oil (MeOH); [α]29D −40.8 (c 0.6, MeOH); IR νmax (KBr)
cm−1: 3240 (OH), 2928 (CH2), 1681, 1601, 1515, 1452, 1383, 1260,
1127, 1034, 821, 555; UV (MeOH) λmax 225, 279 nm; 1H and 13C NMR
data see Table 1; HR-ESI-Q-TOF-MS m/z [M – H2O – H]– 375.1430
(calcd for C20H23O7 [M – H2O – H]–, 375.1444).
2.3.3. (1R,3R,5R)-1,5-epoxy-3-hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-
hydroxyphenyl) heptane 3-O-β-D-glucopyranoside (3)
Yellow oil (MeOH); [α]29D −53.3 (c 0.5, MeOH); IR νmax (KBr)
cm−1: 3189 (OH), 2926 (CH2), 2803, 2382, 1724, 1515, 1460, 1400,
1227, 996, 498; UV (MeOH) λmax 255, 278 nm; 1H and 13C NMR data
see Table 1; HR-ESI-Q-TOF-MS m/z [M – H]– 491.1936 (calcd for
C25H31O10 [M – H]–, 491.1917).
2.3.4. 1-O-4-carbonxylphenyl-(6-O-4-hydroxybenzoyl)-β-D-
glucopyranoside (4)
Brown amorphous powder (MeOH); [α]29 D −65.3 (c 0.5, MeOH); IR
νmax (KBr) cm−1: 3408 (OH), 2925 (CH2), 2804, 1706, 1606, 1513,
1454, 1394, 1276, 1242, 1167, 1071, 858, 778, 699, 523; UV (MeOH)
F. Yao et al. Industrial Crops & Products 125 (2018) 454–461

Table 1 RAW 264.7 cells were seeded at a density of 2 × 104 cells per well in a
1
H and 13C NMR data for compounds of 1–3.a,b 96-well plate, grown for 24 h, then stimulated with 1 μg/mL of LPS in
Position 1 2 3 the presence or absence of test samples. After incubation at 37 °C for
24 h, 50 μL of cell-free supernatant was mixed with 100 μL of Griess
δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC reagent. The optical density was measured at the wavelength of 546 nm
using a microplate reader. The concentration of NO in the medium was
1 4.52, d 72.8 5.09, d 81.0 4.61, d (11.3) 72.5
(10.9) (4.2) measured by assaying the levels of NO2− via the Griess reaction using
2 1.68, mc/ 40.3 3.28, mc 58.4 1.43, t (11.6) 36.5 the reported protocol (Feng et al., 2016). The IC50 was determined and
1.53, mc the values were reported as mean ± SD of three independent experi-
3 4.07, s 62.9 4.67, m 79.8 4.13, s 70.6 ments.
4 1.57, mc/ 38.2 2.03, m/ 36.6 1.78, d (12.9)/ 36.5
In order to find whether inhibition of NO production was due to the
1.37, m 1.81, m 1.92, d (13.4)
5 3.77, m 70.5 3.27, mc 72.4 3.80, m 71.0 cytotoxicity of the tested compounds, cell viability was determined
6 1.64, mc/ 38.0 3.44, m 76.1 1.62, m 38.0 using the MTT method and the lactate dehydrogenase (LDH) activity in
1.56, mc the medium was also measured using an LDH cytotoxicity assay kit
7 2.48, mc 30.4 2.53, m 40.0 2.57, m/2.49, mc 30.0
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according
1' 134.6 135.8 134.5
2' 6.74, s d
113.6 6.96, m c
111.7 6.75, s d
113.6
to the manufacturer’s instructions.
3' 144.9 149.0 144.8
4' 144.2 147.3 144.1 2.6. Statistical analysis
5' 6.66, d (8.0) 115.2 6.80, d 116.3 6.64, mc 115.0
(6.5)
Data were presented as mean ± SD. Analyses were performed
6' 6.55, mc 116.8 6.83, dd 120.8 6.55, d (8.3) 116.8
(1.3, 1.3) using the Graphpad Prism 5 software with one-way ANOVA followed by
1” 132.8 131.3 132.3 Dunnett’s test. Difference with p value < 0.05 was considered to be
2” 6.61, d (8.0) 115.7 6.95, mc 131.3 6.96, d (8.3) 129.1 significant.
3” 145.1 6.67, d 116.0 6.64, mc 115.0
(6.7)
4” 143.1 156.5 155.2
3. Results and discussion
5” 6.55, mc 115.5 6.67, d 116.0 6.64, mc 115.0
(6.7) 3.1. Structural elucidation
6” 6.42, dd 118.9 6.95, mc 131.3 6.96, d (8.3) 129.1
(1.6, 1.7)
Kencur is widely cultivated in Southern China, as an industrial crop
3'-OCH3 3.81, s 56.5
Glc sold in the market. Its rhizome has been used as a flavor spice existing
1 4.24, d (7.8) 101.2 in various cooking, and also used in traditional Chinese medicine for
2 2.98, mc 76.7 relieving pain and digestion. In the process of our study, the chemical
3 3.04, mc 73.4
components and pharmacological activity of kencur were performed.
4 2.98, mc 70.0
5 3.15, mc 76.8
The 75% ethanol extract of the dried rhizomes was partitioned by the
6 3.47, mc/3.65, mc 61.0 various solvents, and these fractions were isolated by the normal-phase
silica gel, ODS MPLC, Sephadex LH-20 and preparative HPLC to yield
a
The 1H NMR data were obtained in DMSO at 400 MHz, 500 MHz for 1, 3, 17 compounds. The chemical structures of all the isolated compounds
respectively. 1H NMR data was obtained in CD3OD at 500 MHz for 2. were elucidated according to the various spectroscopic analysis and
b 13
C NMR were recorded at 100 MHz, 125 MHz and 125 MHz for 1, 2, and chemical methods, including IR, 1D and 2D NMR, MS, GC analyses. The
3.
c chemical structure of compounds 1–17 are shown in Fig. 1.
Overlapped signals, those were assigned by HSQC, 1H-1H COSY, HMBC and
Compound 1 was obtained as an optically active yellow oil, [α]29 D
DEPT experiments.
d
Pseudo-singlet. +26.0 (c 0.5, MeOH). Its molecular formula was determined as
C19H22O6 on the basis of the negative ion HR-ESI-MS peak at m/z
λmax 201, 250 nm; 1H and 13C NMR data see Table 1; HR-ESI-Q-TOF-MS 345.1347 [M–H]– (calcd for C19H21O6, 345.1338) and supported by the
m/z [M – H]– 419.0974 (calcd for C20H19O10 [M – H]–, 419.0978). hydrogen and carbon atom counts in the NMR spectra. The 1H NMR
spectrum (Table 1) of 1 displayed two aromatic hydrogen signals of
typical ABX coupling system of benzene ring (δH 6.55, 1H, ov; 6.66, 1H,
2.4. Acid hydrolysis and GC analysis of sugars d, J = 8.0 Hz; 6.74, s; δH 6.61, 1H, d, J = 8.0 Hz; 6.55, 1H, ov; 6.42,
1H, dd, J = 1.6 Hz, 1.7 Hz). The 13C NMR spectrum (Table 1) of 1 ex-
The hydrolysis and GC analysis of the chiral derivatives of the su- hibited 19 carbon resonances corresponding to three oxygenated car-
gars of compounds 3 and 4 were performed as previously described in bons (δC 72.7, 70.4, 63.0) and four oxygenated olefinic carbons (δC
our lab (Xiang et al., 2016). Each compound (1–2 mg) was dissolved in 145.0, 144.9, 144.2, 143.1). The above NMR spectroscopic data sug-
5 mL 2 M HCl and heated to 90 °C in an ampule. The mixture was ex- gesting it could be a diarylheptanoids structure. In addition, 1H and 13C
tracted with EtOAc, and the solvent was removed in vacuum at 60 °C to NMR spectrum of 1 was similar to phaeoheptanoxide (Ma et al., 2015),
get a residue. After mixing with 600 μL pyridine and 5 mg NH2OH·HCl a cyclic diarylheptanoids isolated from the rhizomes of Curcuma
to end chemical reactions, the residue was heated at 90 °C for 30 min. phaeocaulis, except for the disappearance of one phenolic hydroxyl. The
Finally, Ac2O was added to synthesize acetylated aldononitrile per- molecular formula of C19H22O6 inferred the carbon skeleton required 9
acetates. These samples were analyzed by GC using standard aldono- indices of hydrogen deficiency. The DEPT spectrum of 1 revealed the
nitrile peracetates as references. existences of three methines and four methylenes. Meanwhile, the
1
H-1H COSY (Fig. 2) correlations between H-1/H-2, H-2/H-3, H-3/H-4,
2.5. Nitric oxide inhibitory (NO) and cytotoxicity assay H-4/H-5, H-5/H-6, H-6/H-7 and the correlations between H-3/3-OH,
indirectly suggested the CeC connectivity of the heptane moiety. The
The Griess reaction was used to evaluate the NO inhibitory activity structure of 1 was further demonstrated by analysis of HMBC spectrum
as previously literature described (Cheng et al., 2017). The purified (Fig. 2). The HMBC correlations from H-1 to C-3, C-1′, C-2′, C-6′ in-
compounds were tested on RAW 264.7 macrophage cells, which served dicated one phenyl moiety attached to C-1 of the tetrahydropyran
as inflammatory model stimulated by LPS for releasing NO. Basically, partial structure on the heptane moiety, and from H-3 to C-1, C-5, and

456
F. Yao et al.
Fig. 1. Chemical structures of compounds 1–17.
from H-7 to C-5, C-6, C-1″, C-2″, C-6″ suggested another phenethyl
moiety attached to C-6 of the tetrahydropyran skeleton. Consequently,
the four phenolic hydroxyls were assigned to C-3′, C-4′ and C-3″, C-4″,
which is supported by their chemical shifts and the molecular formula.
For the relative configuration of the tetrahydropyran ring having
chair conformation, H-l [δ 4.52, (d, J = 10.9 Hz)], H-5 (δ 3.77, m) and
the hydroxyl group at C-3 were determined to be all axially oriented by
the means of the NOESY contacts (Fig. 3) and coupling constants (Jiang
et al., 1996). The ECD spectrum (Fig. 4) of 1 displayed a negative
Cotton effect (CE) at 230 nm, similar to those of adrenaline and nya-
sicoside (Chang et al., 1999; Lin et al., 2015; Yin et al., 2013), thus
allowing the assignment of a (1R)-configuration for compound 1. On
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Industrial Crops & Products 125 (2018) 454–461
the basis of the NOESY correlations, the absolute configurations of C-1,
C-3 and C-5 were determined to be R, R and R, respectively. Thus, the
structure of 1 was elucidated as (1R,3R,5R)-1,5-epoxy-3-hydroxy-1-
(3,4-dihydroxyphenyl)-7-(3,4-dihydroxyphenyl) heptane. The 1H and
13
C NMR data of 1 were given in Table 1.
Compound 2 was obtained as an optically active yellow oil, [α]29 D
−40.8 (c 0.6, MeOH). Its HR-ESI-MS data gave a molecular formula of
C20H26O8 based on the presence of a peak at m/z 375.1430
[M–H2O–H]– (calcd for C20H23O7, 375.1444). The 1H NMR spectrum
(Table 1) exhibited a 1,4-bissubstituted (δH 6.95, 2H, ov; 6.67, 2H, d,
J = 6.7 Hz) and a 1,3,4-trisubstituted (δH 6.96, 1H, ov; 6.83, 1H, dd,
J = 1.3 Hz, 1.3 Hz; 6.80, 1H, d, J = 6.5 Hz) benzene rings. The 13C
F. Yao et al.
Fig. 2. Selected key HMBC and 1H–1H COSY correlations of compounds 1–4.
Fig. 3. Key NOESY connectivity for compound 1.
NMR spectroscopic data (Table 1) of compound 2 were similar to 1
except that presence of methoxyl in 2, suggesting that is also a dia-
rylheptanoids structure (Fig. 1). The 13C NMR and DEPT spectra of 2
exhibited a total of 20 carbon signals, including five hydroxymethine
groups, two methylene, one methoxyl and twelve aromatic carbons.
Two aromatic rings accounted for 8 degrees of unsaturation, therefore,
2 was deduced as a linear-diarylheptanoids. The 1H-1H COSY spectra
(Fig. 2) showed the connectivity among H-1, H-2, H-3, H-4, H-5, H-6,
Fig. 4. Experimental ECD spectra of 1 and 3 in MeOH.
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Industrial Crops & Products 125 (2018) 454–461
and H-7. Additionally, the HMBC correlation of H-1 to C-1′/C-2′/C-6′;
and H-7 to C-1″/C-2″/C-6″ established the skeleton of the linear-dia-
rylheptanoids. The location of methoxyl group on the aromatic ring was
determined by the HMBC correlation of C-3′ with 3′-OCH3. Based on the
above evidence, the structure of 2 was determined to be 1-(4-hydroxy-
3-methoxyphenyl)-7-(4-hydroxyphenyl)heptane-1,2,3,5,6-pentaol,
namely galanheptanoxide. The 1H and 13C NMR data of 2 were given in
Table 1.
Compound 3 was obtained as an optically active yellow oil, [α]29 D
–53.3 (c 0.5, MeOH). Its molecular formula of C25H32O10 was de-
termined on the basis of HR-ESI-MS the appearance of an ion peak at m/
z 491.1936 [M–H]– (calcd for C25H31O10, 491.1917), which could be
further deduced via its NMR spectrum. The 1H and 13C NMR spectra
(Table 1) of 3 were very similar to those of 1, except for the dis-
appearance of one phenolic hydroxyl and the presence of one glucosyl
moiety in the molecule of 3 (Fig. 1). The 13C NMR spectra showed 25
carbon resonances, including a diarylheptanoid and six carbons of one
F. Yao et al. Industrial Crops & Products 125 (2018) 454–461

glucosyl moiety. In the HMBC spectrum (Fig. 2), a long-range correla- Table 3
tion was observed from the anomeric proton signal (δH 4.24, 1H, d, Inhibitory effect of the diarylheptanoids isolated from the rhizomes of kencur
J = 7.8 Hz) of the D-glucose to C-3 (δC 70.6) of the tetrahydropyran on NO production induced by LPS in RAW 264.7.
ring moiety. The locations of three phenolic hydroxyl groups were de- Samples IC50 (μM)a
termined by the chemical shift of the 13C NMR and spectroscopic data
of DEPT. The NOESY spectrum showed the mutual correlations between NO inhibitory assay MTT assay
the H-1 and H-5, and to be axially oriented. The ECD spectrum of 3
1 27.85 ± 1.52 > 100
(Fig. 4) showed a negative Cotton effect at 228 nm, similar that of 2 46.98 ± 3.49** > 100
compound 1, thus the absolute configuration of C-1 in 3 was de- 3 79.89 ± 6.11 > 100
termined to be R. Additionally, the NOESY correlations between H-3, H- 4 > 100 > 100
5 26.98 ± 1.39* > 100
4ax, H-4eq and H-2 were observed. Identification of D-glucose, including
6 17.26 ± 1.78** > 100
its absolute configuration, was carried out by acid hydrolysis followed 7 > 100 > 100
by a GC analysis of chiral derivatives after acid hydrolysis in the ex- Cyclohexane extract 20.46 ± 0.92 c
> 100
c
perimental section (Feng et al., 2018). Hence, the combined data es- Chloroform extract 16.28 ± 2.86 > 100
c
tablished the structure of 3 as (1R,3R,5R)-1,5-epoxy-3-hydroxyl-1-(3,4- Ethyl acetate extract 26.38 ± 0.63 > 100
c
n-Butanol extract 49.51 ± 1.15 > 100
dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane 3-O-β-D- glucopyrano-
Indomethacin b 32.26 ± 1.76 > 100
side. The 1H and 13C NMR data of 3 were given in Table 1.
Compound 4 was obtained as a brown amorphous powder. Its HR- a
Results are presented as mean value ± SD, n = 3.
ESI-MS spectrum showed a negative ion peak at m/z 419.0974 [M–H]–, b
Indomethacin was used as positive control.
supporting a molecular formula of C20H20O10 (calcd for C20H19O10, c
The concentration units are μg/mL.
419.0978). The 1H NMR spectrum (Table 2) of 4 in DMSO-d6 displayed * p < 0.05 compared with positive control.
two pairs of characteristic aromatic proton signals for 1,4-trisubstituted ** p < 0.01 compared with positive control.
benzene at δH 7.82 (4H, d, J = 8.1 Hz), 7.10 (2H, d, J = 8.3 Hz), and
6.85 (2H, d, J = 8.2 Hz). The signal of a sugar unit at δH 5.07 (1H, d, to the glucose C-1′ and C-6′, respectively. The D-configuration of the
J = 6.5 Hz) with the six carbonyl signals at δC 99.5, 76.5, 74.0, 73.1, glucopyranosyl moiety was determined by acid hydrolysis followed by
70.1, and 63.7 indicated the presence of glucose (Zhang et al., 2017). a GC analysis of chiral derivatives after acid hydrolysis. Its anomeric
The 13C NMR and HSQC revealed the presence of 20 carbon resonances, proton at δH 5.07 (1H, d, J = 6.5 Hz) indicated β-configuration for the
and showed the signal at δC 166.9 and 165.4, two chemical shifts at- anomeric proton. Thus, compound 4 was determined as 1-O-4-carbon-
tributable to the carbonyl group, respectively. The 1H and 13C NMR xylphenyl-(6-O-4-hydroxybenzoyl)-β-D-glucopyranoside. The 1H and
13
spectra were similar to those of phenolic glycosides, and indicated the C NMR data of 4 were given in Table 2.
presence of a glucose, and two p-hydroxybenzoyl moieties (Hu et al., By comparing spectroscopic data with those reported in the litera-
2015). Analyses of the 1D and 2D NMR including HSQC and HMBC data tures, thirteen known compounds were determined as (3R,5S)-3,5-di-
assigned for the establishment of the structure of 4. The HSQC spectrum hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane (5)
permitted the assignment of all the protons to their bonding carbons. (Yokosuka et al., 2002), phaeoheptanoxide (6) (Ma et al., 2015), he-
The HMBC correlations (Fig. 2) from H-1′ [5.07 (1H, d, J = 6.5 Hz)] to dycoropyran B (7) (Lin et al., 2015), ethyl trans-p-methoxycinnamate
C-4; from H-6′ [4.57 (1H, d, J = 11.6 Hz), 4.12 (1H, dd, J = 7.6 Hz, (8) (Yokosuka et al., 2002), ferulic acid (9) (Zhou et al., 2015), trans-p-
11.1 Hz)] to C-4′ and C-7″; from H-6″ (or H-2″) [7.82 (2H, d, hydroxy-cinnamic acid (10) (Zhou and Li, 2006), trans-p-methox-
J = 8.1 Hz)] to C-7″, which indicated that C-4 and C-7″ were attached ycinnamic acid (11) (Sobolev et al., 2006), methyl (2R,3S)-2,3-dihy-
droxy-3-(4-methoxyphenyl)propanoate (12) (Borah et al., 2012), ethyl
(2R,3S)-2,3-dihydroxy-3-(4-methoxyphenyl)propanoate (13) (Borah
Table 2 et al., 2012), p-hydroxybenzoic acid (14) (Wang et al., 2012), p-meth-
1
H and 13C NMR spectroscopic data for compound 4 in DMSO-d6. oxybenzoic acid (15) (Feng et al., 2005), vanillic acid (16) (Xiao et al.,
4 2016), methyl 3,4-dihydroxybenzoate (17) (Chen et al., 2016), re-
spectively.
Position δH a(J in Hz) δCb

1 124.3 3.2. Inhibitory effects on NO production

2 7.82, d (8.1) 131.5
3 7.10, d (8.3) 115.8 In this study, the anti-inflammatory effects of compounds 1–7 were
4 160.6
5 7.10, d (8.3) 115.8
evaluated by measuring the inhibitory effect on LPS-induced NO pro-
6 7.82, d (8.1) 131.5 duction in RAW 264.7 macrophage cells. MTT reduction and LDH
1' 5.07, d (6.5) 99.5 leakage assays were performed in order to evaluate the cytotoxicity of
2' 3.34, mc 76.5 the tested compounds against the cells. As shown in Table 3 and Fig. 5,
3' 3.34, mc 73.1
all the tested compounds (100 μM) and the extract (100 μg/mL) per-
4' 3.27, mc 70.1
5' 3.80, t (8.5) 74.0 formed no cytotoxicity at the highest tested concentration. In-
6' 4.57, d, 4.12, dd (11.6, 11.1) 63.7 domethacin (IC50 = 32.26 ± 1.76 μM) was used as the positive con-
1” 120.3 trol. The cyclohexane extract, chloroform extract and ethyl acetate
2” 7.82, d (8.1) 131.1 extract showed significant inhibition on production of NO with IC50
3” 6.85, d (8.2) 115.3
4” 162.1
values ranging from 16.28 ± 2.86 to 26.38 ± 0.63 μg/mL, compared
5” 6.85, d (8.2) 115.3 with the positive control. Except for compound 7, most of the isolated
6” 7.82, d (8.1) 131.1 diarylheptanoids exhibited potential inhibitory effects on NO produc-
7” 165.4 tion in dose-dependent manner, compared with indomethacin (Fig. 6).
1-COOH 12.62, br s 166.9
The phenolic glycoside 4 was inactive (IC50 > 100 μM). Compared
1”-OH 10.42, s
with 1 (IC50 = 27.85 ± 1.52 μM), compound 3 exhibited weak in-
a
400 MHz for 1H NMR. hibitory activity (IC50 = 79.89 ± 6.11 μM) suggesting that glycosyla-
b
100 MHz for 13C NMR. tion at C-3 may decrease the inhibitory activity. Hydroxylation at the
c
Overlapped signals. heptane chain and methyl etherification of the phenolic hydroxyl of

459
F. Yao et al.
Fig. 6. Inhibitory effect of compounds 1, 2, 3, 5, and 6 on the LPS-induced NO production in RAW 264.7 macrophages.
Data are expressed as the mean ± SD (n = 3). ∗∗ p < 0.01 compared with the LPS-induced group.
compound 1 led to significant decrease of the activity in compound 7
(IC50 > 100 μM). The similar rule was also observed in compounds 2
(IC50 = 46.98 ± 3.49) and 5 (IC50 = 26.98 ± 1.39 μM). The struc-
ture-activity relationships provided useful clues for understanding the
NO inhibitory activity of diarylheptanoids.
Diarylheptanoids initially isolated in 1815, is a group of compounds
which firmly bears the 1,7-diarylheptane skeleton as special char-
acteristics in natural product estate (Claeson et al., 1995). As found in
previous pharmacological studies, diarylheptanoids possess a variety of
biological and pharmacological activities, including cytotoxic activity
(Per et al., 2002; Yokosuka et al., 2002), anti-proliferative effect (Chen
et al., 2015), anti-allergic activity (Matsumoto et al., 2015), anti-oxi-
dative activity (Tao et al., 2008), and anti-inflammatory activity (Li
et al., 2011). Curcumin a well known diarylheptanoids, has attracted
extensive attention for its wide-ranging biological activities, such an-
tioxidant, anti-inflammatory, and chemopreventive activities. It is
widely used as an herbal supplement, cosmetics ingredient, food fla-
voring or coloring in industry. Results of this study showed that the
diarylheptanoids isolated from kencur have potent NO production in-
hibitory activity and may be partially responsible for their anti-in-
flammatory activities. These provided a scientific basis for further de-
velopment and utilization of kencur as a natural healthy and functional
condiment in food industry, as well as a valuable source of natural anti-
inflammation agent in pharmaceutical industry. The mechanisms of
action of those diarylheptanoids are worthy of further investigation.
4. Conclusion
In conclusion, 17 compounds, including 4 new compounds were
isolated and identified from the rhizomes of kencur. The isolated dia-
rylheptanoids, 1, 2, 5, and 6, exerted potent NO inhibitory activities
(IC50 17 to 46 μM) in RAW 264.7 cells. These results indicate that
diarylheptanoids from kencur may be potential candidates for the
treatment of diseases associated with inflammation. Meanwhile, this
460
Industrial Crops & Products 125 (2018) 454–461
Fig. 5. Effect of the compounds 1–7 and the extract on MTT re-
duction and LDH leakage of RAW 264.7 macrophages.
The compounds were tested at the concentration of 100 μM and the
extract was tested at the concentration of 100 μg/mL. Data are
expressed as the mean ± SD (n = 3). ∗ p < 0.05 and ∗∗ p <
0.01 compared with the LPS-induced group.
study provided a scientific basis for further development and utilization
of kencur as a natural healthy and functional condiment in food in-
dustry, as well as a valuable source of natural anti-inflammation agent
in pharmaceutical industry.
Conflict of interest
The authors declare that there is no conflict of interest for this
manuscript.
Acknowledgments
This study was supported by Guangdong Province Universities and
Colleges Pearl River Scholar Funded Scheme (2017), and the project
(2015A020211027) of Guangdong Provincial of Science and
Technology Department.
Appendix A. Supplementary data
Supplementary data associated with this article can be found. The
data include 1D and 2D NMR, UV, ECD, IR and HR-ESI-MS spectra of
compounds 1–4. The supporting information is available free of charge
in the online version.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2018.09.026.
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