2018

NPC Natural Product Communications Vol. 13

No. 7
Identification of the Chemical Constituents in Ginger 869 - 873
(Zingiber officinale) Responsible for Thermogenesis
Yuto Nishidonoa, Azis Saifudinb, Mikio Nishizawac, Takashi Fujitaa, Masatoshi Nakamotoa and Ken Tanakaa,*
a
College of Pharmaceutical Science, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan
b
Faculty of Pharmacy, Universitas Muhammadiyah Surakarta, Pabelan, KTS Solo, Jawa Tengah 57102,
Indonesia
c
Graduate School of Life Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan

ktanaka@fc.ritsumei.ac.jp

Received: April 20th, 2018; Accepted: May 2nd, 2018

To compare the thermogenic properties of crude drugs derived from ginger, the activities to peroxisome proliferator-activated receptor gamma coactivator 1-
alpha (PGC-1α) of methanol extracts of “Shokyo” (dried rhizome of Z. officinale var. rubens), “Kankyo” (steamed and dried rhizome of Z. officinale var.
rubens), “Red ginger” (Indonesian dried rhizome of Z. officinale var. rubrum) and “White ginger” (Indonesian dried rhizome of Z. officinale var. amarum),
were examined. The extracts of the four specimens were analyzed by liquid chromatography mass spectrometry (LC-MS). The results showed that “Shokyo”
and “White ginger” strongly stimulated PGC-1α and that the amount of [10]-shogaol (6) in these was higher than in “Kankyo” and “Red ginger”. Gingerol-
related compounds were isolated or prepared in order to identify the compounds responsible for stimulating PGC-1α. As a result, [10]-gingerol (3), [10]-
shogaol (6), [10]-gingerdiols (11, 12) and [10]-gingerdiols 3,5-diacetate (17, 18) were identified as the active constituents, while the main constituents, [6]-
gingerol (1) and [6]-shogaol (4), did not show any significant PGC-1α activity. These results suggest that gingerol-related compounds with long alkyl side
chains contribute to the thermogenic properties of ginger.

Keywords: Zingiber officinale, PGC-1α, Thermogenesis, [10]-Shogaol.

Zingiber officinale is a perennial plant of the Zingiberaceae family
and is widely distributed throughout the tropical and subtropical
regions of Asia, Far East Asia and Africa. Its rhizomes, commonly
known as ginger, have a pungent flavor and it has been used as a
spice for at least 2000 years [1a-1c]. It has also been used as
medicine for the treatment of catarrh, rheumatism, nervous diseases,
gingivitis, toothache, asthma, stroke, constipation and diabetes in
the traditional medicines of many countries [2a].
Diarylheptanoids, sesquiterpenes and phenolic compounds have
been identified as the major constituents in ginger [2a-2d]. There
have been several studies indicating that gingerols ([6]-, [8]- and
[10]-gingerol) and shogaols ([6]-, [8]- and [10]-shogaol) have a
number of different beneficial effects. These include
anticarcinogenic, antioxidative, antimicrobial, anti-inflammatory,
antidiabetic, antiobesity, anti-gastric ulcer and antiallergenic effects.
In addition, it has also been reported that the bioactivity of shogaols
is stronger than that of gingerols [2a, 2e].
In many traditional medicines, ginger is used to warm parts of the
body and release the exterior. Several studies on the thermogenic
properties of ginger have been reported. Iwami and Fujiwara
reported that ginger increased the oxygen consumption in rats and
raised the surface temperature of the forehead in humans [3a, 3b].
It is believed that thermogenesis induced by the administration of
ginger is caused by activation of the transient receptor potential
vanilloid subtype 1 (TRPV1) channel, and that [6]-gingerol and [6]-
shogaol are responsible for activating this [4a-4c]. TRPV1
activation improves energy metabolism by upregulating the
peroxisome proliferator-activated receptor gamma coactivator 1-
alpha (PGC-1α) in skeletal muscles [5]. PGC-1α is a coactivator of
the peroxisome proliferator-activated receptor γ (PPARγ) and plays
an important role in thermogenesis through the uncoupling of
oxidative phosphorylation [6a-6c]. The effects of ginger on PPARγ
have been reported [7], but studies on the effects of ginger and/or its
chemical constituents on PGC-1α activity have not yet been
reported.
In this study, we evaluated the effects of the methanol extracts of
four crude drugs, derived from “Shokyo” (dried rhizome of Z.
officinale var. rubens), “Kankyo” (steamed and dried rhizome of Z.
officinale var. rubens), “Red ginger” (Indonesian dried rhizome of
Z. officinale var. rubrum) and “White ginger” (Indonesian dried
rhizome of Z. officinale var. amarum), on the PGC-1α activity. The
four extracts were analyzed by LC-MS and the chemical
constituents identified. The gingerol-related compounds (Figure 1)
were isolated or prepared to identify the compounds responsible for
thermogenesis.
The PGC-1α activity of the methanol extracts of “Shokyo”,
“Kankyo”, “Red ginger” and “White ginger” are shown in Figure 2.
“Shokyo” and “White ginger” strongly stimulated the PGC-1α,
while no activity was apparent for “Kankyo” and “Red ginger”.
Several studies done so far indicate that the major constituents of
ginger, [6]-gingerol and [6]-shogaol, are responsible for the various
effects. In addition, it has also been reported that [6]-shogaol shows
stronger bioactivity than [6]-gingerol [2a]. [6]-shogaol is the
dehydrated product of [6]-gingerol, and [6]-gingerol is easily
transformed to [6]-shogaol during the heating process [8].
Therefore, it is suggested that “Kankyo” has stronger thermogenic
properties than “Shokyo”. However, the present results were not
consistent with previous findings.
To identify the active constituents, the chemical constituents of the
four methanol extracts were analyzed by LC-MS. Figure 3 shows
total ion chromatograms (TIC) with both positive and negative ion
scans and mass chromatograms to monitor the [M˗H]˗ ions of the
gingerols (1-3), shogaols (4-6), and gingerdiols (7-12), as well the
870 Natural Product Communications Vol. 13 (7) 2018 Nishidono et al.

(3S,5S)-[6]-Gingerdiol (7) : R=H

[6]-Gingerdiol 3S,5S-diacetate (13) : R=Ac
[6]-Gingerol (1)

(3R,5S)-[6]-Gingerdiol (8) : R=H

[6]-Gingerdiol 3R,5S-diacetate (14) : R=Ac
[8]-Gingerol (2)

(3S,5S)-[8]-Gingerdiol (9) : R=H

[8]-Gingerdiol 3S,5S-diacetate (15) : R=Ac
[10]-Gingerol (3)

(3R,5S)-[8]-Gingerdiol (10) : R=H

[8]-Gingerdiol 3R,5S-diacetate (16) : R=Ac
[6]-Shogaol (4)

(3S,5S)-[10]-Gingerdiol (11) : R=H

[10]-Gingerdiol 3S,5S-diacetate (17) : R=Ac
[8]-Shogaol (5)

(3R,5S)-[10]-Gingerdiol (12) : R=H

[10]-Gingerdiol 3R,5S-diacetate (18) : R=Ac
[10]-Shogaol (6)
Figure 1: Structures of gingerol-related compounds in ginger.

3 study, a systematic analysis of gingerol-related compounds (1-18)

was performed. Regarding the PGC-1α activity, “Shokyo” and
PGC-1α Activity Fold Induction

2.5 *** “White ginger” showed strong stimulation, while “Kankyo” and
** “Red ginger” showed none. From a comparison of the amounts of
2 gingerol-related compounds, it is considered that [10]-shogaol (6)
contributes to the PGC-1α activity.
1.5 * *
Table 1: Mass spectrometric characterization and retention times (RT) of gingerol-
1 related compounds in ginger detected by LC-MS.
RT (min) Compound Formula Abundant Ions Measured Calculated
-
20.71 [6]-Gingerol (1) C17H26O4 [M-H] 293.1737 293.1758
0.5 24.04 [8]-Gingerol (2) C19H30O4 [M-H]
-
-
321.2042 321.2071
27.10 [10]-Gingerol (3) C21H34O4 [M-H] 349.2366 349.2384
-
24.90 [6]-Shogaol (4) C17H24O3 [M-H] 275.1640 275.1646
-
0 27.95 [8]-Shogaol (5) C19H28O3 [M-H] 303.1946 303.1966
-
30.60 [10]-Shogaol (6) C21H32O3 [M-H] 331.2252 331.2279
Control (A) (B) (C) (D) 19.37 (3S,5S)-[6]-Gingerdiol (7) C17H28O4 [M-H]
-
295.1918 295.1915
-
19.97 (3R,5S)-[6]-Gingerdiol (8) C17H28O4 [M-H] 295.1921 295.1915
Figure 2: PGC-1α activity of the methanol extracts of (A) “Shokyo”, (B) “White ginger”, (C) 22.83 (3S,5S)-[8]-Gingerdiol (9) C19H32O4 [M-H]
-
323.2203 323.2228
-
“Kankyo” and (D) “Red ginger”. 23.47 (3R,5S)-[8]-Gingerdiol (10) C19H32O4 [M-H] 323.2213 323.2228
-
*P < 0.05; ** P < 0.01; *** P < 0.001 vs control (DMSO alone) 25.97 (3S,5S)-[10]-Gingerdiol (11) C21H36O4 [M-H] 351.2535 351.2541
-
26.65 (3R,5S)-[10]-Gingerdiol (12) C21H36O4 [M-H] 351.2546 351.2541
+
26.10 [6]-Gingerdiol 3,5-diacetate (13, 14) C21H32O6 [M+H-2AcOH] 261.1856 261.1849
[M+Na]+ ions of [6]-gingerdiol 3,5 diacetate (13, 14). The
+
[M+H-AcOH] 321.2063 321.2060
+
[M+Na] 403.2097 403.2091
compounds observed in the chromatograms were identified based 28.85 [8]-Gingerdiol 3,5-diacetate (15, 16) C23H36O6 [M+H-2AcOH]
[M+H-AcOH]
+
+
289.2126
349.2359
289.2162
349.2373
on high resolution mass spectral data and a comparison of the [M+Na]
+
+
431.2395 431.2404
31.12 [10]-Gingerdiol 3,5-diacetate (17, 18) C25H40O6 [M+Na] 459.2735 459.2717
retention times with those of standard reference compounds, as well
as comparing the mass spectra with the reported ones shown in Table 2: Weight content of gingerol-related compounds in the methanol extracts.
Table 1 [9a-9c]. The abundant ion in the negative ion mass spectra Compound
Shokyo
Weight content (％) in the methanol extracts
White ginger Kankyo Red ginger
of the gingerols, shogaols and gingerdiols (1-12) is the [M˗H]˗ ion, [6]-Gingerol (1) 6.30 11.57 12.73 10.91
[8]-Gingerol (2) 2.56 3.55 3.87 2.70
while the abundant ions in the positive ion mass spectra of [10]-Gingerol (3) 4.96 5.81 5.86 2.10

gingerdiol 3,5-diacetates (13-18) are [M+Na]+, [M+H-AcOH]+ and

[6]-Shogaol (4) 3.58 3.58 4.11 3.90
[8]-Shogaol (5) 1.20 1.27 1.04 1.11
[M+H-2AcOH]+. Furthermore, the corresponding stereoisomers of [10]-Shogaol (6)
(3S,5S)-[6]-Gingerdiol (7)
2.21
0.58
2.18
0.39
1.08
0.26
1.10
0.30
gingerdiol 3,5-diacetates (13-18) were unable to be separated under (3R,5S)-[6]-Gingerdiol (8)
(3S,5S)-[8]-Gingerdiol (9)
0.73
0.06
0.55
0.07
0.64
0.05
1.25
0.06
the present analytical conditions as shown in Figure 3. (3R,5S)-[8]-Gingerdiol (10)
(3S,5S)-[10]-Gingerdiol (11)
0.06
0.21
0.05
0.23
0.04
0.16
0.11
0.08
(3R,5S)-[10]-Gingerdiol (12) 0.12 0.12 0.07 0.21
[6]-Gingerdiol 3,5-diacetate (13 + 14) 0.23 0.09 0.24 0.57
The amounts of gingerol-related compounds in the methanol [8]-Gingerdiol 3,5-diacetate (15 + 16) trace
a
a
trace
a
a
trace
a
a
0.05
extracts of “Shokyo”, “Kankyo”, “Red ginger” and “White ginger” a
[10]-Gingerdiol 3,5-diacetate (17 + 18) trace trace trace 0.03
Not exceed 0.01 %
are shown in Table 2. “Shokyo” and “White ginger” have higher
amounts of [10]-shogaol (6) than “Kankyo” and “Red ginger”. In Doui and Mikage have said that the ratio of shogaol to gingerol
addition, “Red ginger” contains a larger amount of [6]-gingerdiol ([S/G]) is an important factor that affects the quality of the ginger
3,5-diacetate (13, 14). So far, most of the studies on ginger [11]. In general, it is considered that “Kankyo” has a higher S/G
constituents and the quantification of them have mainly been ratio than “Shokyo” because gingerols are easily transformed to
focused on gingerols (1-3) and shogaols (4-6) [10a, 10b], and there shogaols during the heating process. Recently, Akiba et al., reported
are few reports on chemical profiling of other gingerol-related that the S/G ratio of “Kankyo” in the Japanese crude drug market
compounds (1-18) [10c]. With the analytical conditions used in this had a wide distribution and occasionally the ratio of “Kankyo” is
Thermogenic properties of ginger Natural Product Communications Vol. 13 (7) 2018 871

(x100,000,000) (x100,000,000)

(intensity)

(intensity)
(A) (C)

(1) (1)
1.0 (2) 1.0 (2)
(3) (x 10) (3) (x 
(4) (x 2) ( ) (x 2)
(5) (x 2) ( ) (x 2)
(6) (x 2) ( ) (x 2)
(7) (x 2) ( ) (x 2)
(8) (x 2) ( ) (x 2)
(9) (x 2) ( ) (x 2)
(1 ) (x 2) (10) (x 2)
(1 ) (x 2) (11) (x 2)
(1 ) (x 2) (12) (x 2)
0.0 0.0
10 20 30 (min 40 10 20 30 (min 40
(x100,000,000) (x100,000,000)
(intensity)

(intensity)
(B (D)
(1) (1)
1.0 (2) 1.0 (2)
(3) (x 10) (3) (x 10)
(4) (x 2) (4) (x 2)
(5) (x 2) (5) (x 2)
(6) (x 2) (6) (x 2)
(7) (x 2) (7) (x 2)
(8) (x 2) (8) (x 2)
(9) (x 2) (9) (x 2)
(1 ) (x 2) (10) (x 2)
(1 ) (x 2) (11) (x 2)
(1 ) (x 2) (12) (x 2)
0.0 0.0
10 20 30 (min 40 10 20 30 (min 40
Figure 3: LC-MS total ion chromatograms (TIC) and mass chromatograms of the methanol extracts of (A) “Shokyo”, (B) “White ginger”, (C) “Kankyo” and (D) “Red ginger”. (1)
TIC positive ion mode; (2) TIC negative ion mode; (3) m/z 403.2091 ([M+Na]+ of 13 and 14); (4) m/z 293.1758 ([M-H]- of 1); (5) m/z 321.2071 ([M-H]- of 2); (6) m/z 349.2384 ([M-
H]- of 3); (7) m/z 275.1646 ([M-H]- of 4); (8) m/z 303.1966 ([M-H]- of 5); (9) m/z 331.2279 ([M-H]- of 6); (10) m/z 295.1915 ([M-H]- of 7 and 8); (11) m/z 323.2228 ([M-H]- of 9 and
10); (12) m/z 351.2541 ([M-H]- of 11 and 12).

1.8 *** ***

**
PGC-1α Activity Fold Induction

1.6
** **
1.4 **

1.2

0.8

0.6

0.4

0.2

0
Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Figure 4: PGC-1α activity of gingerol-related compounds. The numbers in the figure indicate the compounds in Figure 1.
*P < 0.05; ** P < 0.01; *** P < 0.001 vs control (DMSO alone).

lower than that of “Shokyo” [12]. The present results support these contribution to the activity. [10]-shogaol (6) stimulated PGC-1α in
findings. a concentration-dependent manner, as shown in Figure 5. As a
consequence (Table 2, Figure 1, 5), it was clarified that [10]-
To identify the constituents responsible for the PGC-1α activity of shogaol (6) contributes to the PGC-1α activity.
ginger, the gingerol-related compounds (1-18) shown in Figure 1
were isolated or prepared. The PGC-1α activities of compounds 1- In this study, the PGC-1α activity of methanol extracts of
18 are shown in Figure 4. [10]-gingerol (3), [10]-shogaol (6), [10]- “Shokyo”, “Kankyo”, “Red ginger” and “White ginger” was
gingerdiols (11, 12) and [10]-gingerdiols 3,5-diacetate (17, 18) investigated. By comparing the constituents in the four ginger
stimulated significant PGC-1α. These results indicate that the samples, we found that [10]-shogaol (6) contributes to the PGC-1α
length of the alkyl side chains in gingerols is a more important activity. The results for the PGC-1α activity of the gingerol-related
factor for PGC-1α activity than the substitutional groups at the 3- compounds (1-18) indicate that [10]-gingerol (3), [10]-shogaol (6),
and 5- positions. Recently, Chen et al., has reported that [10]- [10]-gingerdiols (11, 12) and [10]-gingerdiols 3, 5-diacetate (17,
gingerol (6) is metabolized in the human body into two major 18) stimulate significant PGC-1α. The various bioactivities of
metabolites, (3S,5S)-[10]-gingerdiol (11) and (3R,5S)-[10]- gingerol- related compounds with long alkyl side chains have been
gingerdiol (12) [13]. Our results indicate that both [10]-gingerol (3) reported. van Breemen et al., has reported that [10]-shogaol (6)
and the [10]-gingerdiols (11, 12) showed PGC-1α activity. specifically inhibited COX-2 [14a], and Ho et al., has reported that
Therefore, it is considered that the PGC-1α activity due to [10]- 10-gingerol (3) showed anti-neuroinflammatory properties through
gingerol (3) is retained even if [10]-gingerol (3) metabolizes in the blocking NF-κB activation [14b]. In addition, several studies on
human body. Additionally, the PGC-1α activity of [10]-shogaol (6) 5-HT3 receptor inhibition, NO inhibition and antioxidation by
in different concentrations (1-8 M) was examined to confirm its gingerol-related compounds have been reported [14c-14e]. The
872 Natural Product Communications Vol. 13 (7) 2018
Figure 5: PGC-1α activity of [10]-shogaol.
*P < 0.05; ** P < 0.01; *** P < 0.001 vs control (DMSO alone)
present results and these studies indicated that gingerol-related
compounds also contribute to the bioactivities of ginger.
Experimental
Plant materials and Reagents: Japanese Pharmacopoeia grade
“Shokyo” (dried rhizome of Z. officinale var. rubens) and “Kankyo”
(steamed and dried rhizome of Z. officinale var. rubens) were
purchased from Tochimoto Tenkaido (Osaka, Japan). “Red ginger”
(Indonesian dried rhizome of Z. officinale var. rubrum) and “White
ginger” (Indonesian dried rhizome of Z. officinale var. amarum)
were purchased from M&K Laboratories (Nagano, Japan). All the
specimens were deposited in the Museum of Materia Medica,
College of Pharmaceutical Science, Ritsumeikan University (RIN).
Analytical grade chemicals and LC-MS grades of chromatographic
solvents and reagents were purchased from Wako Chemical Co.
Ltd. (Tokyo, Japan).
Analytical instruments: The LC-MS analyses were performed
using a Shimadzu LC-IT-TOF mass spectrometer (Shimadzu,
Kyoto, Japan) equipped with an ESI interface. The ESI parameters
were as follows: source voltage, +4.5 kV (positive ion mode) or
˗3.5kV (negative ion mode); capillary temperature, 200 °C;
nebulizer gas flow rate, 1.5 l/min. The mass spectrometer was
operated in the positive and negative ion modes, scanning from m/z
150 to 1500. A Waters Atlantis T3 column (2.1 mm × 150 mm, 5
m) was used and the column temperature was maintained at 40 °C.
The mobile phase was a binary eluent of (A) 5 mM (NH4)OAc
solution and (B) CH3CN under the following gradient conditions:
0–30 min, linear gradient from 10% to 100% B, 30–40 min,
isocratic at 100% B. The flow rate was 0.2 ml/min. GC-MS
analyses were performed with a Shimadzu QP 2010 mass
spectrometer (Shimadzu, Kyoto, Japan) equipped with a Shimadzu
GC 2010 gas chromatography system and AOC-20i autosampler. A
DB-5MS capillary column (0.25 mm × 30 m, film thickness 0.25
μm, Agilent Technologies, Santa Clara, CA) was used. The mass
spectrometry conditions were as follows: ionization mode, EI;
ionization current, 300 μA; ionization voltage, 70 eV. The GC
parameters were as follows: the injector and transfer line were
maintained at 270 °C. The oven temperature was programmed as
follows: initial temperature, 50 °C; initial hold time, 3 min;
temperature ramp-up rate, 10 °C/min; final temperature, 300 °C;
final hold time, 5 min. The flow rate of the carrier gas (helium) was
1 ml/min. 1H- and 13C-NMR spectra were measured in CDCl3 using
a JNM-ECS400 NMR spectrometer (JEOL Ltd., Tokyo, Japan) with
tetramethylsilane as an internal standard.
Sample preparation: Pulverized “Red ginger” (15.49 g), “Kankyo”
(21.73 g) and “White ginger” (15.42 g) were extracted with
methanol under reflux for 40 min using an Buchi Extraction System
Nishidono et al.
B-811 LSV (BUCHI, Flawil, Switzerland) yielding 2.06 g of “Red
ginger” extract, 1.78 g of “Kankyo” extract and 2.32 g of “White
ginger” extract. “Shokyo” (500 g) was extracted with methanol
three times under reflux for 1 hr, then concentrated to yield the
methanol extract (50.92 g). To analyze the chemical constituents by
LC-MS, the methanol extracts were dissolved in methanol (10
mg/ml).
Isolation of Gingerols and Shogaols: The methanol extract of
“Shokyo” was suspended in water and extracted with ethyl acetate
(AcOEt), n-BuOH and water to give the AcOEt fraction (26.58 g),
the n-BuOH fraction (5.73 g) and the water fraction (11.94 g). The
ethyl acetate fraction was subjected to Wakogel C-200 column
chromatography (Wako Pure Chemical Industries) using a solvent
of hexane-ethyl acetate (7:3 → 0:10), from which 8 fractions were
obtained. A part of fraction 6 (2.0 g) was subjected to an ODS
column (2L UNIVERSAL COLUMN Packed with High
Performance Silica Gel, Yamazen Corporation, Osaka, Japan) with
a solvent of methanol-water (7:3 → 10:0) to yield compounds 1
(903 mg), 2 (392 mg) and 3 (283 mg). A part of fraction 3 (2.5 g)
was subjected to an ODS column with a solvent of methanol-water
(7:3 → 10:0) to yield compounds 4 (725 mg), 5 (287 mg) and 6
(367 mg). The following compounds were identified by comparing
the EI-MS and 13C-NMR spectral data with those reported [15, 16].
Synthesis of Gingerdiols: [6]-, [8]-, [10]-gingerdiols were obtained
by reduction of the respective [6]-, [8]-, [10]-gingerols using
sodium borohydride. As a result of the reduction, two diastereomers
were obtained. Further subjection to the silica gel column (2L
UNIVERSAL COLUMN Packed with High Performance Silica
Gel, Yamazen Corporation, Japan), separated out the (3S,5S) and
(3R,5S) isomers. The isomers were identified by comparison with
the reported 13C-NMR spectral data [17].
Synthesis of Gingerdiol 3,5-diacetates: The respective gingerdiol
3,5-diacetates were obtained by the acetylation of the corresponding
gingerdiols using acetic anhydride and pyridine followed by
hydrolysis using ammonium hydroxide. These compounds were
identified by the EI-MS and 13C-NMR spectral data.
Quantification: The compounds 1-18 were dissolved in methanol to
make standard solutions (0.1 mg/mL). The [M˗H]˗ ion shown in
Table 1 was selected for quantification of compounds 1-12 and the
individual [M+Na]+ ion was selected for compounds 13-18.
Measurement of the PGC-1α activity using a one-hybrid system:
The PGC-1α activity assay system was established, and the activity
was measured using the method presented in our previous paper
[18]. The cells were allowed to adhere for 3 hr and then treated
with the samples dissolved in DMSO. Sample solutions were
prepared at a final concentration of 0.001 % (w/v) for the methanol
extracts and 5 μM for the gingerol-related compounds. After 20 hr
incubation, the cells were lysed, and the luciferase activity was
measured by a ONE-Glo™ Luciferase Assay System (Promega).
The fold induction was calculated.
Statistical analysis: Data were expressed as mean ± SD (n = 3).
Differences were analyzed using Student’s t test. Statistical
significance was set at *P < 0.05, ** P < 0.01 and *** P < 0.001 vs
control (DMSO alone).
Acknowledgments - This work was supported by a research grant
from the Program for Asia-Japan Research Development,
Ritsumeikan University.
Thermogenic properties of ginger Natural Product Communications Vol. 13 (7) 2018 873

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