molecules

Article
Six New Phenylpropanoid Derivatives from Chemically
Converted Extract of Alpinia galanga (L.) and Their
Antiparasitic Activities
Melanny Ika Sulistyowaty 1,2 , Nguyen Hoang Uyen 1 , Keisuke Suganuma 3 , Ben-Yeddy Abel Chitama 4 ,
Kazuhide Yahata 4 , Osamu Kaneko 4 , Sachiko Sugimoto 1 , Yoshi Yamano 1 , Susumu Kawakami 5 ,
Hideaki Otsuka 5 and Katsuyoshi Matsunami 1, *

1 Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734-8553, Japan; [email protected] (M.I.S.); [email protected] (N.H.U.);
[email protected] (S.S.); [email protected] (Y.Y.)
2 Faculty of Pharmacy, Universitas Airlangga, Surabaya 60286, Indonesia
3 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine,
Inada, Obihiro 080-8555, Hokkaido, Japan; [email protected]
4 Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University,
1-12-4 Sakamoto, Nagasaki 852-8523, Japan; [email protected] (B.-Y.A.C.);
[email protected] (K.Y.); [email protected] (O.K.)
5 Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women’s University,
6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan; [email protected] (S.K.);



[email protected] (H.O.)

 * Correspondence: [email protected]; Tel.: +81-82-257-5335
Citation: Sulistyowaty, M.I.; Uyen,
N.H.; Suganuma, K.; Chitama, B.-Y.A.; Abstract: Chemical conversion of the extract of natural resources is a very attractive way to ex-
Yahata, K.; Kaneko, O.; Sugimoto, S.; pand the chemical space to discover bioactive compounds. In order to search for new medicines
Yamano, Y.; Kawakami, S.; Otsuka, H.; to treat parasitic diseases that cause high morbidity and mortality in affected countries in the
et al. Six New Phenylpropanoid world, the ethyl acetate extract from the rhizome of Alpinia galanga (L.) has been chemically con-
Derivatives from Chemically verted by epoxidation using dioxirane generated in situ. The biological activity of chemically
Converted Extract of Alpinia galanga converted extract (CCE) of A. galanga (L.) significantly increased the activity against Leishmania
(L.) and Their Antiparasitic Activities.
major up to 82.6 ± 6.2 % at 25 µg/mL (whereas 2.7 ± 0.8% for the original extract). By bioassay-
Molecules 2021, 26, 1756. https://doi.
guided fractionation, new phenylpropanoids (1–6) and four known compounds, hydroquinone (7),
org/10.3390/molecules26061756
4-hydroxy(4-hydroxyphenyl)methoxy)benzaldehyde (8), isocoumarin cis 4-hydroxymelein (9), and
(2S,3S,6R,7R,9S,10S)-humulene triepoxide (10) were isolated from CCE. The structures of isolated
Academic Editor: Anna Petruczynik
compounds were determined by spectroscopic analyses of 1D and 2D NMR, IR, and MS spectra.
Received: 3 March 2021 The most active compound was hydroquinone (7) with IC50 = 0.37 ± 1.37 µg/mL as a substan-
Accepted: 18 March 2021 tial active principle of CCE. In addition, the new phenylpropanoid 2 (IC50 = 27.8 ± 0.34 µg/mL)
Published: 21 March 2021 also showed significant activity against L. major compared to the positive control miltefosine
(IC50 = 7.47 ± 0.3 µg/mL). The activities of the isolated compounds were also evaluated against
Publisher’s Note: MDPI stays neutral Plasmodium falciparum, Trypanosoma brucei gambisense and Trypanosoma brucei rhodeisense. Interestingly,
with regard to jurisdictional claims in compound 2 was selectively active against trypanosomes with potent activity. To the best of our
published maps and institutional affil- knowledge, this is the first report on the bioactive “unnatural” natural products from the crude
iations. extract of A. galanga (L.) by chemical conversion and on its activities against causal pathogens of
leishmaniasis, trypanosomiasis, and malaria.

Keywords: Alpinia galanga; Leishmania; Trypanosoma; Plasmodium; chemically converted extract;

Copyright: © 2021 by the authors. unnatural natural product; phenylpropanoid; malaria; NTDs (neglected tropical diseases)
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
1. Introduction
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
Plasmodium, Leishmania, and Trypanosoma are causal microbes of malaria and neglected
4.0/).
tropical diseases (NTDs) and are transmitted by certain species of insects as vector-borne

Molecules 2021, 26, 1756. https://doi.org/10.3390/molecules26061756 https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1756 2 of 12

diseases. Malaria is a life-threatening disease caused by Plasmodium parasites. The World

Health Organization (WHO) estimated 229 million cases and 409,000 deaths by malaria
in 2019. Leishmaniases are grouped into three forms, visceral (kala-azar), cutaneous and
mucocutaneous. The most common leishmaniasis is cutaneous leishmaniasis caused by
Leishmania major and so on. It is estimated that 700,000 to 1 million new cases and 26,000
to 65,000 deaths occur annually [1,2]. In 2018, 977 cases were reported for human African
trypanosomiasis (sleeping sickness), which takes two forms, depending on the parasite
involved, T. b. gambiense or T. b. rhodesiense, and affects the central nervous system [3].
Natural products have been recognized as an important resource in drug discovery
and development due to their structural uniqueness and relatively strong biological activi-
ties. Newman and Cragg emphasized that natural product and natural product structures
continue to play a highly significant role in drug discovery and development. For instance,
of the 175 small molecules approved as anticancer agents from 1981 to 2014, 131 (75%)
were natural or natural products-related compounds. Therefore, natural products and their
derivatives are still playing an essential role in the discovery of drug candidates [4,5].
Natural products chemists have already discovered more than 313,000 natural prod-
ucts as listed in The Dictionary of Natural products so far [6]. Recently, an issue is evoked
that the discovery of novel bioactive compounds is becoming difficult gradually. In the
last decade or so, an attractive approach has been introduced for generating bioactive
compounds having chemical diversity via chemical modification of crude natural extracts.
This approach is an obvious method for altering the chemical composition of crude herbal
extracts, which causes changes in the chemical structure and biological activity in a negative
or positive manner. For example, sulfonylation with p-toluene sulfonyl chloride [7,8], am-
monolysis with hydrazine monohydrate [9–11], bromination with bromine [8,11], oxidation
with Dess–Martin periodinane (DMP) [12], reduction with sodium borohydride [12], epoxi-
dation with m-chloroperbenzoic acid (mCPBA) [12] and methyl(trifluoromethyl)dioxirane
followed by transannulation with a Lewis acid catalyst [13], and ethanolysis with hydroxy-
lamine hydrochloride [14] to natural extracts have been carried out to produce novel or
bioactive compounds.
Alpinia galanga (L.) belongs to a family Zingiberaceae, which is widely distributed in
China, India, and Southeast Asian countries, including Indonesia. The rhizome, fruit, and
flowers of this plant have been used as flavoring agents, food additives, or gastroprotective
agents. In addition, it is traditionally applied for treating dyspepsia and abdominal colic
pain, motion sickness, and gastralgia [15,16]. Up to now, there are numerous articles about
pharmacological activities of A. galanga, for instance antiallergic [17], anticancer [18–20],
anti-inflammatory [21], antimicrobial [21–24], antioxidant [24,25], anti-amnesiac [26], anti-
asthma [27], and also melanogenesis inhibitor [28]. However, there is no report about its
positive activity as an antiparasitic agent [29]. Thus, A. galanga has already been studied
extensively, including chemical constituents and biological activities, and it is unlikely that
any further progress will be obtained easily by the standard method. Therefore, we applied
the chemical derivatization method to generate new bioactive compounds.
In this report, a chemical diversification of A. galanga extract resulted in a generation
of bioactivity against the Leishmania parasite, and new (1–6) and bioactive compounds
were isolated through bioactivity-guided fractionation. The chemical structures were
elucidated by intensive spectroscopic analyses. Activities against leishmaniasis, human
African trypanosomiasis, and malaria are also discussed.

2. Results and Discussion

2.1. Isolation of Chemical Components of Chemically Converted Extract of Alpinia Galanga
The dried rhizomes of A. galanga (2.0 kg) were extracted with MeOH (3 × 10 L) at
room temperature to give MeOH extract. Afterward, this extract was partitioned by solvent
fractionation with ethyl acetate (EtOAc)/H2 O to give EtOAc extract (E). The EtOAc extract
was treated with dioxirane to produce the chemically converted extract (CCE) (Scheme 1).
Dioxirane is a powerful reagent for epoxidation, which is generated in situ by the reaction
 The dried rhizomes of A. galanga (2.0 kg) were extracted with MeOH (3 × 10 L) at
room temperature to give MeOH extract. Afterward, this extract was partitioned by sol-
Molecules 2021, 26, 1756 vent fractionation with ethyl acetate (EtOAc)/H2O to give EtOAc extract (E). The EtOAc 3 of 12
extract was treated with dioxirane to produce the chemically converted extract (CCE)
(Scheme 1). Dioxirane is a powerful reagent for epoxidation, which is generated in situ by
the reaction of potassium peroxomonosulfate with acetone [30]. After being quenched and
of potassium peroxomonosulfate with acetone [30]. After being quenched and evaporated,
evaporated, HPLC analyses and biological assays of CCE and E were carried out. The anti-
HPLC analyses and biological assays of CCE and E were carried out. The anti-Leishmania
Leishmania activity of E and CCE against promastigotes of L. major was conducted by the
activity of E and CCE against promastigotes of L. major was conducted by the colorimetric
colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method [31]. The
method [31]. The HPLC profile showed that major peaks in the original extract have van-
HPLC profile showed that major peaks in the original extract have vanished, and some new
ished, and some new peaks appeared in the CCE (Figure 1). This result exhibited that new
peaks appeared in the CCE (Figure 1). This result exhibited that new chemical components
chemical components were produced by the chemical treatment. Furthermore, the inhib-
were produced by the chemical treatment. Furthermore, the inhibitory activity of CCE was
itory activity of CCE was significantly increased after treating with dioxirane. The CCE
significantly increased after treating with dioxirane. The CCE demonstrated 82.6 ± 6.2 %
demonstrated 82.6 ± 6.2 % of inhibition against L. major, while the original EtOAc extract
of inhibition against L. major, while the original EtOAc extract did not inhibit (2.7 ± 0.8 %)
did
at 25not inhibit
µg/mL (2.7 ± 0.8
(Figure 1). %) at 25 μg/mL (Figure 1).

OH OCH3 OH
1 2'
1' 3' OCH3 OCH3 OCH3
4
Cl OH
HO HO HO
1 2 3
2''

OH OCH3 O 1''
3''

1 O
OCH3 OCH3 1' 2'
4
OH OH 3'
HO HO HO OCH3
4 5 6

OH OH OH O O

O O O
O OH
OH OH
H O
7 8 9 10

Scheme 1.
Scheme 1. Isolation
Isolation of
of compounds
compounds from
from the
the chemically
chemically converted
converted extract
extract of
of A.
A.galanga.
galanga.
 Subsequently, by using the bioassay-guided isolation procedure, the CCE was fur-
ther separated by various column chromatographies and HPLC to provide new com-
pounds 1–6 (Scheme 1). In addition, four known compounds were identified such as hy-
droquinone (7) [32], 4-hydroxy(4-hydroxyphenyl)methoxy)benzaldehyde (8) [33],
isocoumarin cis 4-hydroxymelein (9) [34], and (2S,3S,6R,7R,9S,10S)-humulene triepoxide
Molecules 2021, 26, 1756 4 of 12
(10) [35] by comparing with the spectral data (see Supplementary Materials Figures S4–
S60, Tables S1–S2).

Figure 1. UV-HPLC profiles and anti-Leishmania major activity of A. galanga extract; (A) before (B) after treatment with
Figure 1. UV-HPLC profiles and anti-Leishmania major activity of A. galanga extract; (A) before (B) after treatment with
dioxirane. HPLC: ODS, UV: 215 nm; (C) the percentage of inhibition against L. major before and after chemical treatment.
dioxirane. HPLC: ODS, UV: 215 nm; (C) the percentage of inhibition against L. major before and after chemical treatment.
The
The error
error bar
bar represents SE,nn== 3.
representsSE, 3.

Subsequently, by using the bioassay-guided isolation procedure, the CCE was further
Compound 1 was obtained as a colorless liquid having molecular formula C10H13O3Cl
separated by various column chromatographies and HPLC to provide new compounds 1–6
from an [M + Na]+ ion at m/z 239.0446 with characteristic 3: 1 pattern of isotopic 35Cl and
(Scheme 1). In addition, four known compounds were identified such as hydroquinone (7) [32],
37Cl in the high resolution electrospray ionization (HR-ESI) MS. The IR data exhibited ab-
4-hydroxy(4-hydroxyphenyl)methoxy)benzaldehyde (8) [33], isocoumarin cis 4-hydroxymelein
sorption bands for hydroxy groups (3351 cm−1) and the C-Cl stretching bond (850 cm−1).
(9) [34], and (2S,3S,6R,7R,9S,10S)-humulene triepoxide (10) [35] by comparing with the spec-
The 1H NMR data (Table 1) showed the presence of one methoxy group at 3.36 ppm and
tral data (see Supplementary Materials Figures S4–S60, Tables S1–S2).
a para-disubstituted
Compound 1 wasaromaticobtainedring with ortho-coupled
as a colorless liquid having protons at 7.21
molecular and 6.76
formula C10 Hppm as
13 O3 Cl
doublets (J = 8.6 Hz)
+ each in 1. The 1H and 13C-NMR data were closely similar to those of
from an [M + Na] ion at m/z 239.0446 with characteristic 3: 1 pattern of isotopic Cl 35
the analogous
and 37 Cl in the compound, erythro-2-chloro-1-(4-hydroxyphenyl)propane-1,3-diol.
high resolution electrospray ionization (HR-ESI) MS. The IR data exhibited On the
absorption bands for hydroxy groups (3351 cm ) and the C-Cl stretching bond (850 cm−1in).
basis of the coupling constant of H-1′ and H-2′− 1 (J = 5.9 Hz), 1 was assigned as erythro
The 1 H NMR data (Table 1) showed the presence of one methoxy group at 3.36 ppm and
a para-disubstituted aromatic ring with ortho-coupled protons at 7.21 and 6.76 ppm as
doublets (J = 8.6 Hz) each in 1. The 1 H and 13 C-NMR data were closely similar to those of
the analogous compound, erythro-2-chloro-1-(4-hydroxyphenyl)propane-1,3-diol. On the
basis of the coupling constant of H-10 and H-20 (J = 5.9 Hz), 1 was assigned as erythro in
accordance with a reference (J = 5.9 Hz) [36]. Therefore, the structure of 1 was determined
to be 4-[threo-2-chloro-1-hydroxy-3-methoxypropyl]phenol.
Molecules 2021, 26, x 6 of 13

Molecules 2021, 26, 1756 5 of 12

ppm, and a para-substituted coupling system at δH 6.78 (2H, d, J= 8.5 Hz) and 7.13 (2H, d,
J= 8.5 Hz). The 13C-NMR data (Table 2) exhibited nine carbon peaks that were classified
by chemical1shift values, DEPT and HSQC spectrum as two methoxy carbons (δC 56.8 and
Table 1. H-NMR spectroscopic data for compounds 1–6.
59.2), two sp2 methine carbons (δC 116.2 (2C), 129.9 (2C)), two sp2 quaternary carbons (δC
Position 1 130.6 and 158.5)2 and three oxygenated
3 carbons4(δC 74.6, 75.6, and 5 85.7). Chakraborty 6 et al.
2,6 7.21 d (8.6)recently stereoselectively
7.10 d (8.5) synthesized
7.16 d (8.5) the related compounds,
7.21 d (8.6) threo-
7.13 d (8.5) and erythro-1-(4-hy-
7.21 d (8.6)
3,5 6.76 d (8.6)droxyphenyl)-1-methoxy-2,3-propanediol,
6.77 d (8.5) 6.74 d (8.5) anddthe
6.76 (8.6)coupling6.78
constants
d (8.5) of H-1′6.79
of threo-
d (8.6)and
10 4.80 d (5.9)erythro-
4.19forms were
dd (7.8, 6.1) 7.0 and
4.49 6.4 Hz, respectively.
d (6.6) According
4.52 d (6.3) 4.08tod this
(7.3) report, 4.69
compound
d (8.7) 5
0
1 -OCH3 3.14coupling
s 3.19 s
showing the constant of H-1′ (J = 7.3 Hz) can be concluded as a threo isomer, 4-
4.22 dt-like[threo-2-hydroxy-1,3-dimethoxypropyl]phenol
2.02 m 3.73 td-like 3.84 td-like
[39]. 3.74 ddd 3.89 ddd
20
(5.9, 5.2) 1.78 m (6.6, 3.4) (6.5, 3.4) (7.3, 5.8, 3.2) (8.7, 5.6, 2.8)
3.44 13dt-like 3.14 dd 3.44 dd 3.08 dd 3.52 dd
30 3.61 m (2H) Table 2. C-NMR spectroscopic data for compounds 1–6.
(9.4, 6.5) (10.1, 6.2) (10.1, 6.7) (10.1, 5.8) (11.0, 2.8)
Position 1 2m 3m 3.494 dd 3.476 dd
3.28 3.29 3.26 br5 t (8.4)
1 132.8 133.6 133.9 (10.1,
133.93.4) 130.6 (11.0,
129.55.6)
30 -OCH3 3.36 s 3.30 s 3.26 s 3.34 s 3.24 s 3.35 s
2,6 129.5 129.0 129.1 129.4 129.9 129.3
2” 1.46 s
3,5
3” 115.8 116.2 116.0 115.8 116.2 116.4s
1.51
4 158.2 158.2 158.0 157.9 158.5 158.8
Measured in CD3 OD at 600 MHz (δ in ppm, J in Hz); m = multiplet or overlapped.
1′ 75.2 81.8 75.5 75.6 85.7 80.9
1′-OCH3 56.5 56.8
2′ 65.0 Compound 38.9 2 was obtained 76.0 as a colorless75.0 liquid. Its molecular
75.6 formula showed 83.8 to be
C H O from a pseudo molecular ion at m/z 219.0991 ([M + Na] + ) in the HRESIMS. The IR
3′ 74.6 11 16 3 70.4 74.9 75.1 74.6 72.6
3′-OCH3 59.2 data showed bands for a hydroxyl
58.8 59.2 group (337959.3 cm−1 ) and an aromatic
59.2 ring (1604,59.6
1510, and
1” 1442 cm−1 ). The 1 H-NMR data (Table 1) showed two methoxy groups at 3.14 and110.2 3.30 ppm,
2” and a para-substituted aromatic ring at 7.10 and 6.77 ppm as doublets (J = 8.5 Hz). 27.3 According
3” to the correlation spectroscopy (COSY) and heteronuclear multiple bond connectivity 27.4
correlations (HMBC)
Measured (Figure
in CD 3OD at 2),150
2 was
MHzassigned
(δ in ppm). as 4-(1,3-dimethoxypropyl)phenol.

OH OCH3 OH

OCH3 OCH3 OCH3

Cl OH
HO HO HO
1 2 3

OH OCH3 O
O
OCH3 OCH3
OH OH
HO HO HO OCH3
4 5 6

COSY C H HMBC
Figure 2.
Figure H-H COSY
2. H-H COSY and
and HMBC
HMBC correlations
correlations of
of compounds
compounds 1–6.
1–6.

Compounds6 3was
Compound andisolated
4 were isolated as different
as a colorless liquid peaks by HPLC.formula
with molecular Compounds
C13H183Oand
4 as
4 were colorless
measured by HRESIMSliquids with the same molecular formula C 10 H 14 O 4 by HRESIMS
at m/z 261.1099 ([M + Na] ). The IR data indicated absorption
+ at m/z
221.0781 ([M + Na]+ ). The IR data indicated the presence of hydroxy groups (3: 3384,
bands for a hydroxyl group (3368 cm−1) and the presence of an aromatic ring (1607 and
and 4: 3394 cm−1 1 ) and an aromatic ring (3: 1606, 1514, and 1455, and 4: 1616, 1514, and
1512 cm−).−1 The H NMR data (Table 1) showed peaks caused by one methoxy group at
1455 cm 1 ). The 1 H NMR data (Table 1) displayed signals caused by the presence of one
3.35 ppm, two methyl group at 1.46 and 1.51 ppm, and a para-disubstituted coupling sys-
methoxy group (3: 3.26, and 4: 3.34 ppm), a para-substituted benzene ring at δH 6.74 (2H,
tem at δH 6.79 ppm (2H, d, J= 8.6 Hz) and 7.21 (2H, d, J= 8.6 Hz). The 13C-NMR data (Table
d, J = 8.5 Hz) and 7.16 (2H, d, J = 8.5 Hz) for 3, and δH 6.76 ppm (2H, d, J = 8.6 Hz) and
2) exhibited 11 carbon peaks that13were categorized as two methyl carbons group (δC 27.3
7.21 (2H, d, J = 8.6 Hz) for 4. The C-NMR data (Table 2) exhibited eight carbon peaks that
and 27.4), one methoxy group (δC 59.6), two sp2 methine carbons (δC 116.4 (2C) and
were classified by2 DEPT and heteronuclear single quantum coherence (HSQC) spectrum
129.3(2C)), two sp quaternary carbons (δC 129.5 and 158.8), and three oxygenated carbons
as one methoxy carbon group (3: δ 59.2, and 4: δC 59.3), two sp2 methine carbons (3: δC
(δC 72.6, 80.9, and 83.8). These resultsC suggested a planar structure of 6 as shown in scheme
116.0 (2C), 129.1(2C), and 4: δC 115.8 (2C), 129.4(2C)), two sp2 quaternary carbons (3: δC
133.9 and 158.0, and 4: δC 133.9 and 157.9) and three oxygenated carbons (3: δC 74.9, 75.5,
and 76.0, and 4: δC 75.1, 75.0, and 75.6). These results indicated that compounds 3 and 4
Molecules 2021, 26, 1756 6 of 12

have the same planar structure as shown in Scheme 1. Yang et al. and Li et al. reported
slight but practical differences between erythro- and threo- forms of the related compounds,
1-(4-hydroxyphenyl)-1-methoxy-2,3-propanediol: that is, the erythro- and threo- forms had
different coupling constants of H-10 (6.6 Hz and 6.3 Hz, respectively), and the erythro-form
showed a 0.9 ppm higher-field chemical shift value at C-20 compared to that of threo-form.
In the 1 H and 13 C-NMR spectrum of 3 and 4, the coupling constants of H-10 were 6.6 and
6.3 Hz, respectively, and the relatively lower-field chemical shift value at C-20 of 3 (δC 76.0)
compared to C-20 of 4 (δC 75.0) suggested threo- and erythro-form for 3 and 4, respectively,
as shown in Scheme 1 [37,38].

Table 2. 13 C-NMR spectroscopic data for compounds 1–6.

Position 1 2 3 4 5 6
1 132.8 133.6 133.9 133.9 130.6 129.5
2,6 129.5 129.0 129.1 129.4 129.9 129.3
3,5 115.8 116.2 116.0 115.8 116.2 116.4
4 158.2 158.2 158.0 157.9 158.5 158.8
10 75.2 81.8 75.5 75.6 85.7 80.9
10 -OCH3 56.5 56.8
20 65.0 38.9 76.0 75.0 75.6 83.8
30 74.6 70.4 74.9 75.1 74.6 72.6
30 -OCH3 59.2 58.8 59.2 59.3 59.2 59.6
1” 110.2
2” 27.3
3” 27.4
Measured in CD3 OD at 150 MHz (δ in ppm).

Compound 5 was yielded as a colorless liquid with molecular formula C11 H16 O4
determined by HRESIMS from the peak at m/z 235.0939 ([M + Na]+ ). The IR data indicated
bands for a hydroxy group (3379 cm−1 ) and an aromatic ring (1604, 1509, and 1455 cm−1 ).
The 1 H NMR data (Table 1) showed peaks caused by two methoxy groups at 3.19 and
3.24 ppm, and a para-substituted coupling system at δH 6.78 (2H, d, J= 8.5 Hz) and 7.13 (2H,
d, J= 8.5 Hz). The 13 C-NMR data (Table 2) exhibited nine carbon peaks that were classified
by chemical shift values, DEPT and HSQC spectrum as two methoxy carbons (δC 56.8
and 59.2), two sp2 methine carbons (δC 116.2 (2C), 129.9 (2C)), two sp2 quaternary carbons
(δC 130.6 and 158.5) and three oxygenated carbons (δC 74.6, 75.6, and 85.7). Chakraborty
et al. recently stereoselectively synthesized the related compounds, threo- and erythro-1-(4-
hydroxyphenyl)-1-methoxy-2,3-propanediol, and the coupling constants of H-10 of threo-
and erythro- forms were 7.0 and 6.4 Hz, respectively. According to this report, compound
5 showing the coupling constant of H-10 (J = 7.3 Hz) can be concluded as a threo isomer,
4-[threo-2-hydroxy-1,3-dimethoxypropyl]phenol [39].
Compound 6 was isolated as a colorless liquid with molecular formula C13 H18 O4 as
measured by HRESIMS at m/z 261.1099 ([M + Na]+ ). The IR data indicated absorption
bands for a hydroxyl group (3368 cm−1 ) and the presence of an aromatic ring (1607 and
1512 cm−1 ). The 1 H NMR data (Table 1) showed peaks caused by one methoxy group
at 3.35 ppm, two methyl group at 1.46 and 1.51 ppm, and a para-disubstituted coupling
system at δH 6.79 ppm (2H, d, J= 8.6 Hz) and 7.21 (2H, d, J= 8.6 Hz). The 13 C-NMR
data (Table 2) exhibited 11 carbon peaks that were categorized as two methyl carbons
group (δC 27.3 and 27.4), one methoxy group (δC 59.6), two sp2 methine carbons (δC
116.4 (2C) and 129.3(2C)), two sp2 quaternary carbons (δC 129.5 and 158.8), and three
oxygenated carbons (δC 72.6, 80.9, and 83.8). These results suggested a planar structure of 6
as shown in Scheme 1. The related acetonide, 4-(hydroxymethyl)-5-[3‘,5‘-dihydroxyphenyl]-
2,2-dimethyl-1,3-dioxolane, a threo isomer, has coupling constant J= 8.1 Hz for H-10 and
H-20 [40]. Morikawa et al. explained that threo- and erythro- forms of acetonides have
similar chemical shift values except for the coupling constants of H-10 and H-20 , that is,
erythro- form (J = 2.8 Hz) and threo-form (J = 10.1 Hz) [41]. Therefore, based on the coupling
Molecules 2021, 26, 1756 7 of 12

constants of H-10 and H-20 (J = 8.7 Hz), compound 6 can be assigned as threo isomer as
4-[threo-5-(methoxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]phenol.
All of the isolated compounds did not show significant optical rotation values, proba-
bly because of the formation of the racemic mixture through the chemical derivatization.

2.2. Antiparasitic Activity Examinations of the Isolated Compounds

All the isolated compounds were investigated for their biological activity toward
selected protozoan parasites. Table 3 shows IC50 values of isolated compounds against
L. major, T. b. gambisense, T. b. rhodesiense, and P. falciparum. Among isolated compounds,
hydroquinone (7) had the highest activity against parasites belonging to the family Try-
panosomatidae. The leishmanicidal and trypanocidal activities of hydroquinone deriva-
tives had been reported [41,42]. Our result of 7 further supports the importance of the
hydroquinone motif as an important pharmacophore. Compounds 2, 8 and 9 possessed
significant activities against both L. major and Trypanosoma parasites. On the other hand,
compound 1 had a strong activity on Trypanosoma parasites only. However, all isolated
compounds showed no activity against P. falciparum (Table 3), probably because of the bio-
logical difference at the phylum level; Sarcomastigophora (L. major, T. b. gambiense, and T. b.
rhodesiense) and Apicomplexa (P. falciparum). Alternatively, another possible reason could
be the absence of a peroxide ring system which is important for the antimalarial activity of
artemisinin [43]. Therefore, a chemical modification to introduce peroxide functions may
be a promising method to produce novel antimalarial agents in the future.

Table 3. IC50 of the isolated compounds against parasites (Mean ± SE, µg/mL, n = 3).

Compounds L. major T. brucei gambisense T. brucei rhodeisense P. falciparum

1 >100 4.20 ± 0.78 5.62 ± 1.46 >100
2 27.83 ± 0.34 23.66 ± 0.87 26.85 ± 2.20 >100
3 >100 79.59 ± 0.90 77.80 ± 11.01 >100
4 >100 71.54 ± 0.52 73.57 ± 1.67 >100
5 70.33 ± 0.10 84.54 ± 1.23 80.44 ± 4.91 >100
6 87.60 ± 1.50 38.84 ± 0.92 40.47 ± 3.36 >100
7 0. 37 ± 1.37 3.18 ± 0.09 3.27 ± 0.33 >100
8 51.10 ± 1.19 15.08 ± 0.39 18.01 ± 1.27 >100
9 40.55 ± 0.95 28.37 ± 0.56 30.94 ± 2.28 >100
10 >100 >100 >100 >100
7.47 ± 0.30 1.32 ± 0.68 1.25 ± 0.46 2.64 ± 0.29
Positive control
(Miltefosine) (Niflutimox) (Niflutimox) (Dihydroartemisinin)

To our understanding, there are no reports on the biological activity of 4-hydroxypheny

lpropanols against L. major, P. falciparum, T. brucei gambisense, and T. brucei rhodeisense. There-
fore, our study is the first report on the activities of 4-hydroxyphenylpropanol and related
compounds against those parasites. Ropes et al. reported the derivatives of p-coumaric
acid alkyl esters and their inhibitory activities against Leishmania and Plasmodium [44]. The
presence of alkyl function on C-30 (-OMe) and the relatively non-polar nature of 1 and 2
may be related to the higher activity among 1–6.

3. Materials and Methods

3.1. General Methods
Optical rotations were determined on a P-1030 digital polarimeter (JASCO, Tokyo,
Japan). IR spectra were measured on a FT-710 Fourier transform infrared spectrophotometer
(Horiba, Kyoto, Japan), while UV spectra were taken on a V-520 UV/Vis spectrophotome-
ter (JASCO, Tokyo, Japan). NMR data were obtained by an Ultrashield 600 spectrome-
ter (Bruker, Ettlingen, Germany) with tetramethylsilane (TMS) as an internal standard.
Positive ion HR-ESI-MS measurement was performed with the LTQ Orbitrap XL mass
spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Column chromatography
Molecules 2021, 26, 1756 8 of 12

(CC) was carried out on silica gel 60 (E. Merck, Darmstadt, Germany), and octadecyl silica
(ODS) gel (Cosmosil 75C18-OPN (NacalaiTesque, Kyoto, Japan; Φ = 35 mm, L = 350 mm).
HPLC was performed on ODS gel (Inertsil ODS-3, GL-science, Φ = 6 mm, 250 mm, flow
rate = 1.6 mL/min, GL Sciences, Tokyo, Japan), and the eluate was observed by refractive
index detector with the RI-930 intelligent detector (JASCO, Tokyo, Japan) and a PU-1580
intelligent pump (JASCO, Tokyo, Japan). All chemicals were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan) and TCI (Tokyo, Japan).

3.2. Plant Material

Rhizome of A. galanga (L.) was purchased from Shinwa-bussan Co., Osaka, Japan,
in 2014. A voucher specimen (Ay-AG2014-Shinwa) was deposited in the herbarium of
the Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences,
Hiroshima University, Japan.

3.3. Preparation of Chemically Converted Extract of A. galanga and Isolation of Compounds 1–10
Rhizome of A. galanga (2.0 kg) was cut into small pieces and extracted by maceration
at room temperature with MeOH (10 L) three times and then evaporated to give an MeOH
extract (303 g). This residue was suspended in 1 L of saturated sodium carbonate solution
and partitioned with EtOAc (1 L) three times to produce an EtOAc extract (68 g).
The EtOAc extract (14 g) was dissolved in the mixture of acetonitrile (360 mL) and
water (360 mL). Then, acetone (10.35 mL), sodium bicarbonate (19.6 g) and potassium
peroxosulphate (28.2 g) were added sequentially to the solution at 0 ◦ C. The mixture was
stirred for 3 h at 0 ◦ C and then potassium peroxosulphate (28.2 g) was added again to the
solution with stirring for another 3 h at 0 ◦ C. The mixture was then allowed to rise naturally
to room temperature with stirring for 24 h. The obtained reaction mixture was poured into
water and extracted with ethyl acetate three times. The combined EOAc layer was washed
with water, brine, dried over sodium sulfate, and evaporated to yield a residue, designated
as CCE of A. galanga (10.5 g).
The CCE was separated on a silica gel (50 g) CC and eluted with an increasing
amount of MeOH in CHCl3 (20:1, 10:1, 7:1, 5:1, 3:1, 1:1, and 100% MeOH, 500 mL each),
generating nine fractions. Fraction CCE2 (1.1 g), CCE3 (1.4 g) and CCE4 (4.0 g) were
subjected to ODS CC with a step gradient from 10% aq. MeOH to 100% MeOH, (400 mL
each), leading to eight fractions. The residue of fraction CCE 2.1 (226 mg) was purified
by HPLC using 20% aq. acetone to give 1 (40.1 mg), 7 (hydroquinone, 102 mg), and 8
(4-hydroxy(4-hydroxyphenyl)methoxy)benzaldehyde, 20.2 mg). The residue of fraction
CCE 2.2 (79.5 mg) was purified by HPLC using 30% aq. MeOH to give 1 (1.9 mg), 9
(isocoumarin cis-4-hydroxymelein, 2.2 mg), 2 (2.4 mg), 6 (5.8 mg), and 10 ((2S, 3S, 6R, 7R,
9S, 10S)-humulene triepoxide, 2.8 mg). The residue of fraction CCE 3.1 (297.5 mg) was
purified by HPLC using 20% aq. acetone to give 7 (70.1 mg), 3 (20.1 mg), 4 (6.0 mg), and 5
(12.5 mg) (Supplementary Materials Figures S1–S3).
4-[erytho-2-chloro-1-hydroxy-3-methoxypropyl]phenol (1)
Colorless oil, IR υmax (film) cm−1 : 3351, 2935, 1668, 1607, 1454, 1377, 1236, 1118, 1038,
850; UV λmax (MeOH) nm (log ε): 276 (2.58); 1 H NMR (CD3 OD, 600 MHz), see Table 1;
13 C-NMR (CD OD, 150 MHz), see Table 2. HRESIMS (positive ion) m/z: 239.0446 [M + Na]+
3
(calcd for C10 H13 O3 ClNa: 239.0445).
4-(1,3-dimethoxypropyl)phenol (2)
Colorless oil, IR υmax (film) cm−1 : 3379, 2930, 2854, 1718, 1604, 1510, 1442, 1233, 1104;
UV λmax (MeOH) nm (log ε): 259 (2.57); 1 H NMR (CD3 OD, 600 MHz), see Table 1; 13 C-NMR
(CD3 OD, 150 MHz), see Table 2. HRESIMS (positive ion) m/z: 219.0991 [M + Na]+ (calcd
for C11 H16 O3 Na: 219.0992).
threo-1-(4-hydroxyphenyl)-3-methoxypropane-1,2-diol (3)
Colorless oil, IR υmax (film) cm−1 : 3384, 2987, 2831, 1677, 1606, 1514, 1455, 1240, 1119;
UV λmax (MeOH) nm (log ε): 276 (2.96); 1 H-NMR (CD3 OD, 600 MHz), see Table 1; 13 C-
Molecules 2021, 26, 1756 9 of 12

NMR (CD3 OD, 150 MHz), see Table 2. HRESIMS (positive ion) m/z: 221.0781 [M + Na]+
(calcd for C10 H14 O4 Na: 221.0784).
erythro-1-(4-hydroxyphenyl)-3-methoxypropane-1,2-diol (4)
Colorless oil, IR υmax (film) cm−1 : 3394, 2930, 2360, 1679, 1616, 1514, 1455, 1240; UV
λmax (MeOH) nm (log ε): 276 (2.33); 1 H NMR (CD3 OD, 600 MHz), see Table 1; 13 C-NMR
(CD3 OD, 150 MHz), see Table 2. HRESIMS (positive ion) m/z: 221.0781 [M + Na]+ (calcd
for C10 H14 O4 Na: 221.0784).
4-(threo-2-hydroxy-1,3-dimethoxypropyl)phenol (5)
Colorless oil, IR υmax (film) cm−1 : 3379, 2930, 2360, 1604, 1509, 1455, 1375; UV λmax
(MeOH) nm (log ε): 279 (3.26); 1 H NMR (CD3 OD, 600 MHz), see Table 1; 13 C-NMR
(CD3 OD, 150 MHz), see Table 2. HRESIMS (positive ion) m/z: 235.0939 [M + Na]+ (calcd
for C11 H16 O4 Na: 235.0941).
4-(threo-5-(methoxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)phenol (6)
Colorless oil, IR υmax (film) cm−1 : 3368, 2986, 1732, 1607, 1512, 1375, 1230, 1165, 1078;
UV λmax (MeOH) nm (log ε): 276 (2.95); 1 H NMR (CD3 OD, 600 MHz), see Table 1; 13 C-NMR
(CD3 OD, 150 MHz), see Table 2. HRESIMS (positive ion) m/z: 261.1099 [M + Na]+ (calcd
for C13 H18 O4 Na: 261.1097).

3.4. Biological Assay of Compounds 1–10

The leishmanicidal activities of the isolated compounds were evaluated using MTT
colorimetric assay, as described by Asaumi et al. [31] MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, was reduced to form an insoluble purple precipitate,
formazan, by cell metabolic activity, which is known to correlate to the viable cell number.
In a 96-well plate, 1 µL of sample solutions in dimethylsulfoxide (DMSO) and L. major
(1 × 105 parasite/well) in 99 µL of medium were added to each well. It was then incubated
for 72 h in a CO2 incubator at 25 ◦ C. M-199 medium, supplemented with 10% heat-
inactivated fetal bovine serum and 100 µg/mL of kanamycin, was used. After incubation,
100 µL of MTT solution was added to each well and the plates were incubated for another
8 h. The absorbance of the formazan solution in DMSO was recorded using a microplate
reader at λ540 nm.
The trypanocidal activities of the isolated compounds were performed in 96-well
plates as previously explained with slight modifications [45]. The amount of ATP was
proportional to the number of metabolically active cells. This method utilized the enzymatic
reaction of luciferase and luciferin that requires ATP to generate luminescence. In brief,
each well contained 100 µL of parasite culture (1 × 104 parasites/well) with serial dilutions
of compounds. After incubation for 72 h at 37 ◦ C under 5% CO2 , 25 µL of CellTiter-GloTM
Luminescent Cell Viability Assay reagent (Promega Japan, Tokyo, Japan) was added to
evaluate intracellular ATP concentration according to the instruction.
Antimalarial activity of isolated compounds was evaluated according to the previous
report [46]. The SYBR green assay is widely used for the antimalarial assay. This dye binds
to parasite DNA and emits fluorescence reflecting the number of parasites in the red blood
cells. In brief, 100 µL of P. falciparum parasite culture [47] was plated in a 96-well plate
with various concentrations of the compounds. After incubation for 72 h at 37 ◦ C in a
humidified chamber under a gas mixture of 90%N2 , 5%O2 and 5%CO2 , the parasitemia
was determined by SYBR Green I assay (Lonza Ltd., Basel, Switzerland) with a microplate
reader at 485 and 530 nm. Dihydroartmesinin was used as the positive control and DMSO
as a negative control. Human erythrocytes and plasma were obtained from the Nagasaki
Red Cross Blood Center, and their usage was approved by the ethical committee of the
Institute of Tropical Medicine, Nagasaki University.
The biological assays were performed three times for each assay and the IC50 values
were calculated by linear regression using Microsoft Excel software.
Molecules 2021, 26, 1756 10 of 12

4. Conclusions
The ethyl acetate extract of A. galanga was originally inactive against L. major. How-
ever, the chemical conversion by epoxidation generated significant activity to this parasite.
The HPLC profile clearly indicated the constituent difference between the original and
chemically converted extract (CCE) of A. galanga. Through the intensive fractionation and
purification of CCE, six new phenylpropanoids (1–6) and four known compounds were
obtained as active principles. The most active compound was hydroquinone (7), which
further supports the importance of this chemical motif as shown in our previous report on
the discovery of new anti-Leishmanial benzoquinones, named ardisiaquinones A–H [31].
In addition, the new phenylpropanoid 1 was revealed to inhibit two African Trypanosoma
parasites effectively with high selective manner compared to L. major. Phenylpropanoids
are an important constituent of plant essential oils. Several plant oils have been reported
to show antiparasitic activity by inhibiting proteinase, dehydrogenase, and lipid traffick-
ing [48]. The phenylpropanoids 1–6 isolated in this study may have a similar mode of
action. The chemical synthesis of analogous compounds of 1 and the mechanism analysis
are under investigation.

Supplementary Materials: The following are available online, Figure S1: HPLC chromatogram
for the isolation of 1, 7 and 8, Figure S2: HPLC chromatogram for the isolation of 1, 2, 6, 9 and
10, Figure S3: HPLC chromatogram for the isolation of 3, 4, 5 and 7, Figure S4: HR-ESI-MS of 1,
Figure S5: 1 H NMR spectrum of 1, Figure S6: 13 C NMR spectrum of 1, Figure S7: DEPT 135 spectrum
of 1, Figure S8: COSY spectrum of 1, Figure S9: HSQC spectrum of 1, Figure S10: HMBC spectrum
of 1,Figure S11: IR spectrum of 1, Figure S12: HR-ESI-MS of 2, Figure S13: 1 H NMR spectrum of 2,
Figure S14: 13 C NMR spectrum of 2, Figure S15: DEPT 135 spectrum of 2, Figure S16: COSY spectrum
of 2, Figure S17: HSQC spectrum of 2, Figure S18: HMBC spectrum of 2, Figure S19: IR spectrum of
2, Figure S20: HR-ESI-MS of 3, Figure S21: 1 H NMR spectrum of 3, Figure S22: 13 C NMR spectrum
of 3, Figure S23: DEPT 135 spectrum of 3, Figure S24: COSY spectrum of 3, Figure S25: HSQC
spectrum of 3, Figure S26: HMBC spectrum of 3, Figure S27: IR spectrum of 3, Figure S28: HR-ESI-MS
of 4, Figure S29: 1 H NMR spectrum of 4, Figure S30: 13 C NMR spectrum of 4, Figure S31: DEPT
135 spectrum of 4, Figure S32: COSY spectrum of 4, Figure S33: HSQC spectrum of 4, Figure S34:
HMBC spectrum of 4, Figure S35: IR spectrum of 4, Figure S36: HR-ESI-MS of 5, Figure S37: 1 H
NMR spectrum of 5, Figure S38: 13 C NMR spectrum of 5, Figure S39: DEPT 135 spectrum of 5,
Figure S40: COSY spectrum of 5, Figure S41: HSQC spectrum of 5, Figure S42: HMBC spectrum of
5, Figure S43: IR spectrum of 5, Figure S44: HR-ESI-MS of 6, Figure S45: 1 H NMR spectrum of 6,
Figure S46: 13 C NMR spectrum of 6, Figure S47: DEPT 135 spectrum of 6, Figure S48: COSY spectrum
of 6, Figure S49: HSQC spectrum of 6, Figure S50: HMBC spectrum of 6, Figure S51: IR spectrum
of 6, Figure S52: 1 H NMR spectrum of 7, Figure S53: 13 C NMR spectrum of 7, Figure S54: 1 H NMR
spectrum of 8, Figure S55: 13 C NMR spectrum of 8, Figure S56: 1 H NMR spectrum of 9, Figure S57:
13 C NMR spectrum of 9, Figure S58: 1 H NMR spectrum of 10, Figure S59: 13 C NMR spectrum of 10,

Figure S60: Structures of positive control for the bioassay, Table S1: COSY correlations of compounds
1–6, Table S2: HMBC correlations of compounds 1–6.
Author Contributions: Conceptualization, K.M. and M.I.S.; synthesis and isolation, M.I.S. and
N.H.U.; manuscript preparation, K.M., M.I.S., O.K. and K.S.; facilitated the research and supplied the
materials, S.S. and Y.Y.; Anti-plasmodium assay, O.K, K.Y. and B.-Y.A.C.; Anti-trypanosoma assay,
K.S.; spectrometric measurement, S.K. and H.O.; anti-Leishmania assay, K.M. and M.I.S. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was funded in part by Grants-in-Aid from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, and the Japan Society for the Promotion of Science (Nos.
18K06740, and 17K08336, 17K15465, and 16K18793). This work was also partly supported by the
Joint Usage/Research Center on Tropical Disease, Institute of Tropical Medicine, Nagasaki University
(2019-Ippan-1, 2020-Ippan-18) and Ohyama Health Foundation Inc.
Data Availability Statement: Data is contained within the article or supplementary material.
Acknowledgments: The authors are indebted for superconducting spectroscopic instruments at the
Natural Science Center for Basic Research and Development (N-BARD), Hiroshima University. The
Molecules 2021, 26, 1756 11 of 12

authors are grateful to the Indonesia Endowment Fund for Education (LPDP), Ministry of Finance,
the Republic of Indonesia for doctoral scholarships.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1–6 are available from the authors.

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