Discovery of bioactive phytoconstituentsfrom medicinal plants collected in Myanmar
Name of dispatched researcher（派遣研究者名）

Prema

Demonstrator

Instructor（指導教官，研究指導者）

Hla Ngwe

Professor and Head of
Department of Chemistry

Affiliation of Instructor（所属）
Host Collaborator（受入研究者）

Department of Chemistry,
University of Yangon,Myanmar
Hiroyuki Morita

教授

Abstract
Phytochemical investigation of chloroform extract of rhizomes of the Boesenbergia rotunda led to theisolation of seven known compounds such as pinostrobin (1), 4', 7-dimethyl kaempferol (2), pinocembrin (3),tectochrysin (4), galanal A (5), galanal B (6), and quercentin 3,7,4'-trimethylether (7). Their structures were
established on the basis of NMR spectroscopic analysis and by comparing with the reported data. In addition,
methanol and chloroform crude extracts and four isolates (1, 2, 5, 6) were tested for their cytotoxicity against
Form cancer
2-2（様式２－２）
lung(LK-2, A549), stomach cancer (ECC4), breast cancer (MCF7), cervix cancer (HeLa), prostate

Pinostrobin (1)Tectochrysin (4)4',7-Dimethoxy kaempferol (2)Galanal A (5)Pinocembrin (3)Galanal B (6)Quercetin 3,7,4¢trimethyl ether (7)Fig. 1. Structures of isolated compounds (1-7) from Boesenbergia rotunda.

－ 51 －
Table 2. Antiproliferative activity of crude extract of B. rotunda and isolated compounds (IC50 µg/mL or µM)
against lung (LK-2, A549), stomach (ECC4), breast (MCF7), cervix (HeLa), prostate (DU145) human cancer,

cancer (DU145), and normal human fibroblast (WI-38) cell lines. Among the tested compounds, galanal A (5)and B (6) exhibited cytotoxicity with IC50 values ranging between 5.98 ~38.25 μg/mL.
Keywords: Boesenbergia rotunda, diterpenoids, galanal A, galanal B, flavonoids, cytotoxicity.
1. Introduction
Cancer is a leading cause of disease worldwide. In the anticancer drug discovery, natural products
characterized from medicinal plants play an important role. Vincristine, irinotecan, etoposide, and paclitaxel are
examples of plant-derived compounds that are being employed in cancer treatment.1 According to GLOBOCAN
2012 report, the five most frequent cancers in Myanmar were lung, breast, cervix uteri, stomach, and liver. Manyof Myanmar medicinal plants have been used to prevent alleviate and cure human disease by Myanmar people
for time immemorial. In our ongoing research for the discovery of anticancer agents from Myanmar medicinal
plants, we have screened crude extracts of Myanmar medicinal plants against seven different human cancer celllines, including lung (LK-2, A549), stomach (ECC4), breast (MCF7), cervix (HeLa), prostate (DU145) humancancer, and normal human fibroblast (WI-38) cell lines. Among the crude extracts we have screened, CHCl3soluble extract of Boesenbergia rotunda exhibited potent cytotoxicity. B. rotunda is a perennial plant belongs
to the Zingiberaceae family. In Myanmar, it is locally known as Seik-phoo-chin. The appearance of rhizomes ofB. rotunda looks similar to B. pandurata. However, the taste is a little bit different from that of B. pandurata.
B. rotunda is distributed in tropical Asia including Myanmar, Bangladesh, Thailand, Cambodia, Vietnam,
Malaysia, and Indonesia.2 The rhizomes are considered to be anthelmintic and depurative. They are used totreat a range of conditions including colic, asthma, cough, obesity, and rheumatism. They are used externallyto treat skin conditions such as scurvy and itchy skins. They have been suggested as an application for treating
mental derangement. In Myanmar, Indonesia and Thailand, rhizomes are the chief ingredient of a decoction
given to women after childbirth to accelerate the lochia. Furthermore, the rhizome is popular for the treatmentof stomach cancer. The reported major constituents were cyclohexenyl chalcones and flavonoids. However, ourstudy on this plant suggested that the rhizome was lack of those reported compounds. The major constituents
recently we obtained from this rhizome were labdane diterpenoids and flavonoids.
2. Materials and Methods
2.1 Plant material
The rhizomes of B. rotunda (Seik-phoo-chin) were collected from Kyauk Tan Village, Bago Region
during September, 2016 and were identified and authenticated at Department of Botany, University of Yangon.
The samples were dried under the shade for a week, cut into very small pieces and made powder.
2.2 General experimental procedures
The NMR spectra were recorded at 500 MHz (1H NMR) and 125 MHz (13C NMR), respectively, on
a JEOL ECA500II spectrometer. Chemical shift values were expressed in δ (ppm) downfield from TMS asinternal standard. Open column chromatography was performed with normal-phase silica gel 60N, spherical,
neutral, 40−50 μm, (Kanto Chemical Co., Inc., Japan) and Cosmosil 75C18-OPN (Nacalai Tesque Inc., Kyoto,
Japan). Analytical TLC was carried out on precoated silica gel 60F254 and RP-18 F254 plates (0.25 or 0.50 mm
thickness,Merck). The cell lines LK-2, A549, HeLa, ECC 4 MCF 7, DU 145, and WI-38 were available andmaintained in our laboratory. Cell culture flasks and 96-well plates were from Corning (NY, USA).
－ 52 －

2.3 Preparation of Crude Extracts
The dried rhizome powder of B. rotunda (ca. 200 g) was extracted with 70% ethanol (1 L) undersonication for one hour and filtered. This procedure was repeated five times. The combined filtrate wasconcentrated under vacuum rotatory evaporator to obtain 22.5.g of 70% ethanol crude extract. The ethanol
crudeextract was then extracted with chloroform by sonication to effort the condensed chloroform extract (5.11 g),
which was kept for isolation of phytoconstituents.
2.4 Separation and Isolation of Secondary Metabolites
The CHCl 3 extract (5 g) was separated by column chromatography over silica gel with an-hexane:EtOAc gradient system, (90:10 to 0:100, v/v) followed by MeOH elution to obtain twelve
mainfractions. Fraction 3 (564.8 mg) was rechromatographed on silica gel with n-hexane:CH2Cl2:EtOAc
solvent system to give seven subfractions. Subfraction F3-1 was subjected to normal-phase preparative TLC
with C6H6:CH2Cl2 (2:1 v/v) to give compound 1 (15.8 mg) as yellow amorphous and compound 2 (15 mg) as
yellow needle crystals. Fraction 4 (324.5 mg) was rechromatographed on silica gel open column eluted with thesame solvent system to obtain six further subfractions. Compound 3 (4.2 mg) as colourless crystal was isolated
from subfraction F4-5 using normal-phase preparative TLC with n-hexane:CH2Cl2:EtOAc (20:20:1). Fraction 6(1.35 g) was rechromatographed on cosmosil 75C18-OPN with MeOH:H2O (15:1 v/v) to give 8 subfractions.
Subfraction F6-3 was separated by normal-phase preparative TLC with n-hexane:CH2Cl2:EtOAc (20:20:1) to givecompound 4 (3 mg). Subfraction F6-8 was purified by Sephadex LH 20 with MeOH followed by normal-phase
preparative TLC with n-hexane:CH2Cl2:EtOAc (2:2:1 v/v) to afford compound 5 (25.7 mg) and compound 6(50 mg), and Fraction 7 (2.43 g) was rechromatographed on cosmosil 75C18-OPN with MeOH:H2O (15:1 v/v) to give 3 subfractions. Subfraction F7-2 was separated by RP silica gel with MeOH: H2O (5:1 v/v) to obtaincompound 7 (3.1 mg) as pale yellow amorphous solid, which was recrystallized by CHCl3: MeOH (5:1).
Pinostrobin (1). Yellow amorphous solid, 1H NMR, CDCl3, δ ppm: 2.85 (1H, dd, J = 17.0, 3.0 Hz,H-3a), 3.12 (1H, t, H-3b), 3.81 (3H, s, 7-OCH3), 5.44 (1H, dd, J = 13.0, 3.0 Hz, H-2), 6.08 (2H, d, J = 2.3 Hz,
H-6, 8), 7.47 (5H, m, H-2', 3', 4', 5', 6'), 12.02 (1H, s, 5-OH). The 13C NMR, CDCl3, δ ppm: 43.3 (C-3), 55.1 (7OCH3), 79.2 (C-2), 94.2 (C-8), 95.1 (C-6), 103.1 (C-10), 126.1 (C-2', C-6'), 128.8 (C-3', C-4', C-5'), 138.3 (C-1'),
162.7 (C-5), 164.1 (C-9), 167.9 (C-7), 195.4 (C-4).
4',7-Dimethyl kaempferol (2). Yellow needle, 1H NMR, CDCl3, δ ppm: 3.89 (6H, s, 4', 7-OCH3), 6.38 (1H,d, J = 2.2 Hz, H-6), 6.49 (1H, d, J = 2.2 Hz, H-8), 7.04 (2H, d, J = 9.1 Hz, H-3', 5'), 8.19 (2H, d, J = 9.1 Hz,H-2',6'), 11.72 (1H, s, 5-OH). The 13C NMR, CDCl3, δ ppm: 55.5 (4'-OCH3), 55.9 (7-OCH3), 92.2 (C-8), 97.9 (C-6),
104.0 (C-10), 114.2 (C-2', C-6'), 123.2 (C-1'), 129.4 (C-3', C-5'), 135.7 (C-3), 145.7 (C-2), 156.9 (C-9), 160.8(C-5), 165.8 (C-7), 175.2 (C-4) .
Pinocembrin (3). Colourless crystal, 1H NMR, CD3OD, δ ppm: 2.69 (1H, dd, J=17, 3.0 Hz, H-3cis), 3.02
(1H, dd, J =13, 3.0 Hz, H-3trans), 5.37 (1H, d, J = 13, 3.0 Hz, H-2), 5.79 (1H, d, J = 2.0 Hz, H-8), 5.83 (1H, d, J
=2.0 Hz, H-6), 7.24-7.40 (5H, m, H-2', 3', 4', 5', 6'). The 13C NMR, CDCl3, δ ppm: 44.5 (C-3), 80.4 (C-2), 96.3
(C-8), 97.2 (C-6), 103.3 (C-10), 127.4 (C-2', C-6'), 129.6 (C-3', C-5'), 140.4 (C-1'), 156.9 (C-9), 164.7 (C-5),
165.5 (C-7), 197.3 (C-4).
Tectochrysin (4). White amorphous solid, 1H NMR, CDCl3, δ ppm: 3.84 (3H, s, 7- OCH3), 5.51 (1H, d,
J = 2.5 Hz, H-6), 5.95 (1H, d, J = 2.5 Hz, H- 8), 6.60 (1H, s, H-3), 7.38 (3H, m, H-3', H-4', H-5'), 7.53 (2H, m,
H-2', H-6'). The 13C NMR, CDCl3, δ ppm: 56.0 (-OCH3), 92.9 (C-8), 98.4(C-6), 105.9 (C-10), 106.1 (C-3), 126.5(C-2', C-6'), 129.3 (C-3', C-5'), 131.6 (C-1'), 132.0 (C-4'), 158.1 (C-9), 162.5 (C-5), 164.3 (C-2), 165.9 (C-7),－ 53 －

182.5 (C-4).
Galanal A (5). White amorphous solid, 1H NMR, CDCl3, δ ppm: 0.77 (3H, s, 20-CH3), 0.87 (1H, m,H-1a), 0.89 (3H, s, 19-CH3), 0.93 (1H, m, H-5a), 0.93 (3H, s, 18-CH3), 1.16 (1H, dd, J = 13.4, 4.1 Hz, H-3a),1.40 (1H, m, H-3b), 1.43 (1H, m, H-6b), 1.45 (1H, m, H-2a), 1.56 (1H, m, H-2b), 1.61 (1H, m, H-9), 1.67 (1H, m,H-5b), 1.78 (1H, m, H-1b), 1.78 (1H, m, H-6a), 2.28 (1H, m, H-7), 2.50 (1H, dd, J = 18.5, 8.3 Hz, H-11a), 2.65
(1H, m, H-14), 2.79 (1H, m, H-11b), 4.07 (1H, dd, J = 8.9, 3.1 Hz, H-5), 6.89 (1H, dd, J = 8.3, 1.6 Hz, H-12),
9.38 (1H, s, 16-CHO), 10.11 (1H, s, 17-CHO). The 13C NMR, CDCl3, δ ppm: 16.5 (C-20), 18.3 (C-2), 18.5 (C6), 21.1 (C-19), 23.5 (C-11), 27.5 (C-7), 28.4 (C-14), 33.1 (C-18), 33.3 (C-4), 38.4 (C-10), 38.5 (C-1), 41.6 (C-3),
53.6 (C-9), 55.4 (C-8), 55.6 (C-5), 71.2 (C-15), 142.2 (C-13), 156.1 (C-12), 193.3 (C-16), 206.5 (C-17).
Galanal B (6). White amorphous solid, 1H NMR, CDCl3, δ ppm: 0.76 (3H, s, 20-CH3), 0.77 (3H, s, 19CH3), 0.82 (1H, m, H-1a), 0.82 (1H, m, H-5a), 0.85 (3H, s, 18-CH3), 1.10 (1H, m, H-3a), 1.10 (1H, m, H-6b),1.26 (1H, m, H-5b), 1.38 (1H, m, H-3b), 1.48 (1H, m, H-9), 1.54 (1H, m, H-2a), 1.57 (1H, m, H-2b), 1.64 (1H, m,
H-6a), 1.85 (1H, dd, J = 12.6, 2.5 Hz, H-1b), 2.56 (1H, m, H-7), 2.63 (1H, dd, J = 16.4, 1.0 Hz, H-14b), 2.91 (1H,
dd, J = 16.4, 8.8 Hz, H-14a), 3.12 (1H, m, H-11b), 3.55 (1H, dd, J = 8.8, 1.0 Hz, H-15), 7.03 (1H, dd, J = 8.4, 3.9
Hz, H-12), 9.38 (1H, s, 16-CHO), 10.19 (1H, s, 17-CHO). The 13C NMR, CDCl3, δ ppm: 15.7 (C-20), 18.5 (C-2),
18.9 (C-6), 21.2 (C-19), 24.0 (C-11), 28.4 (C-14), 33.2 (C-18), 33.3 (C-4), 34.1 (C-7), 38.7 (C-1), 38.7 (C-10),
41.5 (C-3), 55.1 (C-8), 55.3 (C-5), 55.4 (C-9), 78.5 (C-15), 140.5 (C-13), 157.9 (C-12), 193.6 (C-16), 208.2 (C17).
Quercetin 3, 7, 4'-trimethyl ether (7). Pale yellow amorphous solid, 1H NMR, CDCl3, δ ppm: 3.88 (6H,s,3, 7-OCH3), 3.39 (3H, s, 4'-OCH3), 6.36 (1H, d, J = 2.2 Hz, H-6), 6.45 (1H, d, J = 2.2 Hz, H-8), 6.98 (1H, d,
J = 8.5 Hz, H-5'), 7.70 (1H, d, J = 2.2 Hz, H-2'), 7.73 (1H, dd, J = 8.5, 2.2 Hz, H-6'), 12.64 (1H, s, 5-OH). The
13

C NMR, CDCl3, δ ppm: 55.9 (4'-OCH3), 56.1 (7-OCH3), 60.2 (3-OCH3), 92.2 (C-8), 97.9 (C-6), 106.2 (C-10),

110.5 (C-5'), 114.5 (C-2'), 120.3 ( C-6'), 121.7 (C-1'), 138.6 (C-3), 145.6 (C-2), 150.2 (C-3'), 155.7 (C-4'), 156.8(C-9), 162.1 (C-5), 165.5 (C-7), 176.5 (C-4).
2.5 In Vitro Cytotoxic Activity
Cytotoxic activity of B. rotunda rhizome crude extracts and some isolated compounds were studied invitro using cancer cell lines such as LK-2, A549 (human lung cancer), ECC4 (human stomach cancer), MCF7
(human breast cancer), Hela (human cervix cancer), DU145 (human prostate cancer), and WI-38 (normal
human fibroblast). Briefly, each cell line was seeded in 96-well plates (2 × 103 per well) and cultured either in
α-MEM or DMEM at 37 °C under a 5% CO2 and 95% air atmosphere for 24 h. After the cells were washed with
PBS (Nissui Pharmaceuticals), serial dilutions of the samples were added. After 72 h incubation, the cells were
washed with PBS, and 100 μL of a-MEM or DMEM, containing 10% WST-8 cell counting kit solution (Dojindo;
Kumamoto, Japan), was added to the wells. After 2 h incubation, the absorbance at 450 nm was measured. The
different concentrations of the serial dilutions of the tested samples were 100-1 μg/mL for the crude extract,100-1 μM for the isolated compounds, and 100-1 μM for the positive control, respectively. Cell viability wascalculated from the mean values of data from three wells by using the following equation, and the cytotoxicity
was expressed as the IC50 (50% inhibitory concentration) value.
(%) Cell viability = 100 ´ [{Abs(test samples) - Abs(blank)}/{Abs(control) - Abs(blank)}]

Results and Discussion
Compound 1 was isolated as a yellow amorphous solid. The 1H NMR spectrum showed singlet of one－ 54 －

hydroxy proton at δH 12.02, one methoxy singal at δH 3.81 and a doublet aromatic proton at δH 6.08 (d, J = 2.3
Hz) attributable to meta coupling protons H-6 and H-8 of ring A. The multiplets of five aromatic protons at δH 7.47
confirmed the non-substitution of the B-ring. A doublet of doublet methine proton at δH 5.44 ( J = 13, 3 Hz), andone triplet and one doublet of doublet methylene protons at δH 3.12 and 2.85 were attributable to H-3 (cis) andH-3 (trans) of the C-ring. The 1H NMR spectrum shows the typical characteristics of flavanone. The 13C NMR
spectrum showed 16 carbon signals, including one carbonyl carbon, five quaternary carbons, eight methinecarbons, one methylene carbon, and one methoxy carbon. The 1H NMR and 13C NMR spectral data of 1 wereconsisted with those of reported pinostrobin.3
Compound 2 was isolated as a yellow needles crystal. The 1H NMR spectrum showed signals forflavonol substituted at H-6 (δH 6.38) and H-8 (δH 6.49) on the ring A and at H-3'/H-5' and H-2'/H-6' (δH 7.04 and8.19) on the ring B, respectively. In addition, a signal at δH 3.89 for two methoxy groups and a signal at δH 11.72
for a hydroxyl group in hydrogen bond were observed. The 13C NMR spectral data displayed signals for fifteen
carbon atoms, which chemical shifts are compatible with C-3 (δC135.7) on the ring C, C-6 (δC 97.9) and C-8 (δC
92.2) on the ring A, and C-3'/5'and C-2'/6' (δC 129.4 and 114.2) on the ring B, as well as one carbonyl (δC 175.2)
and two methoxy groups (δC 55.5 and 55.9) of a flavonol. All of the above mentioned 1H NMR and 13C NMR
spectral data of 2 were consisted with those of reported 4',7-dimethyl kaempferol.4
Compound 3 was isolated and re-crystallized as a colourless crystalline from ethyl acetate. In the 1H
NMR spectrum, two doublets at δH 5.79 (J = 2.0 Hz) and δH 5.83 (J = 2.0 Hz) were attributable to meta coupled
H-6 and H-8. A five protons multiplet at δH 7.24-7.40 confirmed the non-substitution of the B-ring. The 1H NMR
spectroscopic data suggested that the absence of H-3 proton and the presence of an ABX system of the typicalflavanone. The 13C NMR spectrum showed 15 carbon signals, including one methylene, eight methine, and six
quarternary carbons. The six carbon signals at δC 140.4, 127.4, 129.6, and 131.9 were assignable to C-1', C-2'/
C-6', C-3'/C-4', and C-5' of the mono-substituted aromatic ring (ring B), respectively. The six carbon signals at
δC 103.3, 164.7, 97.2, 165.5, 96.3, and 197.3 were assigned to be C-4a, C-5, C-6, C-7, C-8, and C-4 on the ring A,
and two carbon signals, δC 80.4 and 44.5, were located at C-2 and C-3, respectively. The 1H NMR and 13C NMR
completed the elucidation of the structure pinocembrin.5
Compound 4 was isolated as a white amorphous solid. The 1H NMR showed two doublet protons at
δH 5.51 (1H, d, J = 2.5 Hz, H-6) and 5.95 (1H, d, J = 2.5 Hz, H-8), suggesting the presence of meta coupledaromatic protons. A singlet at δH 6.60 was assigned to H-3, while two multiplets at δH 7.38 (3H, m, H-3', H-4',
H-5') and δH 7.53 (2H, m, H-2', H-6') belonged to the protons of the monosubstituted ring B. A singlet proton
signal at δH 3.84 was due to the presence of a methoxy group. Moreover, the 13C NMR spectrum showed thecorresponded signals including a methoxy carbon signal at δC 56.0, and a signal at a very low region (δC 182.5,
C-4), which was definitive of carbonyl carbon of the flavone structure. Four oxygenated aromatic carbon signals
were observed at δC 165.9 (C-7), 164.3 (C-2), 162.5(C-5) and 158.1 (C-9). The rest aromatic carbon signals
were observed from δC 140.0 to 92.9. 1H NMR and 13C NMR spectra data of isolated compound 4 were found to
be consistent with those of tectrochrysin. Therefore, compound 4 was identified as tectrochrysin.6
Compound 5 was isolated as a white amorphous solid. 1H NMR suggested the presence of 30 protons.
Three singlet signals (3H each) at δH 0.77, 0.89, and 0.93 indicated the presence of three CH3 groups. The
two singlet signals at δH 9.38 and 10.11 allow us to confirm the presence of two CHO groups. The doubletdoublet signal at δH 4.07 suggested the presence of a secondary carbinol proton (-CHO-) adjacent to a pair ofdiasterotopic methylene protons. This observation indicated that compound 5 contains the –CH2-CHOH-Cmoiety. The doublet-doublet signal at downfield, δH 6.89, was assigned due to the presence of an olefinic proton－ 55 －

of –C=CH-group, indicating that compound 5 possesses only an olefinic group. 1H-1H COSY spectrum revealed
the connectivity between δH 2.50, 2.79, 2.65, 4.07, and 6.89. An olefinic methine proton at δH 6.89 (dd, J = 8.3, 1.6
Hz, H-12) was coupled with the methylene protons at δH 2.50 (dd, J =18.5, 3.1 Hz, H-11a ) and δH 2.79 (m, Hb11). Oxygenated methine proton at δH 4.07 (d, J = 8.9, 3.1 Hz, H-15) was correlated with methylene proton at
Form 2-2（様式２－２）

δH 2.65 (H-14). One methylene proton at δH 2.79 (H-11b) was coupled with δH 2.50 (dd, J=18.5, 8.3, H-11) and1.61 (C-9). Similarly, one methylne proton at δ 0.93 (H-5) was coupled with δ 1.78 (Hb-6). 13C NMR spectrum
showed 20 signals, including two aldehyde carbons (δC 193.3 and 206.5), two olefinic carbons (δC 142.2 and156.1), and one carbinol carbon (δC 71.2). The remaining 15 carbon atoms were assigned as the sp3 methyl,
methylene, methane, and quarternary carbons. On the basis of 13C NMR, 1H NMR and COSY spectral data,
compound 5 was assumed as galanal A.7
1
Pinostrobin6 (1)kaempferol
Pinocembrin
Compoundwas obtained as a 4',7-Dimethoxy
white amorphoussolid. In (2)H NMR spectrum,
the singlet(3)proton signalat δH 10.22 indicated the presence of the CH-OH proton. The singlet proton signals at δ 9.40 and 10.22 indicatedthe presence of two aldehyde protons. The three quaternary singlet methyl signals at δH 0.76, 0.99, and 0.85 (3Heach) were coincident with those of reported galanal B. 13C NMR spectrum showed twenty carbon signals. The
upfield one methyl group and the downfield one carbonyl signal were simultaneously observed at δC 15.7 (C20) and δC 208.2 (C-17), respectively. 1H NMR, 13C NMR, and COSY spectral data were consisted with those
ofreported galanal B.7

Tectochrysin (4)Galanal A (5)Galanal B (6)Compound 7 was isolated as a pale yellow amorphous solid. The 1H NMR spectrum showed ABX
system at δH 7.70 (d, J = 2.2 Hz, H-2'), 6.98 (d, J = 8.5 Hz, H-5'), and 7.73 (dd, J = 8.5, 2.2 Hz, H-6') and a pairof doublets (J = 2.2 Hz) in the aromatic region at δH 6.36 and 6.45, three methoxyl signals (δH 3.99, 3.88, and3.88), and an OH-phenolic signal at δH 12.64. The 13C NMR spectrum showed 18 carbon signals. Among them,the signals at δC138.6 and 176.5 were characteristic for the flavonol structure and six oxyaryl signals (δC 145.6,
150.2, 155.7, 156.8, 162.1, and 165.5) indicated that compound 7 is a quercetin derivative. The above mentioned
Quercetin
ether(7)of compound 7 were similar to those of quercetin 3,7,4’-trimethyl ether.8
H NMR
and 133,7,4¢trimethyl
C NMR spectral
data

1Fig. 1.In
Structuresof isolated
compounds(1-7)
Boesenbergia
rotundathe cytotoxicity
assay(Table 2),
the from
MeOH
extract and theCHCl. extract of the B.rotunda rhizomes
3

Table 2. Antiproliferative activity of crude extract of B. rotunda and isolated compounds (IC50 µg/mL or µM)
against lung (LK-2, A549), stomach (ECC4), breast (MCF7), cervix (HeLa), prostate (DU145) human cancer,
and normal human fibroblast (WI-38) cell lines
Cell line
Samples

LK-2

A549

ECC4

MCF7

HeLa

DU145

WI-38

Pinostrobin (1)

>100

78.74

>100

>100

>100

>100

>100

4’, 7-Dimethyl
kaempferol (2)

8.79

>100

35.18

>100

>100

91.57

40.71

Galanal A (5)

4.38

28.20

32

38.62

5.10

3.57

2.60

Galanal B (6)

5.75

8.02

5.26

6.79

5.73

7.73

5.68

CHCl3-Extract

65.43

56.12

55.65

73.74

48.70

68.45

70.24

MeOH-Extract

70.02

57.42

60.32

73.06

70.07

70.75

75.88

>10

9.0

>10

>10

7.11

8.05

>10

a

a

5-Fluorouracil
Positive control

9－ 56 －

exhibited mild cytotoxicity against all the tested cancer cell lines. Among the tested compounds, galanal B (6)showed the strong cytotoxicity against all the tested cancer cell lines with IC50 values ranging from 5.26 to 8.02
μM. In contrast, galanal A (5) exhibited selectively cytotoxicity against LK-2 (IC50 4.38 μM), HeLa (IC50 5.10μM), DU145 (IC50 3.57 μM), and WI-38 (IC50 2.60 μM) cells with IC50 values of 4.38 μM, 5.10 μM, 3.57 μM,
and 2.60 μM, respectively. However, two flavonoid compounds, 1 and 2, did not show any activity against all
the tested cancer cells.
CONCLUSION
Pinostrobin (1), 4',7-dimethyl kaempferol (2), pinocembrin (3), tectochrysin (4), galanal A (5), galanal
B (6), and quercetin 3,7,4'-trimethylether (7) were isolated from B.rotunda rhizomes collected in Myanmar.
Furthermore, the cytotoxicity assay revealed that the crude extracts, galanal A (5), and galanal B (6) showed
cytotoxicity against human cancer cell lines. These observations may suggest that the crude extracts and its
phytoconstituents may be useful for the treatment and prevention of cancer.
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