New rotenoids and coumaronochromonoids from the aerial part of Boerhaavia erecta

Article abstract of DOI:10.1248/cpb.c12-01081

From the aerial part of Boerhaavia erecta L., three new rotenoids (3, 8, 10) and two new coumarono-chromonoids (6, 11) were isolated, together with ten known compounds. The structure of the new compounds was established by one dimensional (1D)- and 2D-NMR spectroscopy, as well as high resolution- electrospray ionization (HR-ESI)-MS analysis. The absolute configuration of compound 11 was determined by UV circular dichroism spectroscopy. Compounds were evaluated for their cytotoxic activity against HeLa (human epithelial carcinoma), NCI-H460 (human lung cancer) and MCF-7 (human breast cancer) cell lines at the concentration of 100 μg/mL. Rotenoids 3, 4 and 5 showed a strong cytotoxic activity against HeLa cell line and rotenoids 5 and 8 showed good activity against MCF-7 cell line.

Full text of DOI:10.1248/cpb.c12-01081

6
24
Regular Article
Chem. Pharm. Bull. 61(6) 624–630 (2013)
Vol. 61, No. 6
New Rotenoids and Coumaronochromonoids from the Aerial Part of
Boerhaavia erecta
a
a
b
b
Thi My Lien Do, Anh Vu Truong, Travis George Pinnock, Lawrence Michael Pratt,
c
d
e
,a
Shigeki Yamamoto, Hitoshi Watarai, Dominique Guillaume, and Kim Phi Phung Nguyen*
a
Department of Organic Chemistry, University of Science, National University–Ho Chi Minh City; 227 Nguyen Van
b
Cu Str., Dist. 5, Ho Chi Minh 748355, Vietnam: Department of Physical, Environmental and Computer Sciences,
Medgar Evers College, The City University of New York; 1650 Bedford Ave., Brooklyn, NY 11225, United States:
c
Department of Chemistry, School of Science and Technology, Kwansei Gakuin University; 2–1 Gakuen, Sanda,
d
Hyogo 669–1337, Japan: Institute for Nanoscience Design, Osaka University; 1–3 Machikaneyama-cho, Toyonaka,
Osaka 560–8531. Japan: and School of Pharmacy; 51 rue Cognacq Jay, 51096 Reims Cedex, France.
e
Received December 18, 2012; accepted March 10, 2013
From the aerial part of Boerhaavia erecta L., three new rotenoids (3, 8, 10) and two new coumarono-
chromonoids (6, 11) were isolated, together with ten known compounds. The structure of the new compounds
was established by one dimensional (1D)- and 2D-NMR spectroscopy, as well as high resolution-electrospray
ionization (HR-ESI)-MS analysis. The absolute configuration of compound 11 was determined by UV cir-
cular dichroism spectroscopy. Compounds were evaluated for their cytotoxic activity against HeLa (human
epithelial carcinoma), NCI-H460 (human lung cancer) and MCF-7 (human breast cancer) cell lines at the
concentration of 100µg/mL. Rotenoids 3, 4 and 5 showed a strong cytotoxic activity against HeLa cell line
and rotenoids 5 and 8 showed good activity against MCF-7 cell line.
Key words Boerhaavia erecta; rotenoid; coumaronochromonoid; flavonoid
Boerhaavia is a genus of about 40 species mostly distrib- Boerhaavia erecta aerial part led to the isolation of fif-
1)
uted in tropical and subtropical areas. Boerhaavia erecta L. teen compounds. Among those ten were rapidly iden-
1
2)
12)
Nyctaginaceae, is a puberulous and erect annual herb which tified as boeravinone
G
(1),
boeravinone
H
C
(2),
(5),
2)
13)
14)
grows throughout the tropical regions of Africa and Asia.
boeravinone
C
J
(4),
(7),
10-demethylboeravinone
cucumegastigmane (9),
1
5)
16)
In contrast with B. diffusa whose phytochemistry has been boeravinone
isovitexin
3
–6)
17)
18)
intensively studied,
tochemical studies have been reported on B. erecta
though these studies have evidenced an in vivo antimalarial (14), and kaempferol 3-O-α-l-rhamnopyranosyl-(1→6)-β-d-
to the best of our knowledge little phy- (12),
quercetin 3-O-β-d-glucopyranoside (13), isorham-
7
–10)
even netin 3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside
18)
18)
activity for B. erecta extracts in mice parasitized with Plas- glucopyranoside (15) (Fig. 1) by comparison of their spectral
modium berghei berghei. Betacyanins and phenolics would be data with those reported in the literature. The five remaining
9)
the metabolites responsible for this activity.
unknown compounds included three rotenoids (compounds 3,
As a part of an ongoing research program aimed at study- 8, 10) and two coumaronochromonoids (compounds 6, 11).
ing Vietnamese medicinal plants, we recently started the ex- Their structures were elucidated as follows.
haustive phytochemical analysis of some Boerhaavia species.
Boeravinone K or 8-methoxy-10-demethoxycoccineone E
Preliminary chemical investigation of the aerial part of B. (3) was isolated as pale yellow needles and its specific rotation
2
3
erecta has already led us to isolate 2,6-dimethoxybenzoqui- was [α]D −260 (c=0.001, MeOH). Its positive-ion electrospray
none, (+)-catechin, isorhamnetin 3-O-β-d-glucopyranoside ionization (ESI) mass spectrum showed a pseudomolecular
1
1)
+
1
and rutin. Herein, we report the isolation and identification ion peak at m/z 345.1 [M+H] . Its H-NMR spectrum indi-
13
of five new compounds from the ethyl acetate extract of the cated the presence of 16 protons and its C-NMR spectrum
aerial part of B. erecta. Those compounds are three new displayed resonances for 18 signals. Therefore, the molecular
rotenoids: 8-methoxy-10-demethoxycoccineone
boeravinone K, 3), 6-demethylboeravinone G (named boera- mensional (1D)– H-NMR and H– H correlation spectroscopy
E
(named formula C H O was assigned to compound 3. In the one di-
18 16 7
1
1
1
vinone M, 8), and 10-demethylboeravinone C 11-O-β-d- (COSY) spectra, an ABC signal system was observed [δ 4.68
H
glucopyranoside (named boeravinone N, 10), and two new (1H, dd, 9.0, 7.0Hz, H-6a), 4.50–4.54 (1H, m, H-6β), 4.52
coumaronochromonoids: 10-demethylboeravinone J (named (1H, dd, 9.5, 3.5Hz, H-6α)], indicating the presence of an iso-
1
boeravinone L, 6) and 10-demethyl-6a,12a-dihydroboerhavi- lated –O–CH–CH –O– system. The H-NMR spectrum also
2
none J 3-O-β-d-glucopyranoside (named boeravinone O, 11). showed four signals that were unambiguously assigned to the
Cytotoxic activity against HeLa (human epithelial carcinoma), four aromatic CHs of a rotenoid A-ring at δ 8.33 (1H, dd, 8.0,
H
NCI-H460 (human lung cancer), and MCF-7 (human breast 1.5Hz, H-1), 7.07 (1H, dt, 8.0, 1.0Hz, H-2), 7.33 (1H, dt, 7.5,
cancer) cell lines at the concentration of 100µg/mL was de- 2.0Hz, H-3) and 6.93 (1H, dd, 8.5, 1.0Hz, H-4). Finally, the
1
termined.
H-NMR spectrum of 3 also displayed signals for one chelated
hydroxyl proton (δ 11.72, s), one aromatic proton (δ 6.19, s),
H
H
Results and Discussion
two methoxy groups [δ 3.92 (3H, s) and 3.83 (3H, s)], and one
Chemical examination of the ethyl acetate extract of isolated hydroxyl proton (δH 2.82, s). The C-NMR spectrum
H
13
of 3 showed the presence of a ketone function (δC 192.9),
The authors declare no conflict of interest.
seven aromatic quaternary carbons (δC 161.7, 161.4, 154.9,
*
To whom correspondence should be addressed. e-mail: kimphiphung@yahoo.fr
© 2013 The Pharmaceutical Society of Japan

June 2013
625
Fig. 1. Chemical Structures of Isolated Compounds
Fig. 2. Structures of 3, 5, 6, 8, 10, and 11 with Key HMBC Correlations
19)
1
1
(
52.1, 129.4, 118.6, 101.4), five aromatic methine carbons (δC cineone E, a compound in which positions 9 and 10 are
31.2, 131.1, 121.5, 117.5, 94.1), one aliphatic methine carbon substituted with a methoxy group and position 11 with an OH
δC 76.4), one aliphatic quaternary carbon (δC 66.2), and one group. The heteronuclear multiple bond connectivity (HMBC)
methylene carbon (δ 61.6). It also confirmed the presence of spectrum of 3 (Fig. 2) indicated that the chelated hydroxyl
C
two methoxy groups (δ 61.5, 56.4).
group (δ 11.72) is located at C-11 due to its correlations with
C
H
The spectral pattern of 3 closely resembles that of coc- signals at δC 161.4 (C-11), 101.4 (C-11a), 94.1 (C-10). The sin-

6
26
Vol. 61, No. 6
13,19,22)
glet at δH 6.19 showed correlations with signals at δC 161.4 of 12a-hydroxyrotenoids of the family Nyctaginaceae,
C-11), 101.4 (C-11a), 129.4 (C-8). It was therefore unambigu- the absolute configurations at C-6a and C-12a in 3 are likely
ously attributed to H-10. to be as in boeravinone C, coccineone E and other related
(
The presence of a methoxy group at position C-8 influences natural rotenoids. The structure of compound 3 was deter-
the splitting patterns of protons H-6a and H-6. In coccineone mined as (6aα,12aβ)-6a,12a-dihydro-11,12a-dihydroxy-8,9-
19)
E, chemical shift and multiplicity values (CDCl ) of these dimethoxy[1]benzopyrano[3,4-b][1]benzopyran-12(6H)-one or
3
protons are δ 4.77 (dd, 10.0ax-ax, 5.5ax-eq Hz) for H-6a, 4.48 (t, Boeravinone K.
H
10.0Hz) for H-6β, and 4.44 (dd, 10.0gem, 5.5eq-ax Hz) for H-6α.
Boeravinone L or 10-demethylboeravinone J (6) was ob-
In 3, the chemical shift and multiplicity values (CDCl ) of tained as a white amorphous powder. Its molecular formula
3
H-6a and H-6 protons are δH 4.68 (dd, 9.0, 7.0Hz), 4.50–4.54 determined as C H O through the pseudomolecular ion peak
15
8
6
+
(
m), and 4.52 (dd, 9.5, 3.5Hz), respectively.
m/z 285.0383 [M+H] in the high resolution (HR)-ESI-MS
13
The circular dichroism (CD) and X-ray analyses are used to spectrum was supported by its C-NMR spectrum in which
establish the absolute configuration of natural 12a-hydroxyro- fifteen carbons could be detected (see Table 1). The polariza-
tenoids. However, a literature search showed that the geometry tion transfer (DEPT) NMR spectra of 6 displayed five aromat-
1
of the B/C-ring junction of 12a-hydroxyrotenoids could be as- ic methine and ten aromatic quaternary carbons. The H-NMR
signed by analysis of their chemical shift value of H-1 in their spectrum of 6 showed a singlet of a chelated hydroxyl proton
1
H-NMR spectra as well as the comparison of their optical at δ 12.97, five aromatic methine signals between δ 6.30 and
H
H
rotation value with those of known compounds. Literature re- 7.80. The two doublet signals at δ 7.80 (1H, J=8.0Hz, H-6′),
H
vealed that the chemical shift value of H-1 would be δ 6.6–7.0 7.12 (1H, J=2.0Hz, H-3′) and one double doublet signal at δH
H
for
a
cis-junction as in 4′,5′-dihydro-11,5′-dihydroxy-4′- 7.01 (1H, J=8.0, 2.0Hz, H-5′) were assigned unambiguously
2
0)
21)
3)
13
methoxytephrosin, or in (6aα,12aα)-12a-hydroxyelliptone,
and around δH 8.0 for a trans-junction as in boeravinone C.
to a 1,2,4-trisubstituted A-ring. The C-NMR spectrum of 6
1
showed the presence of a conjugated carbonyl (δ 181.1), nine
C
Further it has been observed that natural trans-junction 12a- aromatic quaternary carbons (δC 165.8, 164.4, 164.1, 157.4,
hydroxyrotenoids would be either 6aα,12aβ-configuration as 156.2, 151.5, 115.3, 104.5, 98.4) and five aromatic methine
19)
22)
in coccineone E and abronione or 6aβ,12aα-configuration carbons (δC 122.4, 114.7, 100.8, 99.6, 95.7). The spectral pat-
2
3)
24)
as in (+)-12a-epimilletosin and usararotenoid C. Those tern of 6 closely resembles that of sophorophenolone, isolated
2
5)
with a 6aα,12aβ-configuration display negative specific rota- from Astragalus membranaceus. Only the methoxy group
13,19,22)
tion,
positive specific rotation.
The chemical shift value for H-1 (δH 8.33) of 3 is strongly
while those with a 6aβ,12aα-configuration display at the position C-7 in sophorophenolone was lacking in 6 sug-
2
3,24)
gesting its replacement by a hydroxyl group. The series of
JH–C correlations detected in the HMBC spectrum of 6 (Fig.
2
,3
deshielded when compared to the value observed for rote- 2) confirmed the presence of a coumaronochromone skeleton
noids with cis-B/C ring junction indicating that the B/C ring and the location of the substituents. Thus 6 was identified
junction in 3 has a trans-geometry. It exhibited negative as 5,7,4′-trihydroxycoumaronochromone or Boeravinone L or
optical rotation, therefore, from biogenetic considerations 10-demethylboeravinone J.
Table 1. NMR Spectroscopic Data of Compounds 6 and 11
Compound 6 (DMSO-d6)
δH, J (Hz)
Compound 11 (C D N)
5 5
Position
δC
δH, J (Hz)
δC
2
3
4
OH
5
6
7
8
9
0
1
2
3
4
5
6
—
—
—
165.8
98.4
181.1
—
164.1
100.8
164.4
95.7
156.2
104.5
115.3
151.5
99.6
6.57 (d, 7.5)
4.38 (d, 7.5)
104.0
76.8
179.3
—
166.4
95.1
162.2
100.4
163.3
105.8
116.7
158.1
114.9
158.1
122.5
123.6
—
—
—
–
12.97 (s)
—
6.36 (d, 2.0)
6.75 (d, 2.0)
—
—
6.59 (d, 2.0)
6.73 (d, 1.5)
—
—
—
—
—
—
—
—
1
′
′
′
′
′
′
7.12 (d, 2.0)
—
7.01 (dd, 8.0, 2.0)
7.80 (d, 8.0)
8.54 (d, 2.0)
—
7.81 (dd, 8.5, 2.0)
7.27 (d, 8.5)
157.4
114.7
122.4
β-Glc
1″
2″
3″
4″
5″
6″
—
—
—
—
—
—
—
—
—
—
—
—
6.35 (d, 7.5)
4.32–4.36 (m)
4.38–4.41 (m)
4.26–4.30 (m)
4.04–4.09 (m)
104.6
76.6
79.0
72.0
79.5
63.1
4.40–4.46 (m), 4.26–4.30 (m)

June 2013
627
Boeravinone M or 6-demethylboeravinone G (8) was iso- spectrum (Table 2) of 8 showed signals for a conjugated car-
lated as white needles. Its molecular formula was determined bonyl (δC 180.0), nine aromatic quaternary carbons (δC 165.5,
as C H O through its pseudomolecular ion peak at m/z 161.8, 157.8, 156.3, 146.4, 136.8, 117.1, 109.0, 105.4), six aro-
17
12
7
+
3
51.0474 [M+Na] in the HR-ESI-MS spectrum. This molecu- matic methine carbons (δ 121.7, 116.6, 116.0, 98.5, 92.7, 87.6),
C
lar formula is well consistent with the presence of 12 protons and one methoxy group (δC 56.2). The HMBC spectrum of 8
the H-NMR spectrum and 17 signals in the C-NMR spec- showed correlations between the hemiacetal signal at δH 6.22
trum of 8. The H-NMR spectrum of 8 (Table 2) showed ten and signals at δ 136.8 (C-4a), 157.8 (C-6a) and 109.0 (C-12a),
1
13
1
C
signals: a chelated hydroxyl proton (δ 12.87, 11-OH), two iso- as well as between the signal of 6-OH at δ 8.14 (d, J=6.5Hz)
H
H
lated hydroxyl protons (δ 9.40, 4-OH and δ 8.14, 6-OH), six and signals at δC 87.6 (C-6), 157.8 (C-6a). Complete analysis
H
H
methine resonances (two double doublets, one triplet and three of the HSQC and HMBC data (Fig. 2) for 8 resulted in its for-
doublets) in the region between δ 6.20ppm and 8.20ppm and mulation as 4,6,11-trihydroxy-9-methoxy[1]benzopyrano[3,4-
H
a methoxy signal at δH 3.88. The two meta-coupled protons b][1]benzopyran-12(6H)-one or 6-demethylboeravinone G.
at δH 6.73 and 6.46, each was a doublet with J=2.0Hz, were
Compound 5 was identified as a 12a-hydroxyrotenoid
attributed to H-8 and H-10, respectively in D-ring. The triplet derivative from mass and NMR spectroscopy data. H– H
1
1
at δH 6.92 (J=8.0Hz, H-2) was ortho-coupled with both the COSY, HSQC, HMBC spectral data of 5 were similar to
2
3
doublet at δH 8.14 (J=8.0, 1.5Hz, H-1) and the doublet at δ
those of (−)-4,11,12a-trihydroxy-9-methoxyrotenoid {[α]
D
14)
H
1
1
6
.85 (J=8.0, 1.5Hz, H-3). The H– H COSY spectrum of 8 −339 (c=1.1, MeOH)}, isolated from B. diffusa and B. coc-
26)
confirmed that these three aromatic methines were arranged cinea, although there was a disparity in the optical rotation
in a continuous sequence in A-ring. The remaining proton values. Based on the 2D-NMR data (Fig. 2), all chemical
resonated at δH 6.22 (d, J=6.0Hz). The heteronuclear single shift values for protons and carbons of 5 are precisely pointed
quantum coherence (HSQC) spectrum of 8 confirmed that out. It turned out that some signals (C-7a and C-11, C-4 and
2
6)
this signal at δH 6.22 was the hemiacetal proton H-6 and its C-4a, C-8 and C-10) had been interchanged in literature.
corresponding carbon resonated at δC 87.6 (DMSO-d ). Such Compound 5 was identified as (6aS,12aR)-4,11,12a-trihydroxy-
6
up-field shifted chemical shift value for C-6 was also observed 9-methoxyrotenoid or 10-demethylboeravinone C.
5
)
13
in boeravinone B (δC 89.5 in pyridine-d5). The C-NMR
Boeravinone N or 10-demethylboeravinone C 11-O-β-d-
Table 2. NMR Spectroscopic Data of Compounds 3, 8, and 10
Compound 3 (CDCl3)
δH, J (Hz)
Compound 8 (DMSO-d6)
Compound 10 (CD OD)
3
Position
1
δC
δH, J (Hz)
δC
δH, J (Hz)
δC
8.33 (dd, 8.0, 1.5)
131.2
118.6
121.5
131.1
—
8.14 (dd, 8.0, 1.5)
—
6.92 (t, 8.0)
6.85 (dd, 8.0, 1.5)
9.40 (s)
116.6
117.1
121.7
116.0
7.40 (dd, 8.0, 1.5)
123.7
121.4
121.7
116.9
—
1
2
3
a
—
7.07 (dt, 8.0, 1.0)
7.33 (dt, 7.5, 2.0)
—
—
6.86 (t, 8.0)
6.81 (dd, 8.0, 1.5)
–
OH
4
—
6.93 (dd, 8.5, 1.0)
—
117.5
154.9
—
—
—
146.4
136.8
—
—
—
146.9
144.5
—
4
OH
6
a
–
—
8.14 (d, 6.5)
6.22 (d, 6.0)
4.52 (dd, 9.5, 3.5)
61.6
87.6
4.46 (dd, 9.5, 4.5)
62.7
4
.50–4.54 (m)
4.39 (dd, 11.5, 10.0)
6
7
a
a
4.68 (dd, 9.0, 7.0)
76.4
152.1
61.5
129.4
56.4
161.7
94.1
—
161.4
101.4
192.9
—
—
—
—
157.8
156.3
—
92.7
56.2
165.5
98.5
—
161.8
105.4
180.0
—
4.66 (dd, 11.5, 4.5)
77.8
163.8
—
97.3
56.4
167.3
99.8
—
162.1
107.5
190.5
—
—
3.83 (s)
—
3.92 (s)
—
6.19 (s)
11.72 (s)
—
—
—
2.82 (s)
—
—
—
–
–
OCH3
8
OCH3
9
0
OH
6.73 (d, 2.0)
3.88 (s)
6.32 (d, 2.5)
3.85 (s)
—
—
1
6.46 (d, 2.0)
12.87 (s)
6.63 (d, 2.5)
–
—
—
—
—
—
—
1
1
1
1
1a
2
—
—
—
—
—
–
OH
2a
β-Glc
1
66.2
109.0
68.8
1′
2′
3′
4′
5′
6′
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.85 (d, 8.0)
3.57 (dd, 9.0, 8.0)
3.50 (t, 9.0)
3.38 (dd, 10.0, 8.5)
3.45–3.49 (m)
104.5
74.9
77.4
71.5
78.7
62.6
3.92 (dd, 12.0, 2.5)
3
.70 (dd, 12.0, 6.0)

6
28
Vol. 61, No. 6
glucopyranoside (10) was isolated as a yellow wax. The mo- mone partial structure in which C-5 and C-7 in the D-ring
lecular formula of compound 10 was determined as C H O were oxygenated carbons and so was C-4′ in the A-ring.
through its pseudomolecular ion peak at m/z 515.1320 [M+ Further analysis showed that C-4′ was not linked to a free
2
3
24 12
+
1
Na] in the HR-ESI mass spectrum. H-NMR spectra of 10 hydroxyl group but to an O-glucose group. This was proved
and 5 exhibited a very similar pattern of a 12a-hydroxyrot- by the chemical shift values of carbons C-3′, C-4′, C5′ were
enoid except for the lack of the signal of the chelated hydroxyl shifted down-field compared to those of 6.
proton (11-OH) and the appearance of signals corresponding to
The coumaronochromone moiety of 11 possesses two chiral
a hexopyranosyl moiety. The presence of a β-d-glucopyranosyl centers at C-2 and C-3, therefore, a possible configuration of
moiety was demonstrated by the doublet signal of an anomeric compound 11 is (2S,3S) or (2R,3R). To determine the absolute
proton at δH 4.85 (d, J=8.0Hz, H-1′) which had COSY cor- configuration of 11, ultraviolet CD spectroscopy was applied.
relation chains with H-2′/H-3′/H-4′/H-5′/H-6′ in the zone from The CD spectrum of compound 11 in methanol showed a
3.3ppm to 4.9ppm. HMBC experiments confirmed the attach- strong positive peak at 251nm, a weak negative at 269nm
ment of the β-d-glucopyranosyl moiety to the rotenone skel- and a broad negative band at around 340nm. The former two
eton at the position C-11 due to the cross peak between H-1′ bands were assigned to the π→π* transitions and the later
(
δ 4.85) and C-11 (δ 162.1). HMBC experiments also showed to the n→π* transition of the acetophenone chromophore
H C
2
7)
that the methoxy group was attached to the aglycone at C-9.
as observed in CD spectra of other flavonoids. Because
The sugar moiety was identified as d-(+)-glucose by the the sign of the n→π* CD band is known to be insensitive
2
8)
acidic hydrolysis and using TLC to compare the hydrolysate to the substitution of phenyl ring, it can be used for the
with the authentic d-(+)-glucose. The B/C-ring junction of determination of the absolute configuration at C2 position in
2
7,29)
the 12a-hydroxyrotenoid aglycone was considered to be trans acetophenone-containing molecule.
Compound 11 with 2S
due to the chemical shift value of H-1 (δH 7.40 in CD OD) configuration possesses a conformation with M helicity so that
3
2
8,29)
and the negative optical rotation value of the aglycone after it will exhibit a negative cotton effect at the n→π* band.
the acidic hydrolysis. The absolute configurations at C-6a and Accordingly, the absolute configuration (2S,3S) was assigned
C-12a in 10 were expected to be the same as those in 3 and to compound 11.
5
, as they were isolated from the same materials and possess-
Combined data allowed to identify compound 11 as
13,19,22)
ing similar spectral data.
The structure of compound (2S,3S)-5,7,4′-trihydroxy-2,3-dihydrocoumaronochromone
1
0 was assigned as 10-demethylboeravinone C 11-O-β-d- 4′-O-β-d-glucopyranoside.
glucopyranoside.
Cytotoxicity assay of nine compounds 3–6, 8–11, and 15
Boeravinone O (11) was obtained as a yellow wax. Its against the HeLa, MCF-7 and NCI-H460 cell lines was evalu-
molecular formula was determined as C H O through the ated by using the Sulforhodamine B (SRB) method (Table
21
20 11
+
pseudomolecular ion peak at m/z 471.0891 [M+Na] in the 3). At a concentration of 100µg/mL, compounds 3, 4, and 5
13
HR-ESI-MS spectrum. The C-NMR spectrum (Table 1) of showed the highest cytotoxic activity against HeLa. Inhibi-
1
1 showed signals for a conjugated carbonyl (δ 179.3), seven tion reached 81 to 91 percent for compounds 5 and 8 against
C
aromatic quaternary carbons (δC 166.4, 163.3, 162.2, 158.1, MCF-7 at the concentration of 100µg/mL. Poor solubility of
158.0, 116.7, 105.8), five aromatic methine carbons (δC 123.6, 3, 4, 5, and 8 in the biological medium prevented an accurate
1
22.5, 114.9, 100.4, 95.1), two acetal carbons (δ 104.6, 104.0) IC50 determination.
C
and 6 carbinol carbons in the region between δ 79ppm and
Our results confirm previous statements suggesting that
3ppm. The H-NMR spectrum of 11 showed the presence of plants of the family Nyctaginaceae are the source of simple
,2,4-trisubstituted and 1,2,3,5-tetrasubstituted aromatic rings: rotenoids. During this study, we have isolated seven rotenoids
C
1
6
1
δH 8.54 (1H, d, J=2.0Hz, H-3′), 7.81 (1H, dd, J=8.5, 2.0Hz, from the aerial part of B. erecta, three dehydrorotenoids (1, 2,
H-5′), 7.27 (1H, d, J=8.5Hz, H-6′), 6.75 (1H, d, J=2.0Hz, 8) and four 12a-hydroxyrotenoids (3, 4, 5, 10). Five rotenoids
1
H-6) and 6.73 (1H, d, J=1.5Hz, H-8). The H-NMR spectrum do not possess a methyl group at the C-10 position (1, 3, 5,
also showed signals for protons of a sugar moiety. This sugar 8, 10) and two do (2, 4), in contrast to the fact that almost
was identified as β-d-glucopyranose by comparison of the all known natural rotenoids contain an isoprenoid-derived
1
3
C-NMR data of 11 with those of other glucosides isolated in substituents usually at the C-8 position and occasionally at
16–18)
23)
the same material.
The β configuration of the glucose resi- the C-10 position. To the best of our knowledge, our study
due was deduced from its anomeric proton at δH 6.35 (1H, d, also constitutes the first report of a rotenoid glycoside (10) in
J=7.5Hz, H-1″) and its corresponding acetal carbon resonated the Boerhaavia genus. We also isolated three coumaronochro-
at δC 104.6. The remaining doublet at δH 6.57 (1H, d, 7.5Hz, mones, a small subclass of the isoflavonoids. Known natural
H-2) correlated with the second acetal carbon at δC 104.0 in coumaronochromones are often prenylated at C-6, C-8 and
2
3)
the HSQC spectrum of 11. This characteristic carbon signal C-3′ and only three O-substituted coumaronochromones:
15)
was assigned to the position C-2 of the coumaronochromone boeravinone J in B. diffusa, sophorophenolone in Astragalus
1
1
25)
30)
moiety. The H– H COSY spectrum of 11 showed that proton membranaceus and coccineone A in B. coccinea were
H-2 at δH 6.57 was adjacent to another proton at δH 4.38 (1H, isolated before.
d, 7.5Hz, H-3) whose carbon resonated at δC 76.8. The large
downfield chemical shift value of this aliphatic methine car- Experimental
bon revealed that it was next to more than one electron–with-
drawing group and was then assigned to the position C-3 of recorded on a Bruker Avance III spectrometer using residual
the aglycone. solvent signal as internal reference: chloroform-d δH 7.24,
Comparison of the 1D and 2D spectral data of 6 and 11 δC 77.23; methanol-d δH 3.31, δC 49.15; DMSO-d δ 2.50,
General Experimental Procedures NMR spectra were
4
6
H
(
Table 1) revealed that they both possessed a coumaronochro- δC 39.51; (CD ) CO δH 2.09, δC 206.31, 30.6; and pyridine-
3 2

June 2013
629
Table 3. Cell Growth Inhibitory Effects of the Purified Compounds
Inhibition of cell growth (%)
NCI-H460
a)
No.
Compound
HeLa
MCF-7
b)
1
2
3
4
5
6
7
8
9
Boeravinone K (3)
Boeravinone C (4)
10-Demethylboeravinone C (5)
Boeravinone L (6)
Boeravinone M (8)
Cucumegastigmane (9)
Boeravinone N (10)
Boeravinone O (11)
Kaempferol 3-O-rutinoside (15)
Camptothecin (positive control)
91.14± 0.17
89.39± 1.35
84.20± 0.90
7.76± 1.64
15.69± 5.50
5.23± 8.41
19.85± 4.89
7.43± 6.02
−4.73± 1.37
61.0± 4.78
54.73± 6.70
4.46± 13.85
5.15± 2.36
7.19± 4.72
48.52± 1.58
2.55± 5.37
31.78± 4.20
7.27± 2.69
3.08± 1.43
77.9± 2.30
30.53± 2.30
74.04± 6.29
81.37± 5.17
8.00± 0.97
85.08± 4.42
0.46± 8.25
35.67± 3.68
5.83± 12.60
−6.15± 1.49
47.0± 1.86
c)
a) The compounds were tested at the concentration of 100µg/mL. b) The presented data are means of three experiments± S.D. c) Camptothecin was tested at the concentra-
tion of 0.01µg/mL for MCF-7 and NCI-H 460 and of 1µg/mL for HeLa.
d5 at δH 8.74, 7.58 and 7.22, δC 123.87, 135.91, 150.35. The absorbance compared to the control (non-treated cells).
HR-ESI-MS were recorded on a HR-ESI-MS MicroOTOF-Q
Plant Material Boerhaavia erecta L. was collected at
mass spectrometer or on a LC-Agilent 1100 LC-MSD Trap Thu Duc District, Ho Chi Minh City, Vietnam in March 2009.
spectrometer. Melting points were determined on Maquenne The material was identified by botanist Vo Van Chi. A vouch-
block and are uncorrected. Optical rotations (MeOH) were er specimen (No US-A002) was deposited at the Herbarium of
measured on a Kruss digital polarimeter. Absorption and CD the Department of Organic Chemistry, Faculty of Chemistry,
spectra were measured on a JASCO V-570 spectrophotometer University of Science, National University, Ho Chi Minh City,
and on a JASCO J-820E spectropolarimeter, respectively. TLC Vietnam.
was carried out on precoated Silica gel 60 F254 or Silica gel 60
RP-18 F254S (Merck). Spots were visualized by spraying with erecta (3.8kg) were exhaustedly extracted with methanol
0% aqueous H SO or 5% ferric chloride solutions followed by maceration at room temperature, then evaporated under
Extraction and Isolation Air-dried aerial parts of B.
1
2
4
by heating. Gravity column chromatography was performed reduced pressure to afford a residue (245g). This latter was
with Silica gel 60 (0.040–0.063mm, Himedia).
suspended in water and partitioned between petroleum ether
31,32)
Biological Assays
Determination of cytotoxic ac- then ethyl acetate to afford a petroleum ether extract (38.1g),
tivities against the HeLa (human epithelial carcinoma), MCF-7 an ethyl acetate extract (25.1g), and the remaining aqueous
human breast cancer) and NCI-H460 (human lung cancer) solution. The ethyl acetate extract was subjected to silica
(
cell lines of tested samples were performed at the concentra- gel column chromatography using gradient elution of ethyl
tion of 100µg/mL using the antiproferative Sulforhodamine acetate–methanol (10:0–0:10) to give fractions EA.A to
B (SRB) assay with camptothecin as the positive control. All EA.F. Fraction EA.B (4.5g) was subjected to silica gel column
cells were cultured in E’MEM medium (Eagle’s Minimal Es- chromatography eluted with chloroform–methanol (98:2) to
sential Medium) supplemented with 10% foetal bovine serum give sub-fractions B.1 and B.2. Sub-fraction B.1 was subjected
(
FBS), 1% of 2mm l-glutamine, 50IU/mL penicillin, 50µg/ to silica gel column chromatography eluted with chloroform–
mL streptomycin and maintained at 37°C in a 5% CO atmo- methanol (98:2) to give 1 (20mg). Sub-fraction B.2 was chro-
2
sphere with 95% humidity. Viable cells were counted and in- matographed with C-18 silica gel eluted with water–methanol
4
oculated in 96-well plate with density of 10 cells/100µL/well. (6:4) to afford 2 (24mg), 3 (8mg), 4 (72mg) and 5 (154mg).
After 24h the cells were treated with pure compound while
Fraction EA.D (7.1g) was subjected to silica gel column
the control wells were added only by 100µL medium. All ex- chromatography eluted with chloroform–methanol (90:10) to
periments were in triplicate. The plates were incubated in an give 3 sub-fractions D.1 to D.3. Sub-fraction D.1 was chro-
atmosphere of 5% CO , 95% humidity at 37°C for 48h. Ad- matographed with C-18 silica gel eluted with water–methanol
2
herent cell cultures were fixed by adding 50µL of cold 50% (6:4) to afford 6 (76mg). Sub-fraction D.2 was chromato-
(
1
w/v) trichloroacetic acid per well and incubated at 4°C for graphed with silica gel C-18 eluted with water–methanol (7:3)
h. The plates were washed five times with distilled water and to afford 7 (22mg), 8 (65mg) and 9 (6mg). Sub-fraction D.3
air dried. Then a solution of 50µL of SRB (0.4% w/v in 1% was chromatographed with silica gel C-18 eluted with water–
acetic acid) was added to each well and allow staining at room methanol (75:25) to afford 10 (10mg), 11 (32mg) and 12
temperature for 30min. The SRB solution was removed out of (20mg). Fraction EA.E (20g) was subjected to silica gel col-
plates by rinsing 4 times with a 1% glacial acetic acid solu- umn chromatography eluted with chloroform–methanol–water
tion (200µL/well). The plates were air-dried for 12–24h. The (80:20:0.2) to give 13 (85mg), 14 (1.4g) and 15 (1.1 g).
bound SRB was solubilised to each well by adding 100µL of
Boeravinone K (3): Pale yellow needles, mp 244–247°C
23
1
0 mm Tris Base (pH 10.5). The plates were shaken gently for (chloroform). [α] −260 (c=0.001, MeOH). ESI-MS m/z 345.1
D
+
1
13
2
0min and the optical density of each well was read using [M+H] (Calcd for C H O +H: 345.097). H- and C-NMR
18
16
7
a scanning multiwall spectrophotometer at a test wavelength (CDCl ) see Table 2.
3
of 492nm and a reference wavelength of 620nm. The optical
density (OD) of SRB in each well is directly proportional to (chloroform). [α]
the cell number. Cell survival was measured as the percentage m/z 353.0602 [M+Na] (Calcd for C H O +Na: 353.0637).
10-Demethylboeravinone C (5): White needles. mp 248°C
23
−513 (c=0.001, MeOH). HR-ESI-MS
+
D
17
14
7

6
30
Vol. 61, No. 6
1
3214–3220 (1989).
Lami N., Kadota S., Kikuchi T., Chem. Pharm. Bull., 39, 1863–1865
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Petrus A. J. A., Siva Hemalatha S., Suguna G., J. Pharm. Sci. Res.,
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(2010).
Hilou A., Nacoulma O. G., Guiguemde T. R., J. Ethnopharmacol.,
103, 236–240 (2006).
H-NMR (acetone-d ), δ ppm: 11.85 (1H, s, 11-OH), 7.79 (1H,
6
6
7
)
)
dd, J=6.0, 3.5Hz, H-1), 6.85 (1H, t, J=8.0Hz, H-2), 6.83 (1H,
m, H-3), 6.11 (1H, d, J=2.5Hz, H-10), 6.09 (1H, d, J=2.0Hz,
H-8), 5.86 (1H, brs, 4-OH), 4.81 (1H, dd, J=10.0, 6.0Hz,
H-6a), 4.48 (1H, t, J=10.0Hz, H-6ax), 4.45 (1H, dd, J=10.0,
(
4
8)
13
6
.0Hz, H-6eq), 3.88 (3H, s, 9-OCH3). C-NMR (acetone-d6),
δ ppm: 195.0 (C-12), 168.8 (C-9), 166.2 (C-11), 162.7 (C-7a),
46.6 (C-4), 143.8 (C-4a), 122.7 (C-1), 121.5 (C-2), 121.4 (C-1a),
9)
1
116.7 (C-3), 102.9 (C-11a), 96.1 (C-10), 94.2 (C-8), 76.9 (C-6a), 10) Stintzing F. C., Kammerer D., Schieber A., Adama H., Nacoulma O.
6
6.8 (C-12a), 62.2 (C-6), 56.3 (OCH3).
G., Carle R., Z. Naturforsch., 59c, 1–8 (2004).
1) Do T. M. L., Nguyen T. M. D., Nguyen K. P. P., Vietnam J. De-
1
Boeravinone L (6): White amorphous powder. HR-ESI-MS
+
1
velop. Sci. Technol., 14, 58–65 (2011).
m/z 285.0383 [M+H] (Calcd for C H O +H, 284.0321). H-
and C-NMR (DMSO-d ) see Table 1.
15
8
6
13
12) Borrelli F., Milic N., Ascione V., Capasso R., Izzo A. A., Capasso
F., Petrucci F., Valente R., Fattorusso E., Taglialatela-Scafati O.,
Planta Med., 71, 928–932 (2005).
6
Boeravinone M (8): White needles, mp 217°C (chloroform).
2
3
[
α] 0 (c=0.001, MeOH), HR-ESI-MS m/z 351.0474 [M+
D
+ 1 13
1
3) Lami N., Kadota S., Tezuka Y., Kikuchi T., Chem. Pharm. Bull., 38,
Na] (Calcd for C H O +Na, 351.0481). H- and C-NMR
(
17
12
7
1558–1562 (1990).
DMSO-d ) see Table 2.
6
14) Borrelli F., Ascione V., Capasso R., Izzo A. A., Fattorusso E.,
Taglialatela-Scafati O., J. Nat. Prod., 69, 903–906 (2006).
c=0.001, MeOH), HR-ESI-MS: m/z 515.1320 [M+Na] (Calcd 15) Belkacem A. A., Macalou S., Borrelli F., Capasso R., Fattorusso E.,
2
3
Boeravinone N (10): White amorphous powder, [α] −198
D
+
(
1 13
for C H O +Na, 515.11654). H- and C-NMR (methanol-
d4) see Table 2.
Scafati O. T., Di Pietro A., J. Med. Chem., 50, 1933–1938 (2007).
16) Kai H., Baba M., Okuyama T., Chem. Pharm. Bull., 55, 133–136
(2007).
17) Rawat P., Kumar M., Sharan K., Chattopadhyay N., Maurya R.,
Bioorg. Med. Chem. Lett., 19, 4684–4687 (2009).
8) Harborne J. B., Mabry T. J., “The flavonoids, Advances in Re-
search,” Chapman and Hall, London, New York, 1982, pp. 85–99.
9) Santos A. S., Caetano L. C., Goulard Sant’Ana A. E., Phytochemis-
2
3
24 12
2
3
Boeravinone O (11): Yellow wax, [α]D +58 (c=0.001,
+
MeOH), HR-ESI-MS: m/z 471.0891 [M+Na] (Calcd for
C H O +Na, 471.09032). H- and C-NMR (pyridine-d5)
see Table 1.
1
13
21
20 11
1
33)
Acidic Hydrolysis of 10
Compound 10 (1mg) was
1
dissolved in 0.5mL solution of 1n HCl–methanol (1:1) and
refluxed at 75°C for 90min. The reaction solution was evapo-
rated under reduced pressure, then the hydrolysate was ex-
tracted with ethyl acetate (1mL×3). The aqueous fraction was
try, 49, 255–258 (1998).
2
0) Jang D. S., Park E. J., Kang Y. H., Hawthorne M. E., Vigo J. S.,
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neutralized with Ag CO and then filtered. The filtrate was 21) Ito C., Itoigawa M., Kojima N., Tan H. T.-W., Takayasu J., Tokuda
2
3
H., Nishino H., Furukawa H., Planta Med., 70, 585–588 (2004).
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ing M. G., O’Neil-Johnson M., Hu J.-F., Rice S. M., Eldridge G. R.,
Phyto. Letters, 4, 72–74 (2011).
concentrated under reduced pressure to afford a residue. This
residue was compared to the authentic d-(+)-glucose (Merck)
by using TLC (ethyl acetate–methanol–water, 10:4:1),
2
(
Rf=0.17). The TLC result showed that the sugar of 10 was
d-(+)-glucose. The ethyl acetate extract was determined to be
0-demethylboeravinone C by comparing it with the authentic
sample (compound 5) by TLC (chloroform–methanol, 98:2),
Rf=0.30).
2
2
3) Veitch N. C., Nat. Prod. Rep., 24, 417–464 (2007).
4) Yenesew A., Derese S., Midiwo J. O., Oketch-Rabah H. A., Lisgar-
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1
(
Acknowledgements This research was supported by 26) Messana I., Ferrari F., Goulard Sant’Ana A. E., Phytochemistry, 25,
2
688–2689 (1986).
7) Slade D., Ferreira D., Marais J. P. J., Phytochemistry, 66, 2177–2215
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2
2
3
Vietnam’s National Foundation for Science and Technology
Development (NAFOSTED) Grant #104.01-2010.04. This work
was also supported by the NSF International Research Experi-
ence for Students (IRES) Grant #INT-0744375 and by JSPS
research fellowship to S.Y.
2
(
8) Snatzke G., Tetrahedron, 21, 413–419 (1965).
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97–598 (1991).
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