Haworforbins A-C, new phenolics from Haworthia cymbiformis

Article abstract of DOI:10.1007/s11418-012-0647-x

Two new isocoumarin glucosides, haworforbins A (1) and B (2), and a new chromone, haworforbin C (3), have been isolated from Haworthia cymbiformis. Their structures and absolute configurations were elucidated on the basis of NMR and CD data. Haworforbin C (3) exhibited moderate inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cell line.

Full text of DOI:10.1007/s11418-012-0647-x

J Nat Med (2013) 67:212–216
DOI 10.1007/s11418-012-0647-x
NOTE
Haworforbins A–C, new phenolics from Haworthia cymbiformis
•
Fumie Yamasaki Shoko Machida Asami Nakata Alfarius Eko Nugroho
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Yusuke Hirasawa Toshio Kaneda Osamu Shirota Naoyuki Hagane
•
•
Tadayoshi Sugizaki Hiroshi Morita
Received: 23 January 2012 / Accepted: 16 February 2012 / Published online: 16 March 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract Two new isocoumarin glucosides, haworfor-
bins A (1) and B (2), and a new chromone, haworforbin C
(3), have been isolated from Haworthia cymbiformis. Their
structures and absolute configurations were elucidated
on the basis of NMR and CD data. Haworforbin C (3)
exhibited moderate inhibition of nitric oxide production in
lipopolysaccharide-stimulated RAW264.7 cell line.
oxide (NO) production in RAW264.7 cell line stimulated
by lipopolysaccharide (LPS). Investigation of the constit-
uents of the chloroform fraction led to the isolation of three
new compounds, haworforbins A–C (1–3), together with
uncinoside A [6]. Structure elucidation and NO production
inhibition activity of the isolated compounds are reported
herein (Fig. 1).
Keywords Haworthia cymbiformis ꢀ Haworforbins A-C ꢀ
iNOS production inhibition activity
Results and discussion
Haworforbin A {1, [a]2D8 -20 (c 1.0, MeOH)} was isolated
as a white amorphous solid, with molecular formula
C17H22O9 as determined by HRESITOFMS [m/z 371.1338
(M?H)?, D -0.4 mmu]. IR absorptions suggested the
presence of a carbonyl (1654 cm-1) and a hydroxy
(3397 cm-1) group. 1H and 13C NMR data (Table 1)
revealed 17 carbon resonances due to six sp2 quaternary
carbons, one sp2 methine, six sp3 methines, two sp3
methylenes, and two methyls. Of them, three sp2 quater-
nary carbons (dC 161.3, 161.5, and 170.8), six sp3 methines
(dC 70.2–100.6), and one sp3 methylene (dC 61.9) are
connected to an oxygen atom. These chemical shifts sug-
gested the structure of 1 to be an isocoumarin glycoside.
Introduction
Haworthia cymbiformis (Haw.) Duval (Xanthorrhoeaceae)
is a succulent plant of South African origin which is
usually grown as an ornamental plant. Recently, the aerial
parts of H. cymbiformis have also been used in foods and
drinks, though there are no reports on the constituents of
this plant. In our screening program for bioactive natural
products [1–5], the chloroform-soluble fraction from the
whole plants of H. cymbiformis showed inhibition of nitric
F. Yamasaki ꢀ S. Machida ꢀ A. Nakata ꢀ A. E. Nugroho ꢀ
Y. Hirasawa ꢀ T. Kaneda ꢀ H. Morita (&)
1
Analysis of the 2D NMR data (HSQC, H–1H COSY,
and HMBC) supported the structure of 1 as a new iso-
coumarin glycoside. The existence of two partial structures
a (from C-9 to C-4) and b (from C-10 to C-60) were
deduced from 1H–1H COSY analysis of 1 in CD3OD/
CDCl3 (1:1, Fig. 2). HMBC correlations of H-3 to C-4a,
H2-4 to C-5 and C-8a suggested the connectivity of partial
structure a to C-4a. HMBC correlations of H-5 to C-6 and
C-7, and C-7-Me to C-6, C-7, and C-8 confirmed the
position of the methyl group at C-7. Finally, HMBC cor-
relations of H-10 to C-6 and C-50 suggested the pyranose
Faculty of Pharmaceutical Sciences, Hoshi University,
Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan
e-mail: moritah@hoshi.ac.jp
O. Shirota
Faculty of Pharmaceutical Sciences at Kagawa Campus,
Tokushima Bunri University, 1314-1 Shido, Sanuki,
Kagawa 769-2193, Japan
N. Hagane ꢀ T. Sugizaki
Sabotensoudanshitsu, 4-23 Chiyoda-cho,
Tatebayashi, Gunma 374-0028, Japan
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J Nat Med (2013) 67:212–216
213
detection of the acid hydrolysate of 2 identified the sugar
unit as D-glucose.
Haworforbin C (3) was isolated as a white amorphous
solid with molecular formula of C12H12O5 as determined
1
by HRESIMS [m/z 237.0765 (M?H)?, D ?0.2 mmu]. H
and 13C NMR data (Table 1) revealed 12 carbon reso-
nances due to eight sp2 quaternary carbons, one sp2
methine, one sp3 methylene, and two methyls. Of them,
five sp2 quaternary carbons (dC 153.9, 156.9, 160.7, 170.1,
and 183.9) and one sp3 methylene (dC 61.0) are connected
1
to an oxygen atom. Further analysis of the H and 13C
NMR revealed that, except for the observation of a meth-
ylene signal instead of a methyl, the NMR data of 3 are
similar to the aglycone unit of uncinoside A [6]. Thus, 3
was assumed to be 5,7-dihydroxy-2-(hydroxymethyl)-6,8-
dimethylchromone.
The structure of 3 was further confirmed through anal-
ysis of the 2D NMR data (Fig. 2). HMBC correlations of
C-6-Me to C-5, C-6, and C-7, and C-8-Me to C-7, C-8, and
C-8a confirmed the methyl positions to be at C-6 and C-8,
and the hydroxymethyl position at C-2 was deduced from
the HMBC correlations of H2-9 to C-2 and C-3. Thus, 3
was determined to be 5,7-dihydroxy-2-(hydroxymethyl)-
6,8-dimethylchromone.
Fig. 1 Structures of compounds 1–3
form of the sugar moiety and the connectivity of C-6 and
C-10 through an oxygen atom.
The ROESY correlations for H-10/H-30 and H-50, and
H-30/H-50 suggested that the hexopyranose took a chair form
with axial orientations for H-10, H-30, and H-50. Anti-rela-
tionships for H-10/H-20 and H-30/H-40 were deduced from
Compounds 1–3 and their aglycones were tested for the
inhibition of NO production in LPS-stimulated RAW264.7
cell line, and only 3 was found to show a moderate
inhibitory activity (IC50 87.5 lM).
To summarize, two new isocoumarin glucosides,
haworforbins A (1) and B (2), and a new chromone,
haworforbin C (3), have been isolated from H. cymbiformis.
Their structures and absolute configurations were elucidated
on the basis of NMR and CD data. Haworforbin C (3)
exhibited moderate inhibition of NO production in LPS-
stimulated RAW264.7 cell line.
3
3
0
0
0
0
the JHꢁ1 =Hꢁ2 and JHꢁ3 =Hꢁ4 values (7.7 and 8.8 Hz,
respectively). Thus, the hexopyranose unit was assigned as
a b-glucopyranoside. The glucose moiety was determined
as the D-form on the basis of HPLC analysis with chiral
detector of the acid hydrolysate of 1. In addition, the
absolute configuration of C-3 was suggested to be S by the
positive Cotton effect at 270 nm in the CD spectrum of 1
and its aglycone [CD (MeOH) kmax 271 (De 1.98) nm]
[7, 8]. Thus, the structure of 1 was deduced to be a
b-D-glucopyranoside derivative of monaschromone [9].
Haworforbin B {2, [a]2D8 -104 (c 0.3, MeOH)} was
isolated as a white amorphous solid with molecular for-
mula of C17H20O9 as determined by HRESIMS [m/z
Experimental section
General experimental procedures
Optical rotations were measured on a JASCO DIP-1000
polarimeter. UV spectra were recorded on a Shimadzu
UVmini-1240 spectrophotometer and IR spectra on a
JASCO FT/IR-4100 spectrophotometer. CD spectra were
recorded on a JASCO J-820 polarimeter. High-resolution
ESI MS were obtained on a LTQ Orbitrap XL (Thermo
1
369.1175 (M?H)?, D -1.1 mmu]. Analysis of the H and
13C NMR data (Table 1) suggested the structure of 2 to be
a 3,4-dehydroderivative of 1, since an exchange of an sp3
methylene and an sp3 methine in 1, an sp2 methine and an
sp2 quaternary carbon were observed in 2.
1
Scientific). H and 2D NMR spectra were measured on a
700 MHz spectrometer at 300 K, while 13C NMR spectra
were on a 175 MHz spectrometer. The residual CD2HOD
chemical shifts used as an internal standard are dH 3.31 and
dC 49.0. Standard pulse sequences were used for the 2D
NMR experiments.
The structure of 2 was further confirmed by analysis of
the 2D NMR correlations (Fig. 2) and acid hydrolysis.
HMBC correlations of H3-9 to C-3 and C-4, and H-4 to
C-4a, C-5, and C-8a confirmed the double bond between
C-3 and C-4. In addition, HPLC analysis with chiral
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J Nat Med (2013) 67:212–216
Table 1 1H and 13C NMR data of haworforbins A–C (1–3) in CD3OD/CDCl3 (1:1) at 300 K
1
2
3
dH (J, Hz)
dC
dH (J, Hz)
dC
dH (J, Hz)
dC
1
2
3
4
170.8
167.4
170.1
105.7
183.9
4.65 (1H, m)
76.4
35.0
153.8
105.4
6.28 (1H, s)
2.83 (1H, dd, 16.3, 11.2)
2.89 (1H, dd, 16.3, 3.1)
6.29 (1H, s)
6.54 (1H, s)
4a
5
138.5
104.9
161.3
113.7
161.5
102.8
20.7
137.4
100.8
162.8
113.7
160.5
101.1
19.3
104.9
156.9a
102.8b
160.7
108.1b
153.9a
61.0
6.43 (1H, s)
6
7
8
8a
9
1.46 (3H, d)
2.09 (3H, s)
2.23 (3H, s)
2.15 (3H, s)
4.49 (2H, s)
2.18 (3H, s)a
6-Me
7-Me
8-Me
10
20
30
40
50
60
7.9c
7.8
8.2
2.10 (3H, s)a
7.7c
4.95 (1H, d, 7.7)
100.6
73.5
76.8
70.2
76.9
61.9
5.01 (1H, d, 7.7)
100.6
73.6
77.1
70.3
77.1
61.9
3.55 (1H, br. t, 7.7)
3.48 (1H, br. t, 8.8)
3.46 (1H, m)
3.57 (1H, dd, 8.9, 7.7)
3.49 (1H, t, 8.9)
3.45 (1H, t, 8.9)
3.44 (1H, m)
3.48 (1H, m)
3.72 (1H, dd, 12.0, 4.7)
3.86 (1H, dd, 12.0, 2.0)
3.73 (1H, dd, 12.1, 5.1)
3.89 (1H, dd, 12.1, 2.3)
a,b,c
Interchangeable
Material
and fraction 5 was further separated by silica gel column
(CHCl3/MeOH/EtOAc, 50:1:1 ? 10:1:1) to give 3 (0.2 mg,
0.00008%).
The botanical identification of the Haworthia cymbiformis
sample was made by Naoyuki Hagane, Sabotensoudan-
shitsu, Japan. A voucher specimen (no. SABO100909) has
been deposited in Sabotensoudanshitsu.
Haworforbin A (1)
White amorphous solid; UV (MeOH) kmax 219 (e 24300),
267 (12500), and 308 (3300) nm; [a]2D8 -20 (c 1.0, MeOH);
CD (MeOH) kmax 211 (De 5.48), 222 (1.01), 224 (0), 228
(-3.82), 245 (-6.74), 267 (0), and 270 (7.40) nm; IR (KBr)
Extraction and isolation
The whole plants of H. cymbiformis (240.4 g) were
extracted with methanol to obtain 6.2 g of extract which
was successively partitioned with n-hexane, chloroform,
ethyl acetate, n-butanol and water. The chloroform-soluble
materials (286.5 mg) were further separated with a silica
gel column (hexane/EtOAc, 1:0 ? 1:1, and then CHCl3/
MeOH, 1:0 ? 0:1) to give 13 fractions. Fraction 13 was
further separated by ODS HPLC (YMC Pack Pro C18 RS
250 9 10 mm; eluted with CH3CN/H2O 22:78, 0.1%
HCO2H; flow rate 2.0 mL/min; UV detection at 254 nm)
to yield 1 (2.2 mg, 0.00091%, tR 21.8 min, purity [99%),
2 (0.6 mg, 0.00024%, tR 30.6 min, purity [99%), and un-
cinoside A (2.1 mg, 0.00087%, tR 24.5 min, purity [99%),
m
max 3397 and 1654 cm-1; 1H and 13C NMR data (Table 1);
ESIMS m/z 371 (M?H)?; HRESITOFMS m/z 371.1338
([M?H]?; calcd for C17H23O9, 371.1342).
Haworforbin B (2)
White amorphous solid; UV (MeOH) kmax 237 (e 25100) and
334 (3400) nm; [a]D28 -104 (c 0.3, MeOH); IR (KBr) mmax
1
3396 and 1689 cm-1; H and 13C NMR data (Table 1);
ESIMS m/z 369 (M?H)?; HRESIMS m/z 369.1175
([M?H]?; calcd for C17H21O9, 369.1186).
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J Nat Med (2013) 67:212–216
215
aqueous layer of hydrolysate of 2 was 9.1 min with posi-
tive intensity. The CHCl3 layer was also dried and the
NMR data of the obtained compound is identical to
6,8-dihydroxy-3,7-dimethylisocoumarin [10].
NO production by RAW264.7 cells
The RAW264.7 cells were cultured in a-MEM medium
supplemented with 10% fetal bovine serum and penicillin/
streptomycin. Cells were seeded onto a 96-well microtiter
plate at 1.5 9 105 cells in 100 lL solution per well and
were preincubated for 12 h at 37°C in a humidified atmo-
sphere containing 5% CO2. The cells were cultured in the
medium containing LPS (5 lg/mL) with or without the test
sample at different concentrations for 24 h. NO production
was then determined by the Griess assay. Supernatant of
the cultured medium (100 lL) was transferred to a 96-well
microtiter plate, and then 100 lL of Griess reagent (1%
sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihy-
drochloride in 2.5% H3PO4 was added). After incubation at
room temperature for 15 min, the absorbance at 540 and
620 nm was measured with a microplate reader (Bench-
mark Plus microplate spectrometer, Bio-Rad). L-NMMA,
an NO synthase inhibitor, was used as a positive control
(IC50 13.8 lg/mL).
Acknowledgments This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan and a grant from The Open Research
Center Project in Hoshi University.
Fig. 2 Selected 2D NMR correlations for haworforbins A–C (1–3)
Haworforbin C (3)
White amorphous solid; UV (MeOH) kmax 205 (e 12700),
262 (7100) and 306 (3200) nm; IR (KBr) mmax 3476 and
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