Two new 5-deoxyflavan-3,4-diol glucosides from roots of Albizia chevalieri

Article abstract of DOI:10.1002/mrc.4504

Phytochemical investigation of the roots of Albizia chevalieri led to the isolation of two new 5-deoxyflavan-3,4-diol glucosides from roots of A. chevalieri, Chevalieriflavanosides A and B. Their structures were established by 2D NMR techniques, UV, IR, CD, and mass spectrometry. Cytotoxicity of the two compounds was evaluated against acute promyelocytic leukemia HL60 cells. The antibacterial activities of 1 and 2 also were evaluated against Pseudomonas aeruginosa and Staphylococcus aureus using the agar diffusion test. Copyright

Full text of DOI:10.1002/mrc.4504

Research article
Received: 12 July 2016
Revised: 13 August 2016
Accepted: 17 August 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4504
Two new 5-deoxyflavan-3,4-diol glucosides
from roots of Albizia chevalieri
Abdou Tchoukoua,a,b Turibio Kuiate Tabopda,a* Shota Uesugi,c
Ken-ichi Kimura,c Eunsang Kwon,d Hiroyuki Momma,d
Bonaventure Tchaleu Ngadjui,a* Takuya Kosekib and Yoshihito Shionob*
ABSTRACT Phytochemical investigation of the roots of Albizia chevalieri led to the isolation of two new 5-deoxyflavan-3,4-diol glu-
cosides from roots of A. chevalieri, Chevalieriflavanosides A and B. Their structures were established by 2D NMR techniques, UV, IR,
CD, and mass spectrometry. Cytotoxicity of the two compounds was evaluated against acute promyelocytic leukemia HL60 cells.
The antibacterial activities of 1 and 2 also were evaluated against Pseudomonas aeruginosa and Staphylococcus aureus using the
agar diffusion test. Copyright © 2016 John Wiley & Sons, Ltd.
Keywords: NMR; 1H; 13C; Leguminosae; Albizia chevalieri; flavan-3,4-diols; cytotoxicity
which showed the pseudo-molecular ion peak at m/z 475.12101
Introduction
([M + Na]+). The IR spectrum displayed absorption band of hydroxyl
groups at νmax 3394 cm-1. The UV spectrum exhibited characteristic
Albizia chevalieri (Mimosaceae) is a tree that grows up to 12 m high
or a shrub under harsher conditions of the dry savannah from
Senegal, Niger, Nigeria, and Cameroon. The leaf extract of A.
absorbance bands of flavan-3,4-diol at λmax 225 and 275 nm.[12] The
1H NMR spectrum of 1 showed an aromatic AB coupled system at
δH 6.83 (1H, d, J = 8.3Hz, H-5) and 6.89 (1H, d, J = 8.3Hz, H-6), and
an AA′BB′ spin system at δH 6.81 (2H, d, J= 8.3 Hz, H-3′, H-5′) and
7.42 (2H, d, J = 8.3Hz, H-2′, H-6′). This observation suggested a
tetrasubstituted A-ring, and hence the AA′BB′ aromatic spin system
was located on B-ring. The location of the AB system at H-5 and H-6
was confirmed by the HMBC correlations (Fig. 2) of H-5 with C-6,
C-7, C-4, C-4a, and C-8a, and of H-6 with C-4a, C-5, C-7, and C-8. The
NMR spectrum of 1 further showed signals of a sugar unit at δH/δC
4.76 (1H, d, J=7.6Hz, H-1″) / 104.4 (C-1″), 3.49 (1H, t, J=7.6Hz, H-2″)
/ 74.9 (C-2″), 3.45 (1H, dd, J= 9.0 Hz, 7.6, H-3″) / 77.6 (C-3″), 3.39 (1H,
m, H-4″) / 71.2 (C-4″), 3.42 (1H, m, H-5″) / 78.3 (C-5″), 3.72 (1H, dd,
J= 12.5, 4.8 Hz, H-6″), and 3.88 (1H, dd, J= 12.5, 2.1 Hz, H-6″) / 62.4
(C-6″). Stereochemistry at anomeric position of the sugar moiety
chevalieri is used for the management of diabetes mellitus by tradi-
tional medical practitioners in some parts of Niger Republic and
Sokoto, Nigeria.[1] The leaf extract has been reported to have a
significant hypoglycemic effect,[1] dysentery,[2,3] hematotoxicity,[4]
hypolipidemic,[5] and antioxidant activity.[5] The bark is used in
Borno-North eastern Nigeria as purgative, taenicide, and coughs.[6]
There are also reports on the local use of the leaves extract for
cancer treatment in Zaria city, Kaduna state.[6] In prior studies on
the genus Albizia, saponins and flavonoids were found as the main
constituents.[7–9] A previous phytochemical investigation on A.
chevalieri stem bark led to the isolation and structure elucidation
of three known pentacyclic triterpenoids: friedelin, friedelan-3-ol,
and lupeol.[10]
In our continuing search for bioactive compounds from
Cameroon medicinal plants, we investigated A. chevalieri from a
phytopharmacological point of view. Therefore, we report herein
the isolation of two previously undescribed flavan-3,4-diol gluco-
sides, chevalieriflavanosides A (1) and B (2) (Fig. 1) from roots of
A. chevalieri. Their structures were determined by spectroscopic
methods including 1D and 2D NMR experiments, IR, UV, CD, and
HR-ESI-MS. Furthermore, they were examined for cytotoxicity
against acute promyelocytic leukemia HL60 cells. The antibacterial
activity of 1 and 2 was also reported.
*
Correspondence to: Turibio Kuiate Tabopda and Bonaventure Tchaleu Ngadjui,
Département de Chimie Organique, Université de Yaoundé 1, BP 812 Yaoundé,
Cameroun. E-mail: ttabopda@yahoo.fr
** Correspondence to: Yoshihito Shiono, Department of Food Life, and
Environmental Science, Faculty of Agriculture, Yamagata University, Tsuruoka,
Yamagata 997-8555, Japan.
a Département de Chimie Organique, Université de Yaoundé 1, Yaoundé,
Cameroun
b Department of Food, Life, and Environmental Science, Faculty of Agriculture,
Yamagata University, Tsuruoka, Yamagata, Japan
Results and Discussion
c
The United Graduate School of Agricultural Sciences, Iwate University, Morioka,
Iwate, Japan
Compound 1 was obtained as a brownish amorphous powder and
gave a dark blue coloration with FeCl3 reagent. Moreover, it showed
a positive Shinoda test,[11] suggesting that 1 is a flavonoid. The
molecular formula C21H24O11 was established from the HR-ESI-MS
d Research and Analytical Center for Giant Molecules, Graduate School of Science,
Tohoku University, Sendai, Miyagi, Japan
Magn. Reson. Chem. (2016)
Copyright © 2016 John Wiley & Sons, Ltd.

A. Tchoukoua et al.
the H-4, correlations with C-2, C-3, C-4a, C-5, and C-8a. Acid hydro-
lysis of 1 afforded a free sugar moiety and (À)-teracacidin.[14] The
HMBC correlation between the anomeric proton of the glucose unit
and C-7 (δC 147.0) suggested that the glucose is fixed at C-7
through an ether linkage. The R-configuration of C-2 was deduced
from the CD spectra which showed a negative cotton effect within
the 1Lb transition ([θ]278 =À4000).[15] The coupling constants
(J2,3 =nearly 0Hz and J3,4 = 2.7 Hz) between H-2 and H-3, and be-
tween H-3 and H-4 of C-ring, confirmed the 2,3-cis-3,4-cis relative
configurations. The trend of small coupling constants compared to
their trans isomers (3J=7.0Hz–10.0 Hz) has been well established
in closely related 2,3-cis-3,4-cis-flavan-3,4,7,3′,4′-pentaol (J2,3 =1.0Hz,
Figure 1. Chemical structures of compounds 1 and 2.
J
3,4 = 4.0 Hz) and 2,3-cis-3,4-cis-4-methoxyflavan-3,7,8,3′,4′-pentaol
1
of 1 was assigned as β on the basis of H–1H coupling constants
(J2,3 =1.0Hz, J3,4 =2.8Hz).[16,17] Thus, the absolute configuration of
the flavan moiety of 1 was elucidated to be 2R,3R,4R as shown in
Fig. 1, named chevalieriflavanoside A.
1
(J1″,2″ =7.6Hz) in the H NMR spectrum of 1. The sugar was identi-
fied as D-glucopyranosyl by the measurement of optical rotation
and retention time and by the evaluation of spin–spin couplings
and chemical shifts.[13] The remaining signals in the 1H and 13C
NMR spectra correspond to those of a propan-1,2,3-triol (chroman
ring) at δH /δC 5.11 (1H, br. s, H-2) / 76.4 (C-2), 3.87 (1H, d,
J= 2.7 Hz, H-3) / 72.5 (C-3), and 4.48 (1H, d, J= 2.7 Hz, H-4) / 69.2
(C-4). They were assigned to H-2, H-3, and H-4, respectively as H-2
showed HMBC correlations with C-1′, C-2′, C-3, C-4, and C-8a, and
Compound 2, obtained as a brownish amorphous powder, also
gave a dark blue coloration with FeCl3 reagent. It showed a pos-
itive Shinoda test suggesting that 2 is a flavonoid. The 1H and 13
C
NMR spectra of 2 (Table 1) were similar to those of 1 with the
differences being the chemicals shifts and coupling patterns of
C-2 [δH 5.00 (1H, d, J = 9.0 Hz); δc 78.3], C-3 [δH 3.97 (1H, dd,
J = 9.0, 3.4Hz); δC 72.0], and C-4 [δH 4.57 (1H, d, J = 3.4 Hz); δC
67.8] in 2 compared to those of corresponding carbons in 1. This
suggested that 2 has different configurations at the C-ring from
those of 1. The gross structure of 2 was clearly elucidated by an-
alyzing 2D NMR experiments (COSY (Fig. S8), HMQC (Fig. S9),
HMBC (Fig. S10)). Furthermore, the absolute configuration of 2
was determined by comparing their coupling constants and CD
data. The CD data of 2 ([θ]270 À1000) established its absolute
configuration to be 2R. By contrast, the large coupling constant
between H-2/H-3 in 2 meant that H-3 must be pseudo-equatorial,
and so the 4-hydroxy must be β. The small coupling constants
between H-3 and H-4 of the C-ring suggested the 3,4-trans
Figure 2. Selected HMBC (H→C) correlations of 1 and 2.
Table 1. 1H and 13C NMR data of 1 and 2
Position
1a
2a
δC
δH (m, J in Hz)
δC
δH (m, J in Hz)
2
76.4
72.5
5.11 (br. s)
3.87 (d, 2.7)
4.48 (d, 2.7)
—
78.3
72.0
5.00 (d, 9.0)
3.97 (dd, 9.0, 3.4)
4.57 (d, 3.4)
—
3
4
69.2
67.8
4a
5
119.0
122.6
111.3
147.0
136.4
145.2
130.7
129.5
115.9
158.2
104.4
74.9
120.4
119.8
111.6
147.7
138.5
145.1
130.5
130.2
116.3
159.3
104.6
74.9
6.83 (d, 8.3)
6.89 (d, 8.3)
—
6.70 (d, 8.9)
6.82 (d, 8.9)
—
6
7
8
—
—
8a
1′
—
—
—
—
2′, 6′
3′, 5′
4′
7.42 (d, 8.3)
6.81 (d, 8.3)
—
7.29 (d, 8.3)
6.78 (d, 8.3)
—
1″
2″
3′′
4″
5″
6″
4.76 (d, 7.6)
3.49 (t, 7.6)
3.45 (dd, 9.0, 7.6)
3.39 (m)
4.74 (d, 7.6)
3.36 (m)
3.42 (m)
3.44 (m)
3.36 (m)
3.69 (dd, 12.4, 4.8)
3.85 (d, 12.4)
77.6
77.6
71.2
71.3
78.3
3.42 (m)
78.3
62.4
3.72 (dd, 12.5, 4.8)
3.88 (dd, 12.5, 2.1)
62.5
aRecorded in 600 MHz (for 1H NMR) and 150 MHz (for 13C NMR) in CD3OD, δ in ppm.
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Copyright © 2016 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2016)

New 5-deoxyflavan-3,4-diol glucosides from Albizia chevalieri
configuration. 2,3-trans-3,4-cis-orientation was further supported
by previously reported compound, 2,3-trans-3,4-cis-7,4′-
of Agricultural Research for Development in December 2013, and
a voucher specimen N° 36696 HNC has been deposited at the
Cameroon National Herbarium.
dimethoxyflavan-3,4-diol
(J2,3 = 9.7Hz,
J3,4 = 3.6 Hz),[18]
(J2,3 =9.5 Hz, J3,4 = 3.5 Hz).[19] On the basis of the evidence, the
absolute configuration of the flavan moiety was established as
2R,3S,4S as shown in Fig. 2, named chevalieriflavanoside B.
Compounds 1 and 2 were evaluated for their cytotoxic activities
against human cancer. Unfortunately, they exhibited no activities
towards HL60 cells at the concentration of 100μM.
The antibacterial activities of 1 and 2 also were evaluated against
Pseudomonas aeruginosa and Staphylococcus aureus using the agar
diffusion test, and the 1 and 2 showed marginal activity at
100 μg/disk.
Extraction and isolation
The powdered roots (1.5kg) of A. chevalieri were extracted with
n-hexane (2 × 5 l), then with MeOH (3 × 6 l) at room temperature. Af-
ter filtration and evaporation procedures, n-hexane (4g) and MeOH
(94 g) extracts were obtained, respectively. The MeOH extract was
dissolved in H2O (400 ml) and successively partitioned with EtOAc
(3× 200 ml) and n-BuOH (3× 200 ml) saturated with H2O. The
n-BuOH extract (33 g) was subjected to VLC using reversed-phase
material (RP-18) employing H2O (1750 ml), H2O–MeOH (8:2,
2750 ml; 6:4, 2250 ml; 4:6, 8000 ml; 2:8, 2500 ml), and MeOH
(750ml) to give 18 main fractions (Fr. 1-1–1-18). Fractions were
combined based on their thin layer chromatography profiles
(TLC). Fr. 1-3–1-5 (2.12 g) were combined and submitted to silica
gel (350 g) CC with the solvent system CH2Cl2–MeOH–H2O (80: 20:
2, 2000 ml; 70: 30: 3, 2500 ml) to give 685 fractions (Fr. 2-1–2-685).
Fr. 2-284–2-393 (89.8mg) was subjected to MPLC (RP-18) using
H2O–MeOH (9:1) to provide 63 fractions (Fr. 3-1–3-63). Fr. 3-19–3-35
(20.9mg) was subjected to open CC using RP-18 (20 g) eluted with
H2O–MeOH (9:1) to give 1 (14 mg). Fr. 2-196–2-276 (190.4mg) was
subjected to MPLC (RP-18) using H2O–MeOH (9:1 and 8:2) to pro-
vide 168 fractions (Fr. 4-1–4-168). Fr. 4-149–4-162 (25.2 mg) was
subjected to open CC using RP-18 (25 g) eluted with H2O–MeOH
(9: 1, 8: 2) to give 2 (10 mg).
Catechins
having
the
same
skeleton
with
the
chevalieriflavanosides are reported to have antibacterial
activity.[20,21] Because of the minimal activity of the
chevalieriflavanosides, one might assume that the absence of hy-
droxyl group at C-5 on A-ring decreases the antibacterial activity,
and the occurrence of a glycosidic unit at C-7 also makes them
weaker because of their high polarity.[22]
Experimental
General experimental procedure
CD spectra were recorded on a JASCO 302-A spectrophotometer in
MeOH. The ESI-MS was measured on a Varian AAT 311A mass
spectrometer, and HR-ESI-MS was taken on a JEOL HX110 mass
spectrometer. 1D and 2D NMR spectra were run on JEOL JNM-
1
Cell culture and cytotoxicity
ECZ600R/S1 600-MHz NMR spectrometer. The H (600 MHz) and
13C (150 MHz) chemical shifts were referenced to the residual sol-
vent peak of methanol-d4 at δH 3.30 for proton and δC 49.0 ppm
for carbon. All sample concentrations ranged from 6 to 10mg/ml
with a total volume of 0.5ml for each sample. The 1H sweep width
was set at 11 282 Hz for all experiments with a 45° pulse for 1H and a
30° pulse for 13C. The pulse programs of the COSY, HSQC, and
HMBC experiments were taken from the Varian Software Library,
and standard pulse sequences were used for 2D spectra. Homonu-
clear 2D spectra and inverse proton-detected heteronuclear 2D
spectra were acquired in the phase-sensitive mode, and HMBC
spectra were acquired in the absolute value mode. The COSY was
acquired with 200F1 increments with 32 scans per increment.
Sinebell weighting was applied to the F2 dimension before zero
filled to 32 K points, and a sinebell was applied to the F1 and zero
filled to 2 K points before Fourier transformation. The NMR note-
book software was used for 1D and 2D spectra processing.
Proton-detected heteronuclear correlations were measured using
HL60 cell (RCB0041, RIKEN BioResource Center, Tsukuba, Japan) was
grown in RPMI 1640 medium supplemented with 10% heat-
inactivated FBS (Sigma Aldrich Corp. St. Louis, USA) and penicillin
(50 units/ml)–streptomycin (50 μg/ml) (Gibco Corp., Carlsbad,
USA) in a humidified atmosphere at 37 °C under 5% CO2. The cyto-
toxicity of the compounds was examined by MTT assay, as de-
scribed previously.[23]
Assay for antimicrobial activity
Antimicrobial activities were determined using the agar diffusion
test using paper disks (8 mm in diameter, thin, ADVANTEC) against
S. aureus NBRC 13276, P. aeruginosa ATCC 15442. An antibiotic
paper disk was loaded with a sample soln. and then dried for 2 h
in vacuo to remove the solvent. Each test sample-loaded disk was
placed on the agar plates inoculated with tester strains, which were
incubated at 25 °C. Antimicrobial activities were estimated by
measuring the diameter of inhibition zone formed on the agar.[24]
1
HSQC (optimized for JCH = 145Hz) and HMBC (optimized for
J
CH =8 Hz). Sinebell weighting was applied to both 1H and 13C di-
mensions and zero filled to 4 K and 1 K, respectively. The chemical
shifts are given in ppm (δ), relative to TMS as internal standard,
and coupling constants are in Hz. Column chromatography (CC)
was carried out on silica gel (70–230mesh, Merck) and vacuum liq-
uid chromatography (VLC) on reversed-phase materials (Lichroprep
RP-18, 25–40μm). TLC was performed on Merck precoated alumi-
num silica gel 60 F254 sheets, and compounds were detected using
sulfuric acid spray reagent.
Acid hydrolysis
Each solution of 1 (5mg) and 2 (5mg) in 0.2M HCl (dioxane-H2O 1:1,
3 ml) was heated at 95 °C for 30 min under argon. After cooling, the
mixture was neutralized by passage through an Amberlite-IRA-
93ZU(Organo, Tokyo, Japan) column andchromatographed (Diaion
HP-20, 40% MeOH followed by Me2CO–EtOH 1:1) to give aglycone
fractions (2.5mg) and a sugar fraction (1.7mg). After the sugar frac-
tion was passed through a Sep-Pak-C18 cartridge (Waters, Milford,
MA, USA; with 40% MeOH) and Toyopak-IC-SP-M-cartridge (Tosoh;
with 40% MeOH), it was analyzed by HPLC (MeCN–H2O 17: 3, flow
rate, 0.9ml.minÀ1; detection, refractive index (RI), and optical reten-
tion (OR): D-glucose (tR 15.84, [α]2D5 +53.3 ° in 1 and 2 (lit.[25] +52.7 °).
Plant material
A. chevalieri was collected in Maroua, Far North Region of
Cameroon and identified by Mr. Tapsou, botanist at the Institute
Magn. Reson. Chem. (2016)
Copyright © 2016 John Wiley & Sons, Ltd.
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Chevalieriflavanoside A (1)
22
D
Brownish amorphous powder; [α]
À190° (c 0.08, MeOH); UV
(MeOH) λmax (logε) nm 225 (5.57), 275 (4.83); IR νmax (KBr) 3394,
2946, 2834, 1654, 1469, 1025 cmÀ1; CD (c 0.1, MeOH) [θ] (nm)
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22
D
Brownish amorphous powder; [α]
À106° (c 0.15, MeOH); UV
(MeOH) λmax (logε) nm 228 (6.58), 275.5 (5.90); IR νmax (KBr) 3367,
2946, 2834, 1654, 1469, 1025 cmÀ1; CD (c 0.1, MeOH) [θ] (nm)
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Supporting information
Additional supporting information may be found in the online ver-
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