Flavonoids from Maclura tinctoria

Article abstract of DOI:10.1016/S0031-9422(99)00126-0

Seven flavonoids, including two new natural products, were isolated from an ethanol extract of the bark of Maclura tinctoria (L.) Gaud. The new compounds are steppogenin 4'-O-β-D-glucoside and orobol 5,3'-di-O-methyl-8- C-glucoside. Orobol, steppogenin, aromadendrin, dihydromorin and orobol 7-O- β-D-glucoside were also isolated.

Full text of DOI:10.1016/S0031-9422(99)00126-0

Phytochemistry 52 (1999) 141±145
Flavonoids from Maclura tinctoria
a,
H.N. El-Sohly *, A. Joshi , X.-C. Li , S.A. Ross
a
a
a,b
a
National Center for the Development of Natural Products, Research Institute of Pharmaceutical Sciences, The University of Mississippi, University,
MS 38677, USA
b
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
Received 11 September 1998; received in revised form 15 January 1999; accepted 15 January 1999
Abstract
Seven ¯avonoids, including two new natural products, were isolated from an ethanol extract of the bark of Maclura tinctoria
L.) Gaud. The new compounds are steppogenin 4'-O-b-D-glucoside and orobol 5,3'-di-O-methyl-8-C-glucoside. Orobol,
(
steppogenin, aromadendrin, dihydromorin and orobol 7-O-b-D-glucoside were also isolated. # 1999 Elsevier Science Ltd. All
rights reserved.
Keywords: Maclura tinctoria; Moraceae; Flavonoids; Orobol; Steppogenin; Aromadendrin; Dihydromorin; Orobol 7-O-b-D-glucoside; Steppo-
genin 4'-O-b-D-glucoside and orobol 5; 3'-di-O-methyl-8-C-glucoside
1. Introduction
5,7,2',4'-tetrahydroxy¯avanone
(steppogenin)
(2),
3
,5,7,4'-tetrahydroxy¯avanonol (aromadendrin) (3),
Maclura tinctoria (L.) Gaud. belongs to the family
3,5,7,2',4'-pentahydroxy¯avanonol (dihydromorin) (4),
orobol 7-O-b-D-glucoside (5), steppogenin 4'-O-b-D-
glucoside (6) and orobol 5,3'-di-O-methyl-8-C-gluco-
side (7). The chemical structures of compounds 1±5
were determined by comparison of their melting points
and UV spectra as well as other spectroscopic data
Moraceae, the mulberry family. The family is widely
distributed in the tropics, subtropics, and some tem-
perate regions of both hemispheres. A great deal of lit-
erature exists on the chemical composition of Maclura
pomifera (Smith & Perino, 1981). The types of com-
pounds isolated belong to the classes of ¯avonoids,
xanthones, triterpenes and stilbenes. However, no
work has been reported on Maclura tinctoria. In this
study we report the isolation and characterization of
seven ¯avonoids from the stem bark of this species.
1
13
( H,
C
NMR, MS) with literature values.
Compounds 6 and 7, on the other hand, appeared to
be new and their structure identi®cation is the subject
of this study. The FABMS of 6 showed a peak at m/z
+
451 [M+1]
C H O . The UV spectrum showed a major absor-
suggesting the molecular formula
21
22 11
bance at 286 nm characteristic of a ¯avanone skeleton
Markham & Marbry, 1975). A singlet at d 12.14,
(
2. Results and discussion
which disappeared on deuterium exchange, indicated a
C-5 hydroxyl.This functionality was further con®rmed
by a bathochromic shift of 24 nm in the presence of
An ethanolic extract of the bark of Maclura tinctoria
was chromatographed on silica gel using MeOH/
CHCl3 mixtures. The fractions eluted with 15±25%
MeOH/CHCl3 when repeatedly chromatographed on
reversed-phase C18 column yielded the following ¯avo-
noids: 5,7,3',4'-tetrahydroxyiso¯avone (orobol) (1),
AlCl . The ¯avanone structure was further substan-
3
1
tiated by H and C NMR analysis (Agrawal, 1989).
13
The proton at C-2 appeared as a double doublet at d
5
.64 (J = 13 and 3 Hz). The equatorial proton of C-3
appeared at d 2.64 as a double doublet (J = 17, 3 Hz).
The C-3 axial proton however, was masked by the res-
onances of the sugar moiety and was not easily deli-
*
Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00126-0

1
42
H.N. El-Sohly et al. / Phytochemistry 52 (1999) 141±145
Table 1
1
13
H and C NMR data for compounds 2, 6 and 7
Position
2
6
7
1
13
1
13
1
13
H d
integral, multi., J (Hz))
C
H, d
(integral, multi., J (Hz))
C
H, d
(integral, multi., J (Hz))
C
(
2
3
5.57 (1H, dd, 13, 2.6)
2.57 (1H, dd, 17, 2.7) eq.,
73.8
42.3
5.64 (1H, dd, 13, 3)
2.64(1H, dd, 17, 3) eq.,
74.2
41.5
8.22 (1H, s)
±
151.4
125.1
3
±
±
.25 (1H, dd, 13, 17) ax.
signal burried ax.
±
4
5
6
7
8
9
196.8
165.5
95.7
166.6
94.9
163.4
101.6
115.3
158.6
102.4
155.7
106.4
128.2
±
196.9
164.0
96.3
167.2
95.4
163.7
102.1
118.9
156.0
104.4
158.6
107.5
128.4
±
±
175.1
160.9
97.8
±
5.88 (2H, s)
±
6.44 (1H, s)
5.85 (2H, s)
±
5.85 (2H, s)
±
5.88 (2H, s)
±
162.0
108.6
158.5
105.9
124.1
114.1
147.9
147.1
115.9
122.4
±
±
±
±
±
1
1
2
3
4
5
6
5
0
±
±
±
'
'
'
'
±
±
±
7.08 (1H, d, 1.9)
±
6.33 (1H, d, 1.9)
±
6.56 (2H, m)
±
±
6.24 (1H, dd, 8.4, 1.9)
±
±
'
6.56 (2H, m)
7.3 (1H, d, 9.2)
12.14 (1H, br, s)
±
6.77 (1H, d, 8.2)
6.91 (1H, dd, 1.7, 8.2)
±
'
-OH
7.26 (1H, d, 8.4)
12.15 (1H, br, s)
Ome
Ome
±
±
±
±
±
±
±
±
±
±
3.79 (6H, s)
56.16
56.22
73.6
±
±
4.8 (1H, d, 7.5)
±
10
20
30
40
50
6
0
±
100.9
73.7
77.1
70.1
77.5
61.1
4.7 (1H, d, 9.5)
±
71.2
79.1
±
3.2 ± 3.5
(4H, m)
3.2±3.4
(4H, m)
±
70.9
81.9
±
±
3.7 (2H, d, 11.6)
3.7 (2H, d, 11.1)
61.6
1
3
13
neated. In the C NMR spectrum, the signals for C-2
and C-3 appeared at d 74.2 and 41.5, respectively. A
two-proton signal at d 5.88 appearing as a singlet was
assigned to H-6 and H-8. A two-proton signal at d
The above data in conjunction with the C NMR
spectrum (DEPT sequence) were found to be consist-
ent with a ¯avanone glucoside structure. Assignments
of the protonated carbons were made using HMQC.
1
13
6
.56 appearing as a multiplet was assigned to H-3' and
H-5' and a one-proton signal at d 7.3 (d, J = 9.2 Hz)
was assigned to H-6'. These H NMR data indicated
that the A ring was hydroxylated at positions 5 and 7,
The results related to H and C NMR spectral in-
terpretations are shown in Table 1 and HMBC corre-
lations are depicted in Fig. 1. The glucose unit was
placed on the C-4' position based on the value of the
proton of the anomeric carbon (d 4.8) (Markham &
Geiger, 1994a) and the HMBC correlation observed
between the anomeric proton of glucose and carbon
'. Thus, 6 was assigned as 5,7,2'-trihydroxy¯avanone-
'-O-b-D-glucoside or steppogenin-4'-O-b-glucoside.
Compound 7, exhibited in the FABMS spectrum a
1
1
3
while the B ring at 2' and 4'. The C NMR data
further established the hydroxylation at C-2' since the
chemical shift of C-2 resonated at d 74.1 (an up®eld
shift of 05 ppm) indicative of the presence of a 2'-oxy
substituent (Agrawal, 1989). A bathochromic shift (37
nm, band II) produced in the UV (methanol) after the
addition of NaOAc indicated a free 7-OH group.
4
4
peak at m/z 477 [M+1] for C H O . An iso¯avone
23 24 11
1
H NMR resonances at d 3.5±3.2 and six signals in
1
3
the C NMR spectrum at 100.9±61.6 indicated the
presence of an O-sugar moiety. A loss of 162 mass
units from the molecular ion in the FABMS and a sig-
nal at d 61.6, shown by DEPT to represent a CH2
group suggested a hexose moiety. The carbohydrate
moiety was con®rmed as glucose by acid hydrolysis
and subsequent TLC sugar analysis. The anomeric glu-
cose proton appeared at d 4.8 as a doublet with a
coupling constant for H-10/H-20 of J = 7.5 Hz, indi-
cating a b-linkage of the glucose unit to the aglycone.
HO
HO
O
O
HO
OH
O
O
OH
OH
OH
H
C
Fig. 1. HMBC correlations of 6.

H.N. El-Sohly et al. / Phytochemistry 52 (1999) 141±145
143
Glc
OMe
OH
Glc
HO
O
H
OMe
OH
HO
H
O
OMe O
H
OMe O
H
C
H
Fig. 2. HMBC correlations of 7.
Fig. 3. Long-range COSY connectivities observed for 7.
skeleton was suggested by a major absorbance in the
1
UV at 259 nm and the appearance in the proton H
NMR spectrum of a one proton singlet at d 8.22. The
UV value of band II (ring A) at d 259 nm was about 6
nm nearer in the UV than the equivalent 5-hydroxyiso-
6
1
1.6 characteristic of a C-glucoside (Nunes et al.,
989). The glucose residue was assigned to position 8
1
13
based on HMBC correlations (Fig. 3). The H and
C
NMR data are presented in Table 1. The conformation
of the anomeric proton was deduced to be b based on
the signal at d 4.7 appearing as a doublet with a coup-
ling constant of 9.5 Hz (Nunes et al., 1989). The struc-
ture of 7 was thus established as orobol 5,3'-di-O-
methyl-8-C-glucoside.
¯
1
(
avone glycoside (1a) (Nunes, Haag, & Bestmann,
989). The absence of a bathochromic shift in the UV
methanol) spectrum after the addition of AlCl was
3
13
evidence of the absence of a free 5-OH group. The
C
NMR spectrum provided additional con®rmation of
the lack of a free 5-OH group as the carbonyl signal
of 7 resonated at d 175.1 instead of 180.0 as shown in
the spectrum of compound 1a (Nunes et al., 1989).
The 7-hydroxyl group was free as indicated by a bath-
ochromic shift in band II (ring A) of 11 nm upon the
1
addition of sodium acetate. The H NMR spectrum
showed a one proton singlet at d 6.44 indicating that
ring A was trisubstituted. From HMBC data this pro-
ton showed correlations with carbons 5 and 8 instead
of 6 and 9, therefore it was assigned to position 6.
Ring B showed a pattern of three one-proton signals
at d 7.08 (1H, d, J = 1.7 Hz), d 6.77 (1H, d, J = 8.2
Hz) and d 6.91 (1H, dd, J = 1.7, 8.2 Hz), the mutipli-
city of which showed one proton coupled to the
remaining two which were, in turn, not coupled to
each other. The size of the coupling constants (1.7 and
8.2 Hz) is characteristic of meta and ortho couplings as
found in a 3',4'-oxygenated ¯avonoids. In addition,
1
3
signals in the
1
CNMR spectrum at d 147.9 and d
47.1 indicated that the oxygenated carbons are adja-
cent due to the shielding e€ect (>d10) each oxygen
exerts on its neighboring ortho carbons. The presence
of two methoxyl groups in the molecule was indicated
1
by a peak in the H NMR spectrum at d 3.79 appear-
ing as a singlet and integrating for 6 protons and in
1
3
the C NMR spectrum as two signals at d 56.16 and
d 56.22. These two methoxyl groups were placed on
carbons 5 and 3' (d 160.9 and d 147.9) based on
HMBC correlations of the methoxyl group with the
above mentioned carbons (Fig. 2). In addition, from
long-range COSY connectivities (Fig. 2) the positions
of the methoxyl group on ring A and ring B were con-
®
rmed since cross-peaks from H-6 to OMe were found
1
3
as well as from H-2' to OMe. The C NMR spectrum
showed signals at d 73.6, 71.2, 79.1, 70.9, 81.9 and

1
44
H.N. El-Sohly et al. / Phytochemistry 52 (1999) 141±145
1
solvent A); identi®ed by comparison (UV, H,
13
3. Experimental
C
NMR) with literature data (Mabry et al., 1970a; Van
Heerden et al., 1980; Markham & Geiger, 1994).
3.1. Plant material
The plant material (stem bark) used in this study
3.5. 5,7,2',4'-Tetrahydroxy¯avanone (steppogenin) (2)
Colorless ®ne needles, m.p. 248±2538C (acetone/
was collected in October, 1994 in Venezuela and ident-
i®ed by Robert F. Smith. Voucher specimens are on
deposit at the National Center for the Development of
Natural Products (Voucher # 13002).
CHCl ) (lit (Sotnikova, Chagovets, & Litvinenko,
1968), 254±2578), R 0.47 (reversed-phase C18, solvent
3
f
C), 0.36 (silica gel, Solvent A); identi®ed by compari-
1
son of UV data (Sotnikova et al., 1968). H and
C NMR spectra are listed in Table 1 and repor-
ted for the ®rst time. CD (c, 0.0135%,
MeOH):[y ]299+5145(max); [y]292 +2857 (sh); [y]236
3
.2. General experimental procedures
13
1
13
H and
C NMR, DEPT,HMQC, HMBC and
long-range COSY spectra were recorded in DMSO-d6
on a Bruker Avance DPX 300 operating at 300 and 75
MHz, respectively, and using the solvent signals at d
� 3696 (max); [y ]2 � 1826 (max); indicating a 2R con-
27
®guration (Gaeld, 1970).
1
13
2.49 ( H) and d 39.5 ( C) as reference. Mass spectra
were obtained on a ZAB HS mass spectrometer (VG
Analytical, Manchester, UK) equipped with an 11/250
J data system. Fast atom bombardment (FAB) exper-
iments were performed using a Xenon gun operated at
3.6. 3, 5, 7, 4'-Tetrahydroxy¯avanonol (aromadendrin)
(3)
Colorless needles, m.p. 229±2318C (acetone/CHCl3).
R 0.54 (reversed-phase C18, solvent C), 0.52 (silica
8
keV energy and 0.8 mA emission. A sample in
f
1
gel, solvent A); identi®ed by comparison (UV, H
13
DMSO was added to glycerol as the matrix. UV spec-
tra were recorded on a Hewlett Packard 8453 equipped
with a Biochemical Analysis System following estab-
lished procedures for the identi®cation of ¯avonoids
C
NMR) with literature data (Mabry, Markham, &
Thomas, 1970b; Shen & Theander, 1985). CD (c,
0.0059%, MeOH): [y]330 +7744 (max); [y]303 � 11997
(max); [y]288 � 38432 (max); [y] +5056 (max); [y]
(
Mabry, Markham, & Thomas, 1970a). Optical ro-
254
230
tations were measured on a Jasco DIP 370 Digital
Polarimeter. Circular dichroism were obtained on a
Jasco J-715 spectropolarimeter. TLC was performed
on precoated polyester backing silica gel G UV254
� 3556 (max); indicating
a
2R,3R con®guration
(Gaeld, 1970).
3.7. 3,5,7,2',4'-Pentahydroxy¯avanonol (dihydromorin)
(4)
plates (Whatman) using 7% MeOH/CHCl (solvent A)
3
and benzene/EtOAc/MeOH (65:26:34, solvent B); or
reversed-phase C18 (E. Merck) using MeOH/H O
Colorless needles, m.p. 232±2358C (CHCl /MeOH)
2
3
(
68:32, solvent C).
(lit. (Deshpande, Srinivasan & Rama Rao, 1975),
288), R 0.68 (reversed-phase C18, Solvent C), 0.24
2
(silica gel, solvent A); identi®ed by comparison (UV,
f
3
.3. Extracton and isolation
1
MS, H NMR) with literature data (Deshpande,
The dried powdered bark (500 g) was extracted sev-
Srinivasan, & Rama Rao, 1975). CD (c, 0.0195%,
MeOH): [y]330 +4255 (max); [y ]309 � 1587 (max);
[y]274 +2743 (max); [y]234 +2013 (max); indicating a
2R,3R con®guration (Gaeld, 1970).
eral times with 95% EtOH. The combined extracts
were evaporated to dryness under red. pres. (34.5 g)
and then a portion of the extract (19 g) was chromato-
graphed on silica gel 60 column (480 g) using MeOH/
CHCl mixtures. Based on TLC similarities, 19 com-
3
3.8. Orobol 7-O-b-D-glucoside (5)
bined fractions were obtained. Fractions from this col-
umn were separated further on reversed-phase C18
column (Bakerbond 40 mm, J.T. Baker) using MeOH/
Cream amorphous solid, m.p. 269±2718C (MeOH/
H O) (lit.(Anhut et al., 1984), 275±276), Rf 0.74
(reversed-phase C18, solvent C), 0.45 (silica gel, solvent
2
H O (1:1) and increasing amounts of MeOH to a€ord
2
1
13
¯
the basis of UV, H NMR, C NMR and MS.
avonoids 1±7. Isolated compounds were identi®ed on
1
B); identi®ed by comparison (UV, H, C NMR) with
literature data (Markham & Geiger, 1994b; Anhut et
al., 1984).
13
3.4. 5,7,3',4'-Tetrahydroxyiso¯avone (orobol) (1)
3
.9. Steppogenin 4'-O-b-D-glucoside (6)
Amorphous solid, m.p. 275±2788C (acetone/CHCl3)
(
0
lit. (Van Heerden, Brandt, & Roux, 1980), 2708), Rf
.43 (reversed-phase C18, solvent C), 0.46 (silica gel,
Colorless
C(MeOH/H O); R 0.62 (reversed-phase C18, solvent
amorphous
solid, m.p.
259±2638
2
f

H.N. El-Sohly et al. / Phytochemistry 52 (1999) 141±145
145
2
5
C), 0.53 (silica gel, solvent B); [a] � 65.58 (MeOH, c,
Grant No. ROI-AI- 27094 from the National Institute
of Allergy and Infectious Diseases, Division of AIDS
and the National Center for the Development of
Natural Products, School of Pharmacy, the University
of Mississippi.
D
MeOH
1
.01); UV lmax nm: 332 (sh), 286; + NaOMe: 320,
43 (sh); +AlCl : 362 (sh), 310; + AlCl ±HCl: 362
(sh), 308; + NaOAc: 323, 280 (sh). FABMS, m/z (rel.
+
int.): 451 [M+1] (35), 154 [A +1] (100), 137 [B
2
3
3
Á]
1
3
1
13
(
90). H NMR and C NMR (see Table 1). CD (c,
.010%, MeOH): [y]327 � 605 (max); [y ] +5871
0
285
(
ing a 2R con®guration (Gaeld, 1970).
max); [y ]237 � 1285 (max); [y] � 5946 (max); indicat-
229
References
3.10. Orobol 5,3'-di-O-methyl-8-C-glucoside (7)
Agrawal, P. K. (1989). In Carbon-13 NMR of ¯avonoids (p. 14; 96).
New York: Elsevier Science Publishing Company.
Fine pale yellow needles, m.p. 233±2378C (MeOH/
H O); R 0.79 (reversed-phase C18, solvent C), 0.33
MeOH
Anhut, S., Zinsmeister, H. D., Mues, R., Barz, W., Machenbrock,
K., Koster, J., & Markham, K. R. (1984). Phytochemistry, 23,
2
f
(
silica gel, solvent B); UV lmax nm: 292 (sh), 259;
1073.
+
+
NaOAc: 332 (sh), 270; +AlCl : 292 (sh), 260;
AlCl /HCl: 292 (sh), 260; +NaOMe: 308 (sh), 275.
3
Deshpande, V. H., Srinivasan, R., & Rama Rao, A. V. (1975).
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3
1
13
FABMS, m/z (rel. int.): 477 [M+1](85).). H and
NMR (see Table 1).
C
Mabry, T. J., Markham, K. R., & Thomas, M. B. (1970a). The sys-
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204.
3.11. Acid hydrolysis of 6
Mabry, T. J., Markham, K. R., & Thomas, M. B. (1970b). The
Systematic Identi®cation of Flavonoids. New York: Springer
Verlag p. 222.
Compound 6 (5 mg) was re¯uxed for 75 min with 4
N HCl in MeOH (8 ml). The HCl hydrolysate was
concentrated, extracted with EtOAc and examined by
TLC on silica gel in 7 % MeOH/CHCl for the liber-
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Markham, K. R., & Geiger, H. (1984b). Advances in research since
3
ated aglycone, which was identi®ed by direct compari-
1986. In J. B. Harborne, The Flavonoids (p. 458). London, New
York: Chapman and Hall.
son with the isolated naturally occurring
steppogenin). The acidic mother liquor was neutral-
ized with Na CO , ®ltered and evaporated to dryness
2
(
Markham, K. R., & Marbry, T. J. (1975). In J. B. Harborne, T. J.
Mabry, & H. Mabry, (p. 50). In The ¯avonoids, 1. New York:
Academic Press.
2
3
for examination of the sugar moiety, which proved to
be glucose by detection on TLC (EtOAc±isopropanol±
H O (65:23:12) and sprayed with p. anisaldehyde±
Nunes, D.S., Haag, A., & Bestmann, H.J. (1989). Liebigs Annalen
der Chemie. 331.
2
Shen, Z., & Theander, O. (1985). Phytochemistry, 24, 155.
Smith, J. L., & Perino, J. V. (1981). Economic Botany, 35, 24.
Sotnikova, O. M., Chagovets, R. K., & Litvinenko, V. I. (1968).
Khimia Prirodni Soedinenia, 4, 82.
H SO reagent followed by heating (Stahl, 1962).
2
4
Stahl, E. (1962). Dunnshicht-Chromatographie. Ein Laboratoriums-
handbuch. Berlin: Springer-Verlag p. 534.
Acknowledgements
Van Heerden, F. R., Brandt, E. V., & Roux, D. G. (1980). Journal
of Chemical Society Perkin Trans. I, 2463.
This work was supported by Public Health Service