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J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000) 169
Flavonoid and Flavonoid glycoside from
Butea superba Roxb. and their cAMP
Phosphodiesterase Inhibitory Activity
Sophon Roengsumran,1 Amorn Petsom,1 Nattaya Ngamrojanavanich,1
Thanatip Rugsilp,1 Pailin Sittiwicheanwong,1 Prapas Khorphueng,1
Wichai Cherdshewasart,2 and Chaiyo Chaichantipyuth3
A flavonoid (3, 7, 3'-Trihydroxy-4'-methoxyflavone) (1) and a flavonoid
glycoside (3, 3'-dihydroxy-4'-methoxyflavone-7-O-β-D-glucopyranoside) (2) were
isolated from the tuber root of Butea superba Roxb. The structures were determined
on the basis of spectral analysis, including 2D-NMR techniques. These compounds
show higher inhibitory effects on cAMP phosphodiesterase than caffeine and
theophylline.
Key Words: Butea superba, flavonoid, flavonoid glycoside, cAMP
phosphodiesterase inhibition
1 Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
2 Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
3 Department of Pharmacognocy, Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok 10330, Thailand.
Sophon Roengsumran, Amorn Petsom, Nattaya Ngamrojanavanich, Thanatip Rugsilp, Pailin Sittiwicheanwong,
Prapas Khorphueng, Wichai Cherdshewasart, and Chaiyo Chaichantipyuth…………………………………….
170 J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000)
ฟลาวอนอยด และฟลาวอนอยดไกลโคไซดจากกวาวเครือแดง และ
ฤทธิ์ตอตานไซคลิกเอเอ็มพีฟอสโฟไดเอสเทอเรส
โสภณ เริงสํ าราญ, อมร เพชรสม, นาตยา งามโรจนวนิชย, ธนาธิป รักศิลป,
ไพลิน สิทธิวิเชียรวงศ, ประภาส ขอพึ่ง, วิชัย เชิดชีวศาสตร และ ชัยโย ชัยชาญทิพยุทธ (2543)
วารสารวิจัยวิทยาศาสตร จุฬาลงกรณมหาวิทยาลัย 25(1)
ฟลาวอนอยด (3, 7, 3'-ไทรไฮดรอกซิ-4'-เมธอกซิฟลาโวน) (1) และฟลาวอนอยด
ไกลโคไซด (3, 3'-ไดไฮดรอกซิ-4'-เมธอกซิฟลาโวน-7-O-β-D-กลูโคไพราโนไซด) (2) แยกได
จากสวนรากของกวาวเครือแดง ไดทํ าการหาสูตรโครงสรางของสารดังกลาวโดยอาศัยผลการ
วิเคราะหทางสเปกโตรสโคป ซึ่งประกอบดวยเทคนิค 2D-NMR สารประกอบทั้งสองชนิด
แสดงผลในการยับยั้งไซคลิกเอเอ็มพี ฟอสโฟไดเอสเทอเรสอยางแรง
คํ าสํ าคัญ กวาวเครือแดง ฟลาวอนอยด ฟลาวอนอยดไกลโคไซด การยับยั้ง
ไซคลิกเอเอ็มพีฟอสโฟไดเอสเทอเรส
Flavonoid and Flavonoid glycoside from Butea superba Roxb. and
their cAMP Phosphodiesterase Inhibitory Activity……………………………………………………………………….
J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000) 171
INTRODUCTION
Butea superba Roxb. is a plant in the
Family Papilionaceae and has the
characteristics of being a crawler that wraps
itself around large trees. One branch has 3
leaves, the flowers are of a yellowish orange
color and the plant grows out in the open.
The long tuber root of the plant is buried
under the ground like the tuber root of a yam.
This plant reproduces through seeds and the
propagation of its tuber root. This plant can
be found growing in forests in Thailand’s
northern regions, eastern regions and along
Kanchanaburi Province. The tuber and stem
of the plant are used in medicines believed to
give strength and power and increase male
sexual performance. Thus, this plant has
come to be known as one type of miracle
herb. Since Butea superba Roxb. helps to
enhance human health, it is therefore very
interesting to investigate the chemical
constituents of this plant and their biological
activity. The bioactivity of each constituent
was tested for an inhibitory effect towards
cAMP phosphodiesterase, which has been
shown to be important in controlling bodily
function and involved a wide number of
diseases [1].
Table 1. 1H NMR spectral data of compounds 1 and 2 (500 MHz DMSO).
12
Position δH (J in Hz) δH (J in Hz)
2
3
4
5 7.96 d (8.8) 8.05 d (8.8)
6 6.94 dd (2.1, 8.8) 7.15 dd (2.1, 8.8)
7
8 6.85 d (2.1) 7.22 d (2.4)
9
10
1'
2' 8.35 d (2.1) 8.40 d (2.0)
3'
4'
5' 6.98 d (8.8) 6.98 d (8.8)
6' 7.50 dd (2.1, 8.8) 7.52 dd (2.1, 8.8)
-OCH33.79 s 3.78 s
1" 5.10 d (7.6)
2" 3.30 dd (7.6, 9.5)
3" 3.35 t (9.5)
4" 3.18 t (9.5)
5" 3.45 m
6"a 3.45 dd (12.0, 2.5)
6"b 3.70 dd (12.0, 4.5)
Sophon Roengsumran, Amorn Petsom, Nattaya Ngamrojanavanich, Thanatip Rugsilp, Pailin Sittiwicheanwong,
Prapas Khorphueng, Wichai Cherdshewasart, and Chaiyo Chaichantipyuth…………………………………….
172 J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000)
Table 2. 13C NMR (125 MHz DMSO) and 2D Long-Range 1H-13C Correlations in the HMBC
Spectra of Compounds 1 and 2.
12
Position δCHMBC δCHMBC
2 158.9 159.1
3 146.8 147.0
4 174.6 174.8
5 127.3 4, 7, 9 127.1 4, 7, 9
6 115.2 5, 7, 8, 10 115.7 5, 7, 8, 10
7 162.6 161.5
8 102.1 6, 7, 9, 10 103.5 6, 7, 9, 10
9 157.4 157.1
10 116.6 118.5
1' 124.2 123.5
2' 153.4 1', 3', 4', 6' 153.6 1', 3', 4', 6'
3' 157.3 157.1
4' 146.9 146.6
5' 113.6 1', 3', 4' 113.7 1', 4', 6'
6' 130.0 1', 2, 4', 5' 130.1 1', 3', 4', 5'
-OCH355.2 55.2
1" 100.1
2" 73.2
3" 76.5
4" 69.8
5" 77.2
6" 60.8
EXPERIMENTAL
General Experimental Procedures
All commercial grade solvents were
distilled prior to use. Melting points were
determined on a Fisher-Johns melting point
apparatus and are reported uncorrected. The
optical rotation was determined on a JASCO
DIP-370 digital polarimeter. Measurements
of UV spectra were carried out on a Milton-
Roy Spectronic 3000 Array UV/VIS
spectrophotometer. IR spectra were recorded
on a Perkin-Elmer model 1760X FT-IR
spectrophotometer. Spectra of solid samples
were recorded as KBr pellets. The 1H and
13C NMR spectra were recorded at 500.00
and 125.65 MHz, respectively, on a JEOL
JNM-A500 NMR spectrometer. LREIMS
were obtained with a Fisons Instruments
model Trio 2000 mass spectrometer at 70 eV.
Plant Materials
The tubers of Butea superba Roxb.
were collected from Amphur Muang,
Lumpang Province, Thailand in May 1997.
Botanical identification was claimed through
comparison with a voucher specimen No.
BKF 70163 in the herbarium collection of
Royal Forest Department of Thailand.
Extraction and Isolation
Powdered sun dried roots (16.0 kg) of
Butea superba were repeatedly extracted
with MeOH (5x10 L). The MeOH extracts
were filtered and evaporated under reduced
pressure to obtain a dark-red gummy residue
Flavonoid and Flavonoid glycoside from Butea superba Roxb. and
their cAMP Phosphodiesterase Inhibitory Activity……………………………………………………………………….
J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000) 173
(106.0 g) of MeOH crude extract. This
MeOH crude extract was subsequently re-
extracted with hexane and then CHCl3 to
leave the final insoluble residue (72.0 g). The
hexane and CHCl3 extract fractions were
evaporated under reduce pressure to give a
hexane crude extract (21.0 g) and CHCl3
crude extract (12.0 g), respectively. The
CHCl3 crude extract (12.0 g) was subjected
to silica gel column chromatography using
gradient elution with CHCl3 and MeOH in a
stepwise fashion. Compound 1 was eluted
with 5% MeOH in CHCl3. Similar fractions
were combined and the solvent was removed
under reduced pressure to give compound 1
(135.0 mg) after recrystallization from
MeOH and CHCl3. The final residue (72.0 g)
was separated by column chromatography on
Silica gel using gradient elution with
increasing amounts of MeOH in CHCl3 to
give compound 2 (60.0 mg) from 10% of
MeOH in CHCl3 fraction. Compound 2 was
recrystallized from MeOH and CHCl3.
Flavonoid (3, 7, 3'-Trihydroxy-4'-
methoxyflavone) (compound 1): pale yellow
needle crystal, mp 258-260°C (d) [lit 288(d),
but no spectroscopic data for verification],
(found: C, 64.0; H, 3.9, C16H12O6 required:
C, 64.0; H, 4.0); UV λmax EtOH nm 254, 316
and 365; IR νKBrmax cm-1; 3340-3000, 2940,
1650, 1594, 1575, 1500, 1450, 1380, 1260,
1090, 1020, 790; 1H and 13C-NMR Table 1;
EIMS m/z (rel. int.) 300[M+] (25), 282 (30),
268 (100), 253 (25), 132 (60).
Flavonoid glycoside (3,5'-Dihydroxy-
4'-methoxyflavone-7-O-β-D-
glucopyranoside) (compound 2); white
amorphous solid; mp 210-212°C, (found: C,
57.0; H, 4.5, C22H22O11 requires: C, 57.1; H,
4.8); [α]25D+9.5 (c 1.05, MeOH); UV λmax
EtOH nm 267, 290 and 355; νKBrmax cm-1;
3600-3100, 2900, 1650, 1550, 1450, 1300,
1260, 1060-1030, 891, 800; 1H and 13C-NMR
Table 1 and 2; EIMS m/z (rel. int.) 282 (20),
268 (100), 253 (23), 133 (12), 132 (90).
1 R = H
Scheme 1
(2) R =
Scheme 2
Bioassay
Phosphodiesterase activity was determined
from the amount of inorganic phosphate liberated
during the reaction by the malachite green
method [2, 3]. Phosphodiesterase assay solutions
were prepared as follows: (a) the enzyme
solution contains phosphodiesterase (0.037
unit mL-1), 5-nucleotidase (1.67 unit mL-1),
MgCl2 (5 mM) and Tris HCl (0.2 M); (b)
reaction mixture A contains malachite green
(0.33 mM), polyvinyl alcohol (3.87g L-1) and
ammonium molybdate in 6 M HCl (8.33
mM). The test sample was dissolved in 1.5 %
dimethyl sulfoxide in water. The reaction
was performed by the addition of cAMP (10
mM, 100 µL) to the enzyme solution (400 µL)
at 30°C. Then the sample solution (500 µL),
reagent mixture A (1.0 mL) and 25% sodium
citrate (200 µL) were added to the above
solution successively at 5 minute intervals.
The absorbance of the colour complex was
O
O
OH
OMe
OH
H
H
RO
H
H
H
H
2
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
O
H
HO
H
HO
H
H
OH
H
OH
HH
1"
2"
3"
4"
5"
6"
Sophon Roengsumran, Amorn Petsom, Nattaya Ngamrojanavanich, Thanatip Rugsilp, Pailin Sittiwicheanwong,
Prapas Khorphueng, Wichai Cherdshewasart, and Chaiyo Chaichantipyuth…………………………………….
174 J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000)
observed at 630 nm using a UV/VIS
spectrophotometer referred against a mixed
reagent blank. A calibration curve, obtained
by this procedure using potassium
dihydrogen phosphate solution of a known
concentration, was used to determine the
amount of phosphate present in the assay.
For control experiment, dimethyl sulfoxide
was added instead of the solution of the
sample to minimize the effects of the solvent
and theophylline and caffeine were used as
positive controls for phosphodiesterase assay.
The IC50 valves of compound (1) and (2)
were determined from the calibration curve
of sample concentration against cAMP
phosphodiesterase activity.
RESULTS AND DISCUSSION
The 3, 7, 3'-Trihydroxy-4'-
methoxyflavonone (compound 1) was
obtained from a chloroform soluble crude
extract from the root of Butea superba by
silica-gel column chromatography using a
gradient elution with chloroform and
methanol. The IR spectrum of 1 showed a
broad absorption band between 3000 and
3340 cm-1 of OH stretching and a strong
absorption band at 1650 cm-1, which was
consistent with a conjugated carbonyl group.
The carbon-carbon double bond stretching
vibration of the aromatic phenyl group was
also observed at 1594, 1575 and 1500 cm-1.
The UV spectrum exhibited absorption
maxima at 254 and 365 nm which are
characteristic absorption bands of a flavone
skeleton [4]. The molecular formula of
C16H12O6 was established for compound 1
from the elemental analysis, LREIMS and 1H
and 13C NMR data (Tables 1 and 2). The 1H
and 13C NMR spectra together with 2D NMR
experiments allowed the complete structure
of compound 1 to be established. The
occurrence of flavonoid (1) was clearly
determined from the 1H 500 MHz NMR
spectrum which displayed six aromatic
protons at δ 6.85 (d, J=2.1 Hz), 6.94 (dd,
J=2.1, 8.8 Hz), 6.98 (d, J=8.8 Hz), 7.50 (dd,
J=2.1, 8.8 Hz), 7.96 (d, J=8.8 Hz), and 8.35
(d, J=2.1 Hz) and one methyl proton singlet
at 3.79 ppm. Detailed analysis of the 2D 1H
and 13C NMR spectrum, including COSY,
NOESY, HMQC and HMBC supported the
structure of 3, 7, 3'-Trihydroxy-4'-
methoxyflavone (1). Although compound 1
is a known compound isolated as a minor
constituents of quebracho tannin extract [5],
its 1H and 13C NMR spectral data have not
been published before.
The 3, 3'-Dihydroxy-4'-
methoxyflavone-7-O-β-D-glucopyranoside
(compound 2) was isolated as an amorphous
white solid powder from MeOH extract
residue by silica gel column chromatography
using CHCl3-MeOH as the eluent. The IR
spectrum of compound 2 displayed
absorption at 3100-3600 cm-1 of OH
stretching, and strong absorption at 2900 cm-
1 of C-H stretching. The absorption band of a
conjugated carbonyl group appeared at 1650
cm-1. The molecular formula of compound 2
was assigned as C22H22O11 based on
elemental analysis and 1H and 13C NMR
(Table 1 and 2) while its EI-MS showed no
molecular ion peak pointing to its glycoside
nature. The 1H NMR spectrum of compound
2 in DMSO showed the presence of a sugar
moiety by one proton doublet at δ 5.10
(J=7.6 Hz, H-1''), one proton doublet of
doublets at δ 3.30 (J=7.6, 9.5 Hz, H-2''), one
proton triplet at δ 3.35 (J=9.5 Hz, H-3''), one
proton triplet at δ 3.18 (J=9.5 Hz, H-4'') and
one proton multiplet at δ 3.45 (H-5''). Two
protons of C-6'' included one proton showed
doublet of doublets at δ 3.45 (J=12.0, 2.5 Hz,
H-6''a) and another one proton showed
doublet of doublets at δ 3.70 (J=12.0, 4.5 Hz,
H-6''b). The chemical shifts at δ 6.98, 7.15,
7.22, 7.52, 8.05 and 8.40 were assigned to
flavonoid protons at C-5', C-6, C-8, C-6', C-5
and C-2' respectively, on the basis of their
similarity to signals observed for compound 1.
Their 1H and 13C-NMR spectra indicate
that glycoside 2 has a glycone portion
identical to that in compound 1. In the 1H
NMR spectra (Table 1), an unusual pattern of
7-O-glycosylation was indicated by
downfield shifts of H-6 (ca. +0.21 ppm) and
H-8 (ca. +0.36 ppm) with respect to
Flavonoid and Flavonoid glycoside from Butea superba Roxb. and
their cAMP Phosphodiesterase Inhibitory Activity……………………………………………………………………….
J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000) 175
compound 1. Similarly, in the 13C-NMR
spectra of compound 2 (Table 2), 7-O-
glycosylation was confirmed by the
diagnostic [6] upfield shift of C-7 (-0.24
ppm) and by downfield shifts of the ortho-
related C-8 (+1.45 ppm) and C-6 (+0.55
ppm) and para-related C-10 (+1.99 ppm)
carbon with respect to compound 1. The 1H
and 13C NMR data indicated the β-
configuration at an anomeric position for the
glucopyranosyl unit (Table 1 and 2).
Therefore, the structure of compound 2 was
assigned as 3,3'-dihydroxy-4'-
methoxyflavone-7-O-β-D-glucopyranoside.
From the bioactivity testing of
compound 1 and compound 2, it was found that
both of these compounds were effective in
inhibiting cAMP phosphodieterase. These two
compounds have IC50 = 190 and 58 µg/mL,
respectively. The cAMP phosphodiesterase
inhibition of both these compounds was more
effective than those of theophylline (IC50 = 615
µg/mL) and caffein (IC50 = 420 µg/mL). The
cAMP phosphodiesterase enzyme has a main
function in the hydrolysis of the intracellular
cAMP. Substances that inhibit cAMP
phosphodiesterase are therefore capable of
stimulating the functioning of the central
nervous system (CNS) and stimulating the
functioning of cells [7]. Thus, papaverin,
dipyridamole, caffeine and theophylline are
effective when the cAMP phosphodiesterase
is inhibited [8, 9]. Furthermore, substances
that have an effect in inhibiting cAMP
phosphodesterase also take part in controlling
numerous severe diseases, including diabetes
[10-13], hypertension [14, 15], asthma [16],
hepatomas [17], psoriasis [18] and possibly
cancer [19, 20]. In addition, substances that
inhibit phosphodiesterase are shown to have
effects in controlling platelet-aggregation
inhibition [21- 26].
Therefore, Butea superba Roxb. is
composed of flavonoids that are effective in
inhibiting the cAMP phosphodiesterase
enzyme, which are very beneficial to the
human body. At least, when taking this herb,
the body will begin to feel healthier. This
herb will also help control the numerous
diseases mentioned beforehand.
ACKNOWLEDGEMENTS
We thank the Graduate School and
Department of Chemistry, Faculty of
Science, Chulalongkorn University, for
financial support and the staff of the
Scientific and Technology Research
Equipment Centre, Chulalongkorn
University, for recording highfield NMR
spectra and performing 2D NMR
experiments. We also thank Mr. Thanase U.
Patriyakul of Eiwlee Industrial for providing
some plant materials.
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Sophon Roengsumran, Amorn Petsom, Nattaya Ngamrojanavanich, Thanatip Rugsilp, Pailin Sittiwicheanwong,
Prapas Khorphueng, Wichai Cherdshewasart, and Chaiyo Chaichantipyuth…………………………………….
176 J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000)
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Received: August 6, 1999
Accepted: February 1, 2000
Flavonoid and Flavonoid glycoside from Butea superba Roxb. and
their cAMP Phosphodiesterase Inhibitory Activity……………………………………………………………………….
J. Sci. Res. Chula. Univ., Vol. 25, No.1 (2000) 1
