Bioactive flavonoids of the flowers of Butea monosperma

Article abstract of DOI:10.1248/cpb.57.428

One new dihydrochalcone, dihydromonospermoside (7), was isolated from the flowers of Butea monosperma together with three known chalcones, butein (2), monospermoside (4) and isoliquiritigenin (8), one flavone, 7,3',4'- trihydroxyflavone (6), four flavanon

Full text of DOI:10.1248/cpb.57.428

4
28
Notes
Chem. Pharm. Bull. 57(4) 428—432 (2009)
Vol. 57, No. 4
Bioactive Flavonoids of the Flowers of Butea monosperma
a
a
a
b
Ratchanaporn CHOKCHAISIRI, Channarong SUAISOM, Siriluk SRIPHOTA, Anon CHINDADUANG,
a
,a
Thipphawan CHUPRAJOB, and Apichart SUKSAMRARN*
a
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng
b
University; Bangkok 10240, Thailand: and National Nanotechnology Center, National Science and Technology
Development Agency; Phaholyothin Road, Pathumthani 12120, Thailand.
Received December 17, 2008; accepted January 21, 2009; published online January 26, 2009
One new dihydrochalcone, dihydromonospermoside (7), was isolated from the flowers of Butea monosperma
together with three known chalcones, butein (2), monospermoside (4) and isoliquiritigenin (8), one flavone,
7
,3ꢀ,4ꢀ-trihydroxyflavone (6), four flavanones, (ꢁ)-butin (1a), (ꢁ)-butrin (3a), (ꢂ)-isomonospermoside (5b) and
(
ꢁ)-liquiritigenin (9a), and three isoflavones, formononetin (10), afrormosin (11) and formononetin-7-O-b-D-glu-
copyranoside (12). The structure of the new compound was elucidated by spectroscopic techniques whereas those
of the known compounds were identified by comparisons of spectroscopic and some physical data with those of
reported compounds. The absolute configurations at the 2-position of the flavanones 1a, 3a, 5b and 9a were es-
tablished to be 2S, 2S, 2R and 2S, respectively, by circular dichroism spectral measurements and were confirmed
by comparison of the optical rotations with those of reported values and by enzymic hydrolysis of the glucosides
to the corresponding aglycones. The isolated flavonoids exhibited varying antimycobacterial activity with the
chalcone 2 being the most active compound (MIC 12.5 mg/ml).
Key words Butea monosperma; flavonoid; circular dichroism; antimycobacterial activity
Butea monosperma (LAM.) TAUB. (Fabaceae), known in side and nonacosanoic acid were also among those isolated
1
5,20)
Thai as Tong-kwoaw, is a medium sized deciduous tree of up compounds.
As part of an ongoing project on bioactive
to 50 feet high with stunning flower clusters, very conspicu- compounds from Thai medicinal plants for the treatments of
ous when in flower, and the trunk is usually crooked and tropical diseases, the plant species was investigated and it
twisted with irregular branches and rough, grey bark. The was found that the EtOAc and MeOH extracts exhibited an-
1)
flowers are bright orange red, large and in rigid racemes.
timycobacterial activity. The present report deals with the
This plant species has been found to display a wide variety of
biological activities. The stem bark is used in indigenous
medicine for the treatment of dyspepsia, diarrhea, dysentery,
2)
diabetes, ulcers, sore throat and snake bites. The flower ex-
3)
tract showed antidiabetic effect in rats. The plant leaves ex-
4)
hibited antiinflammatory effect and exerted effect on stress,
5)
anxiety and cognition in rats. Anti-diarrhoeal activity of the
6)
ethanolic extract of the stem bark and anticonvulsive activ-
7)
ity of the petroleum ether extract of the flowers of this plant
species in experimental animals have been reported. The
plant extracts exhibited other biological activities including
8
)
9,10)
aphrodisiac activity in male rats, anthelmintic activity,
11)
and dermal wound healing in rats.
Isolation of (ꢀ)-
medicarpin with antifungal activity from this part of the plant
12)
has also been reported. From the flowers of this plant
species the flavonoids butin (1), butein (2), butrin (3),
isobutrin, palasitrin, coreopsin, isocoreopsin, sulphuresin,
monospermoside (4), isomonospermoside (5) and 7,3ꢁ,4ꢁ-tri-
1
3—15)
hydroxyflavone (6) have been isolated.
Antihepatotoxic
16)
activity of the flower extract and of compound 3 and
isobutrin has been observed. Compound 1 which was iso-
17)
lated from the seeds of this plant species showed estrogenic
18)
and postcoital anti-conceptive activity in rats. A lectin was
also isolated from the seeds and was found to agglutinate
human erythrocytes. The activity was inhibited by N-acetyl
19)
galactosamine and did not require a divalent ion. The eu-
phane triterpenoid 3a-hydroxyeuph-25-ene and the alcohol
2
,14-dihydroxy-11,12-dimethyl-8-oxo-octadec-11-enylcyclo-
20)
hexane have been isolated from the stem. The imide
palasimide has been isolated from the pods of this plant
species. Stigmasterol, stigmasterol-3-O-b-D-glucopyrano-
21)
∗
To whom correspondence should be addressed. e-mail: s_apichart@ru.ac.th
© 2009 Pharmaceutical Society of Japan

April 2009
429
Fig. 1. Structure and Selected HMBC Correlations of Dihydromonosper-
moside (7)
glucosidic moiety as evident from the presence of the
anomeric proton at d 4.50 (d, Jꢃ7.2 Hz, H-1ꢄ), and other
proton signals at d 3.36—3.50 (m, H-2ꢄ, H-3ꢄ and H-4ꢄ),
3
.29 (m, H-5ꢄ), 3.73 (dd, Jꢃ12.1, 4.0 Hz, H-6ꢄa) and 3.78
dd, Jꢃ12.1, 2.9 Hz, H-6ꢄb). The anomeric proton signal of
isolation and antimycobacterial activity of the isolated new H-1ꢄ attributable to that of b-glucosyl unit from the coupling
(
1
13
compound 7 and the known compounds 1a, 2, 3a, 4, 5b, 6, 8, constant. The H- and C-NMR data of 7 were similar to
a, and 10—12. those of monospermoside (butein-3-O-glucopyranoside,
Investigation of the flowers of Butea monosperma resulted 4) ; the significant differences being the presence of two
9
22)
in the isolation of twelve flavonoids. These included a new methylene signals at d 2.88 and d 3.07, and the absence of
dihydrochalcone, dihydromonospermoside (7), together with olefinic protons of the a,b-unsaturated keto system. Assign-
1
3
1
13
three known chalcones, butein (2),
monospermoside ments of the H- and C-NMR signals were confirmed by
14,22)
23,24)
(4)
and isoliquiritigenin (8),
one flavone, 7,3ꢁ,4ꢁ-tri- 1D and 2D NMR techniques (see also the selected HMBC
hydroxyflavone (6), four flavanones, (ꢀ)-butin (1a),
25)
26,27)
correlations in Fig. 1). The point of attachment of the 1-posi-
and tion of the glucosidic moiety to the 3-position of the dihy-
17,27)
14)
(ꢀ)-butrin (3a),
(ꢂ)-isomonospermoside (5b)
23)
(
ꢀ)-liquiritigenin (9a),
and three isoflavones, for- drochalcone was established by HMBC correlation (see Fig.
mononetin (10), afrormosin (11) and formononetin-7-O- 1). The ROESY spectrum showed cross peak between H-2
b-D-glucopyranoside (12). The spectroscopic and physical and H-1ꢄ confirming the existence of C-3–O–C-1ꢄ linkage.
data of the flavanones 1a, 3a, 5b and 9a are presented in Ex- The structure of compound 7 was thus concluded as the a,b-
perimental. The structure of the new compound was eluci- saturated analogue of monospermoside (4) and named dihy-
dated by spectroscopic techniques whereas those of the dromonospermoside.
28)
29)
29)
known compounds were identified by comparisons of spec-
The absolute configuration at C-2 of flavanones could not
troscopic data with those of reported compounds. The ab- be determined from the coupling constants of H-2 and H-3 in
1
solute configurations of the isolated flavanones 1a, 3a, 5b the H-NMR spectrum, since natural flavanones exist in the
and 9a have been determined by circular dichroism (CD) and thermodynamically favoured conformation with the C-2 aryl
were confirmed by comparison of the optical rotations with substituent in the equatorial orientation and it follows that
30)
those of reported values and by enzymic hydrolysis of the J2,3ax for both S and R isomers are large. The C-2 configu-
glucosides to the corresponding aglycones.
ration of flavanones was determined by CD spectral measure-
2
6
31,32)
Compound 7 was obtained as pale yellow gum, [a]
ment.
The UV absorptions of flavanones at 270—290 nm
D
ꢀ
33.9° (cꢃ0.15, MeOH). The IR spectrum showed absorp- and 320—330 nm have been assigned to the p→p* and
ꢀ1
tion for the hydroxyl (3385 cm ) and a chelated aromatic n→p* acetophenone chromophore transitions, respectively.
ꢀ1
keto (1636 cm ) groups. The electrospray-time of flight Flavanones with 2S configuration possessing a conformation
ꢂ
(ES-TOF)-MS (positive ion mode) gave an [MꢂNa] ion at with P-helecity of the heterocyclic ring and having a C-2
m/z 459.1269, corresponding to a molecular formula of equatorial aryl group exhibit a positive Cotton effect at the
1
C H O . The H-NMR spectrum of compound 7 exhibited n→p* absorption band and a negative Cotton effect at the
2
1
24 10
32)
an a,b-saturated keto function as a triplet-like signal p→p* absorption band. The absolute configurations at C-
(
Jꢃ7.1 Hz) of two b-protons at d 2.88 and a multiplet signal 2 of the isolated flavanones 1a, 3a, 5b and 9a were estab-
31,32)
of two a-protons at d 3.07. The 2ꢁ,4ꢁ-dihydroxyl substituted lished by CD spectral measurements
and comparison of
pattern was confirmed by heteronuclear multiple bond con- the specific rotations with those of the reported compounds.
nectivity (HMBC) correlation as shown in Fig. 1. Placement The C-2 aryl substituent of the flavanone (ꢀ)-butin (1a) was
of the 2ꢁ-hydroxyl group was confirmed by the presence of a in the equatorial orientation, as evident from the large J2,3ax
1
chelated signal at d 12.80 in the H-NMR spectrum of 7. The (12.5 Hz). The CD spectrum of 1a showed negative Cotton
1
H-NMR spectrum of 7 revealed ABX systems in both the effect at the p→p* absorption band ([q] ꢀ11600), which
2
91
A- and B-rings at d 6.25 (br s, H-3ꢁ), 6.27 (br dd, Jꢃ8.9, indicated the 2-position to be the S orientation. The specific
2
H-2), 6.76 (apparent br s, H-5 and H-6). Upon addition of ble to the reported value ([a] ꢀ36.1° (cꢃ1.0, MeOH)) of
C D , the apparent broad singlet signal of H-5 and H-6 re- (ꢀ)-butin. The C-2 configuration of flavanone glycosides
2
6
.3 Hz, H-5ꢁ), and 7.47 (d, Jꢃ8.9 Hz, H-6ꢁ); and d 6.84 (br s, rotation of 1a ([a] ꢀ39.9° (cꢃ0.40, MeOH) was compara-
D
22
D
27)
6
6
solved to two doublets (Jꢃ8.1 Hz) at d 6.88 (d) and 6.84 can be determined in the same manner to that of the agly-
32)
(
br d), respectively, whereas the H-2 signal appeared as a cone. The CD spectra of (ꢀ)-butrin (3a) showed positive
broad singlet signal at d 6.99. The fourth substituent was a Cotton effect at the n→p* absorption band ([q]340 ꢂ83327),

4
30
Vol. 57, No. 4
Table 1. Antimycobacterial Activity of the Isolated Flavonoids
recorded in KBr on a Perkin-Elmer FT-IR Spectrum BX spectrophotometer.
1
13
H- and C-NMR spectra were recorded on a Bruker AVANCE 400 FT-
a,b)
1
13
Compound
MIC (mg/ml)
NMR spectrometer, operating at 400 ( H) and 100 ( C) MHz. For the spec-
tra taken in CD OD, CDCl and DMSO-d the residual nondeuterated sol-
3
3
6
1
2
3
4
a
a
25
12.5
50
25
25
50
50
25
25
vent signals at d 3.30, 7.24 and 2.49, and the solvent signals at d 49.0, 77.0
1 13
and 39.5, were used as references for H- and C-NMR spectra, respec-
tively. ES-TOF-MS and ES-MS spectra were measured with a Bruker mi-
crOTOF and a Finnigan LC-Q mass spectrometer. Unless indicated other-
wise, column chromatography and TLC were carried out using Merck silica
gel 60 (finer than 0.063 mm) and precoated silica gel 60 F254 plates, respec-
tively. Spots on TLC were detected under UV light and by spraying with
anisaldehyde–H SO reagent followed by heating.
5b
6
7
8
9
0
1
2
2
4
a
Plant Material The flowers of B. monosperma were purchased from
Tai-un-jan herbal store, Bangkok in 2006. A voucher specimen is deposited
at the Faculty of Science, Ramkhamhaeng University (Apichart Suksamrarn,
No. 054).
1
1
1
50
25
100
Extraction and Isolation The dried flowers (15.0 kg) were milled and
soaked successively with n-hexane, EtOAc and MeOH. The hexane, EtOAc
and MeOH extracts were evaporated under reduced pressure to give the
crude hexane extract (brownish syrup, 32.8 g), the EtOAc extract (dark
brownish sticky solid, 102.7 g) and the MeOH extract (dark brownish sticky
solid, 527.6 g).
a) MIC at ꢆ200 mg/ml is regarded as inactive. b) The standard drugs, kanamycin
sulphate, isoniazid and rifampicin, showed MIC values of 2.5, 0.06 and 0.004 mg/ml,
respectively.
which indicated the 2S configuration. The specific rotation of
3
a was also comparable to the reported value of (ꢀ)-
The EtOAc extract (100.0 g) was fractionated by quick column chro-
27)
butrin. It should be noted that, though it was established matography (Merck silica gel 60 PF254, 500 g), eluting with n-hexane, n-
hexane–EtOAc and EtOAc with increasing amounts of the more polar sol-
that the laevorotatory flavanones possess a 2S configura-
tion, the optical rotation of the corresponding glycosides
does not necessarily follow this trend and this was due to the
chirality of the carbohydrate moiety. The C-2 configuration vent to give 6 subfractions. Subfraction 3 was chromatographed eluting
of 3a was further confirmed by enzymic hydrolysis of 3a
33)
vent. The eluates were examined by TLC and 10 combined fractions (C1—
C10) were obtained. Fraction C7 was rechromatographed over silica gel
0.063—0.200 mm, 125 g) with n-hexane–EtOAc and EtOAc as eluting sol-
(
32)
under isocratic condition (20% EtOAc in n-hexane) to yield formononetin
2
8)
23,24)
(
10) as orange solid (10.4 mg), isoliquiritigenin (8)
as pale yellow
with b-glucosidase to (ꢀ)-butin (1a) (see Experimental).
Flavanones with 2R configuration with a C-2 equatorial aryl
group exhibit a negative Cotton effect at the n→p* absorp-
29)
solid (6.8 mg) and afrormosin (11) as white solid (11.0 mg). The position
of the methoxyl group of compound 10 was confirmed by HMBC correla-
tion between the methoxyl proton signal with C-4ꢁ. The positions of the two
tion band and a positive Cotton effect at the p→p* absorp- methoxyl groups of compound 11 were at the 6- and 4ꢁ-positions from NOE
32)
experiments of the 6-OMe and H-5, and of the 4ꢁ-OMe and H-3ꢁ/H-5ꢁ, re-
spectively. Subfraction 4 was chromatographed using n-hexane–EtOAc and
EtOAc as eluents, with increasing amount of the more polar solvent, to af-
tion band. The CD spectra of (ꢂ)-isomonospermoside (5b)
showed negative maximum in the long wavelength region
(
[q] ꢀ55403), which indicated the 2R configuration. The
23)
3
42
ford liquiritigenin (9a) (34.7 mg). Fraction C9 was combined and chro-
2
6
specific rotation of 5b ([a] ꢂ6.4° (cꢃ0.30, MeOH) was matographed over silica gel (0.063—0.200 mm, 500 g) using CH Cl2 and
D
2
not compared with the literature value, since no report has CH
Cl –MeOH as eluents, with an increasing amount of the more polar sol-
2
2
vent, to give 3 subfractions. Subfraction 2 was chromatographed over silica
gel and eluted under isocratic condition (2% MeOH in CH Cl ) to give butin
been found. The configuration of 5b was confirmed by en-
zymic hydrolysis to (ꢂ)-butin (1b) (see Experimental). The
configuration of 1b was determined to be R from the negative
2
2
14,26)
(
(
1a).
Subfraction 3 was subjected to repeated column chromatography
3ꢅ) with similar eluting solvent systems to give butein (2) as orange
13)
Cotton effect at the n→p* absorption band ([q] ꢀ20156). solid (270.0 mg).
3
45
2
6
Compound 1b gave positive specific rotation with [a]
The MeOH extract (520.0 g) upon standing white solid separated out,
D
which was recrystallized from MeOH and CH Cl to give 42.3 g of (ꢀ)-
ꢂ19.0° (cꢃ0.05, MeOH). The S configuration of compound
a was similarly determined from CD spectral measurement
[q] ꢀ6558). It should be noted that the J2,3ax of the 2R fla-
2
2
14,17)
1
13
butrin (3a).
The H- and C-NMR data were consistent with the re-
9
(
27)
ported values. The mother liquor was evaporated and the residue (475.5 g)
was chromatographed over silica gel (0.063—0.200 mm, 650 g) eluting with
2
80
vanones 5b and 1b are approximately the same order of mag- CH Cl , CH Cl –MeOH and MeOH with increasing amount of the more
2
2
2
2
polar solvent. The eluates were examined by TLC and 13 groups of eluting
fractions (fractions M1—M13) were obtained. Fraction M8 was chro-
matographed over silica gel (0.063—0.200 mm, 30 g) and eluted under iso-
cratic condition (2% MeOH in CH Cl ) to give 7,3ꢁ,4ꢁ-trihydroxyflavone
nitude as those of the 2S flavones 1a and 9a (see Experimen-
tal).
All the isolated flavonoids were subjected to antimycobac-
terial evaluation and the results are presented in Table 1. The
chalcone butein (2) exhibited highest activity with MIC of
2
2
25)
(6) (4.5 mg).
Fraction M9 was chromatographed over silica gel (0.063—0.200 mm,
0 g) and eluted under isocratic condition (2% MeOH in CH Cl ) to give 11
3
1
2.5 mg/ml. The activity of the other flavonoids was between
2
2
subfractions. Subfraction 6 was rechromatographed over silica gel and
eluted under isocratic condition (3% MeOH in CH Cl ) to give for-
2
5 and 100 mg/ml. The relationships between the flavonoid
2
2
structures and their antimycobacterial activity could not be
29)
mononetin-7-O-b-D-glucopyranoside (12)
as white amorphous solid
deduced from the existing data. However, the assay results (5.2 mg). The position of the methoxyl group was confirmed by HMBC cor-
indicated that the presence of glucosidic moiety tended to de- relation of the methoxyl proton signal with C-4ꢁ. Subfraction 8 was rechro-
matographed twice eluting with CH Cl and CH Cl –MeOH with increasing
crease the activity of the flavonoid aglycone and that the ab-
sence of a,b-olefinic system decreased the biological activ-
ity.
2
2
2
2
14)
amount of the more polar solvent to give (ꢂ)-isomonospermoside (5b) as
white amorphous solid (104.5 mg), dihydromonospermoside (7) as pale yel-
14,22)
low gum (61.2 mg) and monospermoside (4)
596.4 mg).
(ꢀ)-Butin (1a): White amorphous solid, [a] ꢀ39.9° (cꢃ0.40, MeOH)
as yellow amorphous solid
(
2
6
Experimental
D
27)
22
25
General Procedures Melting points were determined with an Elec-
trothermal melting point apparatus and are uncorrected. Optical rotations
(lit. [a]D ꢀ36.1° (cꢃ1.0, MeOH)); CD (cꢃ0.06, MeOH) [q] (nm):
ꢀ1
ꢀ11600 (291) (negative maximum); IR (KBr) cm : 3481, 3366, 3119,
were recorded on a JASCO-1020 digital polarimeter and CD spectra were 1664, 1607, 1584, 1508, 1361, 1326, 1287, 1266, 1232, 1170, 1113, 994,
1
recorded in methanol on a JASCO J-810 spectropolarimeter. IR spectra were 819; H-NMR (400 MHz, DMSO-d ) d: 2.62 (1H, dd, Jꢃ16.7, 2.9 Hz, H-
6

April 2009
431
ꢀ
3
eq), 3.03 (1H, dd, Jꢃ16.7, 12.5 Hz, H-3ax), 5.36 (1H, dd, Jꢃ12.5, 2.9 Hz, 2ꢁ, H-6ꢁ), 7.86 (1H, d, Jꢃ8.6 Hz, H-5); ES-MS m/z: 255 [MꢀH] .
H-2), 6.32 (1H, d, Jꢃ2.1 Hz, H-8), 6.47 (1H, dd, Jꢃ8.7, 2.1 Hz, H-6), 6.72
Enzymic Hydrolysis of (ꢁ)-Butrin (3a) A solution of 3a (12 mg) in
(
9
2H, s-like, H-5ꢁ, H-6ꢁ), 6.86 (1H, s, H-2ꢁ), 7.61 (1H, d, Jꢃ8.7 Hz, H-5), water (4 ml) was treated with b-glucosidase from almonds (Fluka, 2 mg) and
1
.02 (2H, brs, OH); H-NMR (400 MHz, CDCl ꢂ5 drops CD OD) d: 2.67 the mixture was stirred at ambient temperature for 4 h. Water (10 ml) was
3
3
(1H, dd, Jꢃ16.9, 2.9 Hz, H-3eq), 2.93 (1H, dd, Jꢃ16.9, 12.9 Hz, H-3ax),
added and the mixture was extracted with EtOAc (3ꢅ15 ml). The organic
5
.23 (1H, dd, Jꢃ12.9, 2.9 Hz, H-2), 6.31 (1H, d, Jꢃ2.1 Hz, H-8), 6.44 (1H,
phase was evaporated and the residue was passed through a Sephadex LH-20
26
dd, Jꢃ8.7, 2.1 Hz, H-6), 6.73 (1H, dd, Jꢃ8.1, 1.6 Hz, H-6ꢁ), 6.78 (1H, d, column eluting with MeOH to yield (ꢀ)-butin (5 mg), [a] ꢀ44.8°
Jꢃ8.1 Hz, H-5ꢁ), 6.87 (1H, d, Jꢃ1.6 Hz, H-2ꢁ), 7.70 (1H, d, Jꢃ8.7 Hz, H-5);
ES-MS m/z: 271 [MꢀH] .
D
2
5
(cꢃ0.30, MeOH), CD (cꢃ0.10, MeOH) [q] (nm): ꢂ11909 (341) (positive
maximum). H-NMR spectrum (in CDCl ꢂ5 drops CD OD) were identical
ꢀ
1
3
3
ꢀ1
Butein (2): Orange solid; IR (KBr) cm : 3301, 2928, 1638, 1558, 1457, to that of the natural 1a.
1
1
352, 1235, 1175, 859. H-NMR (400 MHz, CD OD) d: 6.28 (1H, d,
Enzymic Hydrolysis of (ꢂ)-Isomonospermoside (5b) Compound 5b
(6 mg) was subjected to enzymic hydrolysis in the same manner employed
for compound 3a to yield (ꢂ)-butin (1b) (2 mg).
3
Jꢃ2.4 Hz, H-3ꢁ), 6.40 (1H, dd, Jꢃ8.8, 2.4 Hz, H-5ꢁ), 6.81 (1H, d, Jꢃ8.4 Hz,
H-3), 7.10 (1H, dd, Jꢃ8.4, 1.6 Hz, H-2), 7.17 (1H, d, Jꢃ1.6 Hz, H-6), 7.52
2
6
(
1H, d, Jꢃ15.4 Hz, H-a), 7.71 (1H, d, Jꢃ15.4 Hz, H-b), 7.92 (1H, d,
(ꢂ)-Butin (1b): [a] ꢂ19.0° (cꢃ0.05, MeOH); CD (cꢃ0.21, MeOH)
D
25 ꢀ1
ꢂ
Jꢃ8.8 Hz, H-6ꢁ). ES-MS m/z: 567 [2MꢂNa] .
ꢀ)-Butrin (3a): White solid, mp 193—194 °C from MeOH–CH Cl
[q] (nm): ꢀ20156 (345) (negative maximum); IR (KBr) cm : 3453, 3229,
(
1660, 1600, 1576, 1533, 1467, 1356, 1281, 1247, 1195, 1160, 1128, 1074,
2
2
27)
26
27)
21
1
(
lit. 190—191 °C); [a]D ꢀ65.3° (cꢃ0.45, MeOH) (lit. [a]D ꢀ73.0° 867; H-NMR (400 MHz, CDCl ꢂ5 drops CD OD) d: 2.71 (1H, obscured
3
3
2
5
(cꢃ1.0, pyridine)); CD (cꢃ0.21, MeOH) [q] (nm): ꢂ83327 (340) (posi-
signal, H-3eq), 2.94 (1H, dd, Jꢃ16.9, 12.9 Hz, H-3ax), 5.27 (1H, dd,
ꢀ1
tive maximum); IR (KBr) cm : 3367, 2921, 2873, 1669, 1612, 1575, 1520, Jꢃ12.9, 2.8 Hz, H-2), 6.33 (1H, d, Jꢃ2.2 Hz, H-8), 6.45 (1H, dd, Jꢃ8.7,
1
1
442, 1279, 1252, 1191, 1170, 1109, 1073, 1038, 1014; H-NMR (400 MHz,
2.1 Hz, H-6), 6.77 (1H, dd, Jꢃ8.1, 1.7 Hz, H-6ꢁ), 6.80 (1H, d, Jꢃ8.1 Hz, H-
DMSO-d ) d: 2.67 (1H, dd, Jꢃ16.8, 2.6 Hz, H-3eq), 3.20 (1H, obscured sig-
5ꢁ), 6.89 (1H, d, Jꢃ1.7 Hz, H-2ꢁ), 7.72 (1H, d, Jꢃ8.7 Hz, H-5); ES-MS m/z:
nal, H-3ax), 3.15 (2H, m, H-5ꢄ, H-5ꢇ), 3.25 (2H, obscured signal, H-2ꢄ, H- 271 [MꢀH] .
6
ꢀ
2
ꢇ), 3.40 (4H, obscured signal, H-3ꢄ, H-4ꢄ, H-3ꢇ, H-4ꢇ), 3.43 and 3.68 (each
Antimycobacterial Assay Antimycobacterial activity was assessed
2
H, each m, H-6ꢄ, H-6ꢇ), 4.54 and 4.59 (each 1H, each dd, Jꢃ5.5, 5.4 Hz, against Mycobacterium tuberculosis H Ra using the Microplate Alamar
3
7
35)
OH-6ꢄ, OH-6ꢇ), 4.71 (1H, d, Jꢃ7.1 Hz, H-1ꢇ), 4.97 (1H, d, Jꢃ7.2 Hz, H- Blue Assay. In our system, the standard drugs, kanamycin sulphate, isoni-
1
4
ꢄ), 5.02 (2H, d, Jꢃ4.6 Hz, OH-2ꢄ, OH-2ꢇ), 5.09 (2H, br d, Jꢃ4.6 Hz, OH- azid, and rifampicin showed MIC values of 2.5, 0.06, and 0.004 mg/ml, re-
ꢄ, OH-4ꢇ), 5.35 (1H, br d, Jꢃ4.1 Hz, OH-3ꢄ), 5.45 (1H, obscured signal, spectively.
OH-3ꢇ), 5.45 (1H, dd, Jꢃ12.8, 2.6 Hz, H-2), 6.60 (1H, d, Jꢃ2.2 Hz, H-8),
.70 (1H, dd, Jꢃ8.7, 2.2 Hz, H-6), 6.83 (1H, d, Jꢃ8.2 Hz, H-5ꢁ), 7.02 (1H,
dd, Jꢃ8.2, 1.5 Hz, H-6ꢁ), 7.30 (1H, d, Jꢃ1.5 Hz, H-2ꢁ), 7.70 (1H, d, Strengthening Grant of the National Center for Genetic Engineering and
6
Acknowledgments This work was supported by the Research Team
1
3
Jꢃ8.7 Hz, H-5), 8.78 (1H, br s, OH-4ꢁ); C-NMR (100 MHz, DMSO-d ) d: Biotechnology, National Science and Technology Development Agency.
6
4
7
7
1
1
9
2.9 (C-3), 60.4 and 60.7 (C-6ꢄ and C-6ꢇ), 69.4 and 69.8 (C-5ꢄ and C-5ꢇ),
3.0 and 73.2 (C-2ꢄ and C-2ꢇ), 75.9 and 76.3 (C-3ꢇ and C-3ꢇ), 76.9 and (PERCH-CIC), Commission on Higher Education, Ministry of Education is
Support from the Center of Excellence for Innovation in Chemistry
7.1 (C-4ꢄ and C-4ꢇ), 79.2 (C-2), 99.6 (C-1ꢁ), 101.9 (C-1ꢇ), 103.4 (C-8),
10.9 (C-6), 115.3 and 115.6 (C-2ꢁ and C-5ꢁ), 115.7 (C-10), 121.6 (C-6ꢁ),
27.8 (C-5), 129.7 (C-1ꢁ), 145.0 (C-3ꢁ), 147.1 (C-4ꢁ), 162.8 (C-7), 163.4 (C-
gratefully acknowledged.
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), 190.4 (C-4); ES-MS m/z: 595 [MꢀH] .
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6
(
ꢂ)-Isomonospermoside (5b): White amorphous solid; [a] ꢂ6.4°
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25
(
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1
1
335, 1284, 1162, 1119, 1071, 1034, 990, 799; H-NMR (400 MHz, DMSO-
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ꢂ
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2
6
D
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1
1
133, 1071, 895; H-NMR (400 MHz, CDCl ꢂ5 drops CD OD) d: 2.88
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3
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2
6
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6
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3
3
a
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(
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2
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1
24 10
4
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6
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26
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5
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1
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