Antioxidant study and assignments of NMR spectral data for 3′,4′,7-trihydroxyflavanone 3′,7-Di-O-β-D-glucopyranoside (butrin) and its hydrolyzed product

Article abstract of DOI:10.1023/B:CONC.0000039134.46227.1f

The NMR spectral data including high resolution ¹H, ¹³C and 2D NMR for butrin, 3′,4′,7-trihydroxyflavanone 3′,7-di-O-β-D-glucopyranoside, isolated from flowers of Butea monosperma, are reported here for the first time. Butrin was hydrolyzed using b-glucosidase to butin in high yield. They were subjected to free radical scavenging test using 2,2-diphenyl-1-picrylhydrazyl (DPPH) spectrophotometric assay. At a dose of 4 × 10^-8 mol of tested compounds, the percentage of reduced DPPH for butin was 14.5% while no reduction was observed for butrin (0%).

Full text of DOI:10.1023/B:CONC.0000039134.46227.1f

Chemistry of Natural Compounds, Vol. 40, No. 3, 2004
ANTIOXIDANT STUDY AND ASSIGNMENTS OF NMR SPECTRAL DATA
FOR 3 ,4 ,7-TRIHYDROXYFLAVANONE 3 ,7-DI-O-
′ ′ β-D-GLUCOPYRANOSIDE
′
(BUTRIN) AND ITS HYDROLYZED PRODUCT
Amir Reza Jassbi1, Pahup Singh2, Vivek Krishna2,
Pradeep K. Gupta2, and Satoshi Tahara3
UDC 547.972:66.094
The NMR spectral dataincludinghighresolution1H, 13C and 2D NMR for butrin, 3′,4′,7-trihydroxyflavanone
3′,7-di-O-β-D-glucopyranoside, isolated from flowers of Butea monosperma, are reported here for the first
time. Butrin was hydrolyzed using b-glucosidase to butin in high yield. They were subjected to free radical
scavenging test using 2,2-diphenyl-1-picrylhydrazyl (DPPH) spectrophotometric assay. At a dose of 4 × 10-8
mol of tested compounds, the percentage of reduced DPPH for butin was 14.5% while no reduction was
observed for butrin (0%).
Key words: Butrin, butin, antioxidant, 3′,4′,7-trihydroxyflavanone3′,7-di-O-β-D-glucopyranoside, Buteamonosperma,
NMR spectra.
Butrin, 3′,4′,7-trihydroxyflavanone 3′,7-di-O-β-D-glucopyranoside (1), is a major flavonoid found in Butea
monosperma [1, 2], B. frondosa [3, 4], and B. superba [5]. Butea monosperma is used in India as a medicinal plant to cure
hepatic disorders and viral hepatitis [2]. Isobutrin and butrin (1) were reported as the antihepatotoxic components in a butanolic
fraction of methanolic extracts of the flower of B. monosperma [2]. Despite the early identification of this compound [3], to the
best of our knowledge there is no report on its 13C NMR and high resolution 2D NMR in the literature. In this paper we report
the detailed NMRassignment ofbutrin. Butrin ishydrolyzedcompletelytobutin (2) and their antioxidant activities were assayed
by radical scavenging test using DPPH.
OH
H
H
O
HO
O
1' ' '
O
HO
OH
H
H
5'
3'
H
OH
OH
H
OH
O
OH
H
1'
H
HO
HO
O
O
9
O
9
5'
7
3'
HO
1'
7
1' '
H
OH
3
3
H
5
5
1
2
O
Butrin was separated as yellowneedles from the flowers ofButea monosperma. The UV and FABnegative mass spectra
1
were similar to those reported previously [2]. The high resolution H NMR spectrum recorded on a 500 MHz instrument is
presented in Table 1. The 13C NMR spectral data are assigned with the help of 2D NMR spectra including HMQC and HMBC
spectra. In the 1H NMR spectrum (in DMSO-d6) the signals at δ 5.45 (2H, bdd, J = 2.2, 12.5 Hz, H-2, OH-3″ or OH-3′″), 2.67
(1H, dd, J = 2.2, 14.5 H-3β), and 3.2 (m, H-3α) represented the C ring proton signals for a flavanone structure and the signals
at δ 7.70 (1H, d, J = 8.6 Hz, H-5), 6.70 (1H, bd, J = 8.6 Hz, H-6), and 6.66 (1H, bs, H-8) are assigned for the A ring. The signals
at δ 7.28 (1H, bs, H-2′), 6.83 (1H, d, J = 8.3 Hz, H-5′), and 7.02 (1H, bd, J = 8.3 Hz, H-6′) represent the 3′, 4′ di-substituted B
ring. The anomeric protons at 4.97 (1H, d, J = 7.4 Hz) and 4.71 (1H, d, J = 7.1 Hz) represent two β-glycosidic bonds.
1)Department ofPhytochemistry, Medicinal PlantsResearch Institute, ShahidBeheshti University, Evin, Tehran, Iran,
fax: (+9821) 241 86 79, e-mail: a-jassbi@cc.sbu.ac.ir; 2) Department of Chemistry, University of Rajasthan, Jaipur, India;
3) Laboratoryof Ecological Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Kita-ku, Sapporo 060-8589, Japan. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 212-214, May-June, 2004.
Original article submitted April 16, 2004.
0009-3130/04/4003-0250 ©2004 Plenum Publishing Corporation
250

TABLE 1. 1H and 13C NMR Spectral Data for Butrin (δ, ppm, J/Hz)
C atom
H (in DMSO-d6)
H (in DMSO-d6+D2O)
C (in DMSO-d6)
2
3α
5.45 (bdd, J = 2,2, 12.5)
5.44 (d, J = 2.2, 12.5)
79.2 (CH)
42.9 (CH2)
3.2 (m)
3.10-3.45 (m)
3β
2.67 (bdd, J = 2.2, 15.5)
2.66 (dd, J = 2.5, 17.5)
4
5
6
7
-
-
190.4
127.9 (CH)
110.9 (CH)
162.8
7.70 (d, J = 8.6)
6.70 (bd, J = 8.6)
7.69 (d, J = 8.6)
6.70 (dd, J = 2.3, 8.6)
-
-
8
9
6.66 (bs)
6.62 (d, J = 2.3)
103.5 (CH)
163.5
-
-
10
1′
-
-
-
-
115.7
129.7
2′
3′
7.28 (bs)
7.24 (d, J = 1.6)
115.4 (CH)
145.1
-
-
4′
-
-
147.1
5′
6′
1″
1′″
6.83 (d, J = 8.3)
7.02 (bd, J = 8.3)
4.97 (d, J = 7.4)
4.71 (d, J = 7.1)
3.25 (m)
6.82 (d, J = 8.2)
7.00 (dd, J = 1.6, 8.2)
115.3 (CH)
121.7 (CH)
99.7 (CH)
101.3 (CH)
73.3, 73.1 (CH)
76.4, 76.0 (CH)
77.2, 77.0 (CH)
69.9, 69.4 (CH)
60.7, 60.5 (CH2)
4.94 (d, J = 7.3)
4.71 (d, J = 7.3)
3.10-3.45 (m)
3.10-3.45 (m)
2″,2′″
3″,3′″
4″,4′″
5″,5′″
6″,6′″
OH-2″,2′″
OH-3″,3′″
OH-4″,4′″
OH-6″,6′″
OH-4′
3.30 (m)
3.30 (m)
3.15 (m)
3.10-3.45 (m)
3.10-3.45 (m)
3.10-3.45 (m), ca 3.7 (m)
3.68 (m), 3.43 (m)
5.02 (d, J = 4.4)
5.35 (bd, J = 4.2), 5.45 (bd)
5.09 (bd)
4.54 (t, J = 5.6), 4.59 (t, J = 5.4)
8.78 (bs)
-
-
-
-
-
OH
H
H
H
O
OH
H
H
O
HO
1' ' '
O
HO
HO
1' ' '
O
HO
OH
H
H
H
OH
OH
H
H
H
OH
H
H
H
H
H
H
OH
H
H
3'
O
H
H
OH
H
HO
HO
2 '
O
1' '
O
HO
HO
O
2' '
O
1' '
O
7
8
2
H
OH
2
5
7
H
H
OH
6
H
H
H
H
H
H
H
H
H
O
H
O
The important HMBC correlations for Butrin.
The important ROESY correlations for Butrin.
Fig. 1. The HMBC and ROESY correlations of Butrin.
The relative position of the sugar parts were deduced according to HMBC and ROESY spectral data (Fig. 1). In the
HMBC spectrum the cross peaks between H-1″ and C-7 and H-1′″ and C-3′ suggested the position of the sugar parts at C-7 and
C-3′ respectively. On the other hand the ROESY spectrum showed the cross peaks between H-1″ and H-6 and H-8, and H-1′″
and H-2′ which confirmed the position ofthe glucosidation at C-7 and C-3′ respectively. Table 1 shows the chemical shift values
of the 13C NMR spectrum of butrin; the correlation of 1H and 13C NMR signals was achieved by interpretation of its HMQC
spectrum.
251

%
30
C atechin
25
20
15
10
5
2
1
0
0
1
2
3
4
5
−
8
10 m ol
x
Fig. 2. Scavenging activity of butrin and butin on
DPPH radical. The DPPH reduction percentage is
proportional tothe amount ofinitial radical scavengers
added to the DPPH solution.
The 1H NMR spectrum of 1 was well resolved after addition of a few drops of D2O to the DMSO-d6 solution
(Table 1). The H-2 signal at δ 5.45 that overlapped with OH-3″ and OH-3′″ was simplified to a double doublet at 5.44 (J = 2.5,
12.5 Hz).
To confirm the stereochemistryat C-2, butrin (1) (6 mg) was hydrolyzed with β-glycosidase, which gave butin (2) and
glucose. The 1H NMR spectrum and optical rotation of the aglycone was measured in acetone-d6 and methanol respectivelyand
compared to those reported in the literature for (–)-butin [6, 7]. The presence of the glucose was confirmed by co-TLC on silica
gel using a mobile phase, acetone: 2 mM sodium acetate (17: 3), Rf 0.52 visualized by thymol reagent.
The free radical scavenging activity of compounds 1 and 2 was measured by the method of Blois [8] and compared to
the standard compound, (+)-catechin. Figure 2 compares the activity of 1, 2, and standard antioxidant, (+)-catechin. At a dose
of 4 × 10-8 mol of tested compounds the percentage of reduced DPPH for 2 and (+)-catechin were 14.5 and 27.1% respectively
while no reduction was observed for 1 (0%). This indicates that the antioxidant activity of 2 is mostly due to the free 3′ and
4′-hydroxy groups in the C ring, and glucosidation at one of them decreased the activity [9, 10].
EXPERIMENTAL
General Procedures: 13C NMR spectra (broad band and DEPT experiments) were measured on a JEOL, JNM-EX270
instrument at 67.5 MHz. 1H NMR, 1H-1H COSY, ROESY, HMQC, and HMBC experiments were performed on a 500 MHz
Bruker AMX500. Sample concentration was 10 mg per 0.6 ml DMSO-d6 and 2.8 mg per 0.6 ml acetone-d6 for compounds 1
and 2, respectively. MS spectra and optical rotations were recorded respectively on a JEOL JMS-SX102A spectrometer and a
JASCO DIP-370 digital polarimeter. The absorptions of DPPH solutions were measured at 517 nm on a Spectronic 20D+
UV/VIS spectrophotometer. (+)-Catechin and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained commercially from
Extrasynthese and Aldrich chemical companies, respectively.
Isolation of Butrin. Butea monosperma (Lam.) Kuntze was collected from Tonk district of Rajasthan-India on March
2001. The plant material was identified by Prof. P. S. Jain at the Department of Botany, University of Rajasthan, Jaipur. A
voucher specimen, with herbarium number RUBL 19871, has been deposited in the herbarium of the same university. The air-
dried flowers of the plant were extracted with hot methanol. Butrin was separated from the methanolic extract, after
concentration followed by recrystallization from methanol, as pale yellow needles, m.p. 190–191°C, [α]D21 –73° (c, 1 in py),
lit., [α]D30 –81.7° (c, 1.681 in py) [11].
Hydrolysis of Butrin. Butrin (6 mg) was dissolved in 12 ml distilled water and incubated with about 0.5 mg of
β-glucosidase from almond (Sigma Co) at 25° for 3 hr. The water layer was concentrated and extracted with n-butanol to
separate the aglycone, which was identified by1HNMRand optical rotation as (–)-butin (2.8 mg), [α]D22 –36.1° (c, 1 in MeOH),
30
lit.,[α]D –18.7° (c, 0.5 in MeOH) [7].
252

1H NMR spectral data of butin (270 MHz, in acetone-d6, δ, ppm, J/Hz): 7.71 (1H, d, J = 8.6, H-5), 7.03 (1H, d, J = 1.3,
H-2′), 6.86 (2H, m, H-5′, 6′), 6.56 (1H, dd, J = 2.3, 8.6, H-6), 6.41 (1H, d, J = 2.3, H-8), 5.39 (1H, dd, J = 3.0, 12.5, H-2), 3.00
(1H, dd, J = 12.5, 16.9, H-3α), 2.67 (1H, dd, J = 3.0, 16.9, H-3β).
Determination of the Free Radical Scavenging Activity of 1 and 2 by Spectrophotometric Method. To 5 ml of
a 100 mM solution of DPPH in methanol was added 50–400 ml of a solution of (100 mM) compounds 1 and 2 and (+)-catechin.
After 30 min shaking of the solutions under dark conditions, the absorptions of the test solutions were measured at 517 nm.
Figure 2 shows the results of correlation between the percentage of DPPH reduction after addition of the radical scavenging
compounds and the initial amount of the antioxidants (×10-8 mol). The percentage of the reduced DPPH was calculated by the
following equation:
Percentage of reduced DPPH = [(A0–A1)/A0] × 100,
where A0 is the absorbance of the control (5 ml DPPH solution + 50–400 ml methanol), and A1 is the absorbance in the
presence of sample.
ACKNOWLEDGMENT
A. R. J. is thankful to the Japan Society for the Promotion of Science for granting the postdoctoral fellowship.
The authors are thankful to Dr. Eri Fukushi and Mr. Kenji Watanabe for their skilful measuring of NMR and MS spectra.
P. K. G. is thankful to the CSIR, New Delhi for award of a senior research fellowship.
REFERENCES
1.
2.
3.
S. R. Gupta, B. Ravindranath, and T. R. Seshadri, Phytochemistry, 9, 2231 (1970).
H. Wagner, B. Geyer, M. Fiebig, Y, Kiso, and H. Hikino, Planta Med., 2, 77 (1986).
J. B. Lal and S. Dutt, J. Ind. Chem. Soc.,12, 262 (1935).
4.
5.
K. G. Shah, A. J. Baxi, V. J. Shukla, K. K. Dave, S. De, and B. Ravishanker, Ind. J. Pharm. Sci., 52, 272 (1990).
V. S. Rao and T. R. Seshadri, J. Sci. Ind. Res., 8B, 178 (1949).
6.
7.
T. G. Fourie, D. Ferreira, and D. G. Roux, Phytochemistry, 13, 2573 (1974).
D. G. Roux and E. Paulus, Biochem. J., 80, 62 (1960).
8.
M. S. Blois, Nature, 181, 1199 (1958).
9.
10.
11.
F. Nanjo, K. Goto, R. Seto, M. Suzuki, M. Sakai, and Y. Hara, Free. Radical Biol. Med., 21, 895 (1996).
M. Wang, Y. Jin, and C. Ho, J. Agric. Food Chem., 47, 3974 (1999).
J. B. Lal, J. Chem. Soc., 1562 (1937).
253