Introduction The genus Betula Betulaceae comprises more than 35 scientifically recognized species found in temperate and boreal zones of the northern hemisphere Fuchino et al., 1995; Kras
Trang 1Three new dammarane glycosides from Betula alnoides
a Faculty of Chemistry, College of Natural Science, Vietnam National University, Hanoi, 19 Le Thanh Tong Street, Hanoi, Viet Nam
b Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
1 Introduction
The genus Betula (Betulaceae) comprises more than 35
scientifically recognized species found in temperate and boreal
zones of the northern hemisphere (Fuchino et al., 1995; Krasutsky,
2006) A comprehensive review (Krasutsky, 2006) revealed the
concentration of lupanes (major) and oleananes (minor) in the
outer bark of Betula plants Dammarane triterpenoids were
reported predominantly from the leaves of Betula species such
as B ermanii, B platyphylla var japonica, B maximowicziana, B
davurica, B ovalifolia, and B schmidtii (Fuchino et al., 1995, 1996a,b,
1998a,b,c) The great number of occurrences of lupanes–oleananes
in the outer bark and dammaranes in the leaves is considered to be
significant due to their chemosystematic relevance There has been
also an increase of chemosystematic interest in the phenolic
(flavonoid, lignan, and diarylheptanoid) constituents of the leaves
(Keina¨nen et al., 1999; Keina¨nen and Julkune-Tiitto, 1998) and
inner bark (Fuchino et al., 1995, 1996a,b, 1998a,b) of Betula species
B alnoides Buch -Ham ex D Don (Betulaceae) is the only Betula
species recorded in the Flora of Vietnam (Pham, 1993) The
expected constituents lupeol, 3-O-acetoxyoleanolic acid, betulinic
acid, and betulin were found in a previous report from the bark of B
alnoides (Kamperdick et al., 1995) Our present study investigated
the distribution of triterpenoids and flavonoids in the leaves, twigs,
and stem bark of B alnoides
2 Results and discussion Twenty compounds were isolated from B alnoides, of which 13 (1–13) were from the leaves, three (2, 16, and 17) from the twigs, and seven (14–20) from the stem bark Compounds 7, 8, and 11, named betalnosides A–C, are new dammarane glycosides The structures of the known compounds, pentacosanoic acid (1),b -sitosterol (2) (Goad and Akihisha, 1997), ovalifoliolide B (3) (Fuchino et al., 1998b), rhamnocitrin (4) (Harborne, 1994), chrysoeriol (5) (Agrawal, 1989), 1-O-(tetracosanoyl)glycerol (6) (Sultana et al., 1999), b-sitosterol 3-O-b-D-glucopyranoside (9), quercetin 3-O-b-D-glucopyranoside (10) (Harborne, 1994), rutin (12) (Harborne and Mabry, 1982), quercetin (13) (Harborne, 1994), taraxeryl acetate (14) (Jin et al., 2007), taraxerone (15) (Sakurai
et al., 1987), lupeol (16) (Fuchino et al., 1995), betulin (17) (Fuchino
et al., 1995), betulinic acid (18) (Jin et al., 2007), oleanolic acid (19) (Fuchino et al., 1995), and ursolic acid (20) were determined by comparing their spectroscopic data (EIMS, HRESIMS,1H, and13C NMR) with the reported literature values or those of the authentic samples
Compound 7 was isolated as a white amorphous powder Its molecular formula was determined to be C35H60O7by positive-ion HRESIMS (m/z 615.4230 [M+Na]+) The IR spectrum showed a hydroxyl absorption band at 3381 cm1 Acid hydrolysis of 7 with
1 M HCl gave D-xylose, which was identified by HPLC analysis (Matsunami et al., 2009) In the1H NMR spectrum of 7 (Table 1) signals for eight tertiary methyl groups, of which three were linked
to oxygenated carbons [dH1.15 (3H, s), 1.17 (3H, s), and 1.19 (3H, s)], two oxygenated methine groups [dH3.15 (1H, dd, J = 12.0 Hz, 4.5 Hz) and 3.77 (1H, t, J = 7.5 Hz)], and protons of a sugar moiety
A R T I C L E I N F O
Article history:
Received 26 December 2010
Received in revised form 16 February 2011
Accepted 25 February 2011
Available online 21 March 2011
Keywords:
Betula alnoides
Betulaceae
Dammarane glycoside
A B S T R A C T Twenty compounds including three new dammarane glycosides, named betalnoside A (7), betalnoside B (8), and betalnoside C (11) were isolated from Betula alnoides Buch -Ham ex D Don (Betulaceae), of which 13 (1–13) were from the leaves, seven (14–20) from the stem bark, and three (2, 16, and 17) from the twigs Their structures were determined using spectroscopic analyses
ß2011 Phytochemical Society of Europe Published by Elsevier B.V All rights reserved
* Corresponding author Tel.: +84 4 38351439.
E-mail address: phanminhgiang@yahoo.com (M.G Phan).
Contents lists available atScienceDirect
Phytochemistry Letters
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / p h y t o l
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe Published by Elsevier B.V All rights reserved.
Trang 2(dH3.20–4.29) were observed The13C NMR spectrum of 7 (Table 1)
showed the presence of 35 signals After subtraction of five carbons
for a xylopyranosyl moiety (dC66.7, 71.3, 75.5, 78.0, and 107.4)
(Fuchino et al., 1998c) 30 signals left belonged to a tetracyclic
triterpenoid moiety containing an epoxide ring (dC84.8 and 87.8)
On the basis of the NMR data the aglycone of 7 was identified as
ocotillol (Fu et al., 2005) The xylopyranosyl moiety of 7 was
determined to be linked to C-3 of the aglycone on the basis of the
significant downfield shift of C-3 (dC90.7) on going from ocotillol
(dC79.0) The chemical shift of C-3 was also indicative of the 3aH
orientation of 7 (Li et al., 2007); glycosidation of the 3a-hydroxyl
group caused a chemical shift to ca.dC82–83 of C-3 (Fuchino et al.,
1996b) The coupling constant of the anomeric proton [dH4.29 (1H,
d, J = 7.5 Hz)] indicated thebconfiguration at C-10for the xylose
Therefore 7 was determined to be 3-O-b-D-xylopyranosyl ocotillol
(Fig 1) which was given a trivial name betalnoside A
Compound 8 was isolated as a white amorphous powder Its
molecular formula was determined to be C35H60O7by positive-ion
HRESIMS (m/z 615.4229 [M+Na]+) The IR spectrum showed a
hydroxyl absorption band at 3385 cm1 Acid hydrolysis of 8 with
1 M HCl gaveD-xylose, which was identified by HPLC analysis
(Matsunami et al., 2009) The1H and13C NMR spectra of 8 (Table 1)
indicated that the structures of 8 and 7 differed only in the side
chain at C-24 The side chain, which contained a tertiary (dC75.8) and a secondary (dC77.3) hydroxyl groups, two methylenes (dC
30.2 and 38.2), and an isopropenyl group (dC 17.8, 111.2, and 147.0), was determined as depicted inFig 1(S2) by comparing its NMR data with those of 20(S),24(S)-dihydroxydammara-25-en-3-one (Malinovskaya et al., 1980) and notopanaxoside A (Komakine
et al., 2006) The absolute stereochemistries of C-20 and C-24 of 8 could not be determined in this study Therefore 8 was determined
to be 3-O-b-D-xylopyranosyl 3b,20,24-trihydroxydammar-25-ene which was given a trivial name betalnoside B
Compound 11 was isolated as a white amorphous powder Its molecular formula was determined to be C40H68O12by positive-ion HRESIMS (m/z 763.4594 [M+Na]+) The IR spectrum showed a hydroxyl absorption band at 3392 cm1 Acid hydrolysis of 11 with
1 M HCl gaveL-arabinose, which was identified by HPLC analysis (Matsunami et al., 2009) The13C NMR spectrum of 11 (Table 1) showed the presence of 40 signals, of which 30 were assigned to an ocotillone-type aglycone (Fu et al., 2005) and ten to two arabinopyranosyl moieties (dC66.4, 69.7, 72.8, 73.5, and 100.7; and 65.1, 68.8, 71.7, 73.3, and 99.9) (Fu et al., 2001) Accordingly, the signals observed atdC82.4 and 75.6 were assigned to two glycosylated methines at C-3 and C-11, respectively, by comparing the13C NMR spectroscopic data of 11 with those of 3-epi-ocotillol
Table 1
1
H (500 MHz) and 13
C NMR (125 MHz) spectroscopic data of 7, 8, and 11.
C/H 7 (CD 3 OD) a
8 (CD 3 OD) a
11 a , b
a-Arabinose
a
Assignments were based on DEPT, HSQC, and HMBC (compound 11) spectra.
b 1
H NMR was measured in CDCl 3 and 13
C NMR in CD 3 OD + CDCl 3
Trang 3(Fuchino et al., 1996b) (A ring),
20(S),24(R)-epoxydammarane-3a,11a,25-triol (Fuchino et al., 1995) (B, C, D rings, and the side
chain), and their analogous compounds (Fuchino et al., 1995, 1996b)
Based on the coupling constants of H-3 [dH3.29 (1H, br s)] and H-11
[dH4.04 (1H, ddd, J = 10.5 Hz, 10.5 Hz, 5.5 Hz)], H-3 and H-11 were
assigned asa-oriented (Fig 1) On the basis of the NMR chemical
shifts (Fu et al., 2005; Sugimoto et al., 2009) the absolute
configurations at C-20 and C-24 of this ocotillone-type triterpenoid
were determined as S and R, respectively The linkages of the sugar
moieties were confirmed by HMBC correlations between H-10 (dH
4.26) and C-3, between H-3 and C-10(dC100.7), and between H-100(dH
4.28) and C-11 (Fig 2) The a-anomeric configurations for the
arabinoses were determined by their3J coupling constant (7.5 Hz)
between H-1 and H-2 Therefore 11 was determined to be
3,11-di-O-a-L-arabinopyranosyl 20(S),24(R)-epoxydammarane-3a,11a
,25-tri-ol which was given a trivial name betalnoside C
Full 1H NMR assignments and the revised stereostructure of
ovalifoliolide B (3) based on 2D NMR techniques (1H–1H COSY,
HMQC, HMBC, and NOESY) were also reported by us The
stereochemistry of the isopropenyl group at C-5 of 3 was revised
asb-oriented by the NOESY correlations between H-28a (dH4.69)
and H3-19 (dH1.13) and between H3-29 (dH1.73) and H-1b (dH1.79)
3 Experimental
3.1 General procedures
Optical rotations were determined using a Jasco P-1030 digital
polarimeter HRESIMS spectra were measured on a Thermo Fischer
Scientific LTQ Orbitrap XL mass spectrometer.1H,13C NMR, DEPT,
1H–1H COSY, HSQC, HMBC, and NOESY spectra were recorded on a Bruker Avance 500 NMR spectrometer Silica gel Merck 60 (Darmstadt, Germany) and Diaion HP-20 (Mitsubishi, Japan) were used for open-column chromatography (CC) and flash chromatog-raphy (FC) TLC was performed on precoated silica gel Merck 60
F254plates
3.2 Plant materials The leaves, twigs, and stem bark of B alnoides Buch -Ham ex D Don (voucher specimen: No 426) were collected in June 2007 from district Dong Van, province Ha Giang, Vietnam by Dr Tran Ngoc Ninh of the Institute of Biological Resources and Ecology, Vietnam Academy of Science and Technology, Hanoi, Vietnam
3.3 Extraction and isolation The dried powdered leaves (580 g), the stem bark (270 g), and the twigs (800 g) of B alnoides were extracted separately with MeOH at room temperature The combined MeOH extracts were concentrated and then successively partitioned between water and organic solvents of increasing polarities to give n-hexane-, CH2Cl2-, EtOAc-, and n-BuOH-soluble fractions
The leaves The n-hexane-soluble fraction (14 g) was subjected
to silica gel CC (n-hexane–acetone 15:1 to 2:1) to give 13 fractions
Fr 3 (2.06 g) was washed with n-hexane to yield 1 (18 mg) Fr 6 (1.52 g) was subjected to silica gel CC (n-hexane–EtOAc 19:1, 9:1, and 4:1) to give 2 (10.8 mg) Fr 7 (1.74 g) was separated by silica gel CC (n-hexane–acetone 9:1, 6:1, and 4:1) to give 3 (53.8 mg) The CH2Cl2-soluble fraction (27.6 g) was chromatographed by silica gel CC (n-hexane–acetone 15:1 to 2:1) to give five fractions Frs 1 (0.95 g) and 2 (2.3 g) were washed with n-hexane or acetone, respectively, to give 1 (35.2 mg), 2 (10.8 mg), and 3 (143.5 g) The EtOAc-soluble fraction (9 g) was subjected to silica gel CC (CH2Cl2– MeOH 29:1 to 3:1) to give three fractions Fr 2 (0.64 g) was separated by silica gel CC (CH2Cl2–EtOAc 9:1 to 2:1) and then purified by repeated silica gel FC (n-hexane–EtOAc, CH2Cl2–EtOAc,
or CH2Cl2–MeOH gradients) to give a mixture of 4 and 5 (5.3 mg), 6 (3.2 mg), 7 (3 mg), 8 (3.3 mg), and 9 (13.9 mg) Fr 3 (7.62 g) was chromatographed by silica gel CC (CH2Cl2–acetone 9:1 to 1:2) to give 9 (28.3 mg), 10 (45.3 mg), and 11 (5.3 mg) The 1-BuOH-soluble fraction (5.5 g) was separated by Diaion HP-20 CC (MeOH–
H2O 20%, 40%, and 60%) Compounds 12 (5 mg) and 13 (8.1 mg) were obtained from frs 3 and 4 eluted with MeOH–H2O 40% and MeOH–H2O 60%, respectively, by silica gel CC with CH2Cl2–MeOH 4:1 or 6:1
1
2
3
4
5
6 7 8
9 10
11 12 13
14 15 16
17 18 19
20
21 22
2425 26 27
28
23 O
O
3
O OH
7 R = S1
8 R = S2 O
17 16 15 14
13 12 11 19 1 2
3 4 5 6 7 8 9
30
28 29
10
1'
O HO
OH
4' 3'
5' 2' HO
O OH HO
OH 18
R
O
O
O OH
O
OH
27 26 25 24 23 22
21 20
HO
OH HO
OH
Fig 1 Chemical structures of compounds 3, 7, 8, and 11.
O OH HO
OH
O
OH
O OH HO
OH
Fig 2 HMBC (H ! C) correlations of 11.
Trang 4The stem bark The CH2Cl2-soluble fraction (9.21 g) was
subjected to silica gel CC (n-hexane–acetone 29:1 to 4:1) to give
six fractions Fr 1 (0.15 g) was separated by silica gel CC
(n-hexane–CH2Cl2 29:1 and 19:1) to give 14 (16.2 mg) and 15
(22 mg) Fr 2 (0.8 g) was washed with acetone to give 16 (3 mg) Fr
5 (1.6 g) was chromatographed on silica gel (CH2Cl2–acetone 29:1
and 19:1) to give 17 (45.1 mg), 18 (20.9 mg), and a mixture of 19
and 20 (9.2 mg)
The twigs The n-hexane-soluble fraction (2.4 g) was subjected
to silica gel CC (n-hexane–acetone 19:1 to 2:1) to give seven
fractions Fr 2 (0.77 g) was chromatographed by silica gel CC
(n-hexane–acetone 90:1) to give 16 (4 mg) Fr 3 (0.36 g) was washed
with n-hexane to give 2 (40 mg) The CH2Cl2-soluble fraction
(7.87 g) was chromatographed twice by silica gel CC (CH2Cl2–
EtOAc 19:1 to 4:1) to give 17 (3.5 mg)
3.3.1 Ovalifoliolide B (3)
Colorless needles; ½a24D +92.3 (c 0.2, CHCl3); mp 196–198 8C; IR
(film):nmaxcm13483, 1702, 1633, 1445, 1371, 1292, 1165, 1082,
1028; HRESIMS: m/z 495.34335 [M+Na]+, calc for C30H48O4Na:
495.34448;1H NMR (CDCl3):d0.93 (3H, s, H3-18), 0.95 (3H, s, H3
-30), 1.12 (3H, s, H3-26), 1.13 (6H, s, H3-19, H3-21), 1.13 (1H, m,
H-15a), 1.20 (3H, s, H3-27), 1.22 (1H, m, H-7a), 1.39 (1H, t, J = 13.5 Hz,
H-1a), 1.47 (1H, m, H-6a), 1.49 (1H, m, H-15b), 1.58 (1H, m, H-16a),
1.59 (1H, m, H-7b), 1.65 (1H, m, H-22a), 1.70 (1H, m, H-22b), 1.73
(2H, m, H-12a, H-13), 1.73 (3H, s, H3-29), 1.77 (2H, m, 16b,
H-23a), 1.79 (1H, m, H-1b), 1.81 (1H, m, H-5), 1.82 (1H, m, H-9), 1.84
(1H, m, H-17), 1.86 (1H, m, H-23b), 1.91 (1H, m, H-6b), 2.38 (1H, t,
J = 15.5 Hz, H-12b), 2.40 (1H, dd, J = 15.5 Hz, 8.0 Hz, H-2a), 2.60
(1H, dd, J = 15.5 Hz, 12.5 Hz, H-2b), 3,72 (1H, t, J = 7,0 Hz, H-24),
4.51 (1H, q, J = 8.5 Hz, 11), 4.69 (1H, s, 28a), 4.86 (1H, s,
H-28b)
3.3.2 Betalnoside A (7)
White amorphous powder; ½a24D +0.83 (c 0.06, CH3OH); IR
(film):nmaxcm13381, 1456, 1375, 1164, 1075, 1042; HRESIMS:
m/z 615.4230 [M+Na]+, calc for C35H60O7Na 615.4231;1H and13C
NMR: seeTable 1
3.3.3 Betalnoside B (8)
White amorphous powder; ½a24D +108 (c 0.03, CH3OH); IR (film):
nmaxcm13385, 1455, 1374, 1164, 1070, 1042; HRESIMS: m/z
615.4229 [M+Na]+, calc for C35H60O7Na 615.4231; 1H and 13C
NMR: seeTable 1
3.3.4 Betalnoside C (11)
White amorphous powder; ½a24D 4.58 (c 0.31, CH3OH); IR
(film):nmaxcm13392, 1456, 1385, 1160, 1071, 1041; HRESIMS:
m/z 763.4594 [M+Na]+, calc for C40H68O12Na 763.4603;1H and13C
NMR: seeTable 1
3.4 Sugar analysis
Compound 7 (about 500mg) was heated in 1 M HCl (1.0 ml) at
90 8C for 2 h After cooling, the reaction mixture was extracted with
EtOAc and the aqueous layer was subjected to HPLC analysis
[column: Shodex Asahipak NH 2P-50 4E,F= 4.6 mm, L = 25 cm,
mobile phase: MeCN–H2O (4:1, v/v), detection: optical rotation
detector (JASCO 2090Plus), and flow rate: 1.0 ml/min] to detectD
-xylose, which was identified by comparison of its retention time
with that of authentic sample, D-xylose (tR: 8.6 min, positive
optical rotation) HPLC analyses under the same conditions as
above revealed the presence ofD-xylose for 8 andL-arabinose for
11 The sugars were identified by comparison of their retention times with those of authentic samples,L-arabinose (tR: 8.4 min, positive optical rotation)
Acknowledgement This work was supported by the National Foundation for Science and Technology Development (NAFOSTED, Hanoi, Viet-nam)
References
Agrawal, P.K (Ed.), 1989 Carbon-13 NMR of Flavonoids Elservier, Amsterdam Fuchino, H., Satoh, T., Tanaka, N., 1995 Chemical evaluation of Betula species in Japan I Constituents of Betula ermanii Chem Pharm Bull 43, 1937–1942 Fuchino, H., Konishi, S., Satoh, T., Yagi, A., Saitsu, K., Tatsumi, T., Tanaka, N., 1996a Chemical evaluation of Betula species in Japan II Constituents of Betula platy-phylla var japonica Chem Pharm Bull 44, 1033–1038.
Fuchino, H., Satoh, T., Tanaka, N., 1996b Chemical evaluation of Betula species in Japan III Constituents of Betula maximowicziana Chem Pharm Bull 44, 1748– 1753.
Fuchino, H., Satoh, T., Shimizu, M., Tanaka, N., 1998a Chemical evaluation of Betula species in Japan V Constituents of Betula davurica Chem Pharm Bull 46, 166– 168.
Fuchino, H., Satoh, T., Shimizu, M., Yokochi, M., Tanaka, N., 1998b Chemical evaluation of Betula species in Japan V Constituents of Betula ovalifolia Chem Pharm Bull 46, 169–170.
Fuchino, H., Satoh, T., Hida, J., Terada, M., Tanaka, N., 1998c Chemical evaluation of Betula species in Japan VI Constituents of Betula schmidtii Chem Pharm Bull.
46, 1051–1053.
Fu, H., Koike, K., Zheng, Q., Mitsunaga, K., Jia, Z., Nikaido, T., Lin, W., Guo, D., Zhang, L.,
2001 Fargoside A–F, triterpenoid saponins from Holboellia fargesii Chem Pharm Bull 49, 999–1002.
Fu, L., Zhang, S., Li, N., Wang, J., Zhao, M., Sakai, J., Hasegawa, T., Mitsui, T., Kataoka, T., Oka, S., Kiuchi, M., Hirose, K., Ando, M., 2005 Three new triterpenes from Nerium oleander and biological activity of the isolated compounds J Nat Prod.
68, 198–206.
Goad, L.J., Akihisha, T., 1997 Analysis of Sterols Chapmann & Hall, London Harborne, J.B (Ed.), 1994 The Flavonoids Advances in Research since 1986 Chapman & Hall, London.
Harborne, J.B., Mabry, T.J (Eds.), 1982 The Flavonoids Advances in Research Chapman & Hall, London.
Jin, W.J., Cai, X.F., Na, M.K., Lee, J.J., Bae, K.H., 2007 Triterpenoids and diarylhepta-noids from Alnus hirsuta inhibit HIF-1 in AGS cells Arch Pharm Res 30, 412– 418.
Kamperdick, C., Thuy, T.T., Sung, T.V., Adam, G., 1995 Triterpenoids from Betula alnoides Planta Med 61, 486.
Keina¨nen, M., Julkune-Tiitto, R., 1998 High performance liquid chromatographic determination of flavonoids in Betula pendula and Betula pubescens leaves J Chromatogr 793, 370–377.
Keina¨nen, M., Julkunen-Tiitto, R., Rousi, M., Tahvanainen, J., 1999 Taxonomic implications of phenolic variation in leaves of birch (Betula L.) species Biochem Syst Ecol 27, 243–254.
Komakine, N., Okasaka, M., Takaishi, Y., Kawazoe, K., Murakami, K., Yamada, Y.,
2006 New dammarane-type saponin from roots of Panax notoginseng J Nat Med 60, 135–137.
Krasutsky, P.A., 2006 Birch bark research and development Nat Prod Rep 23, 919– 942.
Li, Q., Yao, Z.H., Shi, Y.H., Liu, X., Yao, X.S., Ye, W.C., 2007 Determination of the three-dimensional structure of gynoside A in solution using NMR and molecular modelling Molecules 3, 907–916.
Malinovskaya, G.V., Novikov, V.L., Denisenko, V.A., Uvarova, N.I., 1980 A new triterpene from the leaves of Betula mandschurica Chem Nat Compd 16, 257–261.
Matsunami, K., Otsuka, H., Kondo, K., Shinzato, T., Kawahata, M., Yamaguchi, K., Takeda, Y., 2009 Absolute configuration of (+)-pinorenol 4-O-[6 00 -O-galloyl]-b
-D -glucopyranoside, macarangiosides E, and F isolated from the leaves of Macar-anga tanarius Phytochemistry 70, 1277–1285.
Pham, H.H., 1993 Illustrated Flora of Vietnam Published by the author, Montreal Sakurai, N., Yaguchi, Y., Inoue, T., 1987 Triterpenoids from Myrica rubra Phyto-chemistry 26, 217–219.
Sugimoto, S., Nakamura, S., Matsuda, H., Kitagawa, N., Yoshikawa, M., 2009 Chemi-cal constituents from seeds of Panax ginseng: structure of new dammarane-type triterpene ketone, panaxadione, and HPLC comparisons of seeds and flesh Chem Pharm Bull 57, 283–287.
Sultana, N., Armstrong, J.A., Waterman, P.G., 1999 Benzopyran derivatives from the aerial parts of Eriostemon rhomboideus Phytochemistry 52, 895–900.