Post on 20-Dec-2015
A Photo of Viburnum speciesA Photo of Viburnum species
Abstract
Bioassay-guided fractionation of the methanolic extract of Viburnum awabuki afforded two new lupane triterpene derivatives; 6b -hydroxyl-3,20-dioxo-30 norlupane-28-oic acid (1) and 3,4-secolup-4,20-dihydroxy-3,28-dioic acid-3-oic acid methyl ester (2), along with seven known lupane and oleanane-type triterpenes (3–9). The structure of the isolated compoundswas assigned using different spectroscopic techniques including 1D and 2D NMR. The 13CNMR data of compounds 5 and 9 is reported for the first time. Triterpenes 1, 2, and 7–9 showed in vitro cytotoxic activity against several tumor cell line
ResultsDifferent chromatographic techniques utilizing normal
and reversed phase silica gel have been used for fractionation of the methanol extract of the leaves and twigs of V. awabuki. The result of this separation yielded two new triterpenes derivatives, norlupane (1) and secolupane (2). In addition, seven known lupan- and oleanane-type triterpenes were also identified. The known oleanane-type triterpenes were idenitified as oleanolic acid (3-hydroxyolean-12-en-28-oic acid) (3) and sumareesinolic acid (3,6-dihydroxyolean-12-en-28-oic acid) (4), while the known lupane type were identified as 3-hydroxylup-12-en-28-oic acid (5), 3,20-dihydroxy lupane-28-oic acid (6), 6-hydroxy-3-oxolup 20(29)-en-28 oic acid (7), 6,30-dihydroxy-3-oxolup 20(29)-en-28-oic acid (8), and 20 hydroxy-3-oxolup-28-oic acid (9). The 13CNMR data of compounds 5 and 9 is reported for the first time. Triterpenes 1, 2, and 7–9 showed in vitro cytotoxic activity against several tumor cell line
Air dried leaves and twigs of Vibrnum awabuki were extracted with MeOH (10Lx3) to give 127g of a solid
CC, Sil. gel using CHCl3. and CHCl3-MeOH gradient to give fifteen fractions
CHCl3/MeOH
100%
5 3 6 7, 84
2-HPLC (Rp-18), MeOH-H2O (3:2)
1, 2
CC, Sil. gel using n-hex./acetone (6.7:3.3)
Fraction F1 Fraction F2 Fraction F4
CC, Sil. gel using n-hex./acetone (4:1)
100%
CC, Sil. gel using n-hex./acetone (7.5:2.5))
Fraction F3
CC, Sil. gel using n-hex./acetone (7:3))
Fraction F5
3%MeOH
-CC, Sil. gel using n-hex./EtOAc (3:1)-CC, Sil. gel using CHCl3./MeOH (99:1)
4%MeOH
Fraction F6
1-CC, Sil. gel using n-hex./acetone (3:2)
100% 100%
Extraction, Separation and Purification Scheme of Extraction, Separation and Purification Scheme of viburnum awabuki viburnum awabuki leaves and twigsleaves and twigs
O
COOH
H
O Compound 1
OH
COOH
HO
H
OH
MeOOC
H
Compound 2
R
HO
COOH
Compound 3 R = H Compound 4 R = OH
COOH
HOCompound 5
COOH
HO
H
R
Compound 6 R = OH , HCompound 9 R= O
CH2R
COOH
H
O
OH
Compound 7 R = HCompound 8 R= OH
12
34 5 6 7
89
10
11
12
13
14
1516
17
18
19
20
21
22
2324
25 26
27
28
29
Triterpenes isolated from Triterpenes isolated from vibrnum awabukivibrnum awabuki
11HNMR of Compound 1HNMR of Compound 1
O
COOH
H
O
OH
12
34 5 6 7
89
10
11
12
13
14
1516
17
18
19
20
21
22
2324
25 26
27
28
29
661919 22 22’’
2929
77
24242626
25252323
2727
DEPT Experiment of Compound 1DEPT Experiment of Compound 1
2727
25252626
1111
24242323
2929
1212212115151616222222
1313
7711
1919999
99
HSQCHSQC of Compound 1of Compound 1
O
COOH
H
O
OH
12
34 5 6 7
89
10
11
12
13
14
1516
17
18
19
20
21
22
2324
25 26
27
28
29
66 1919 222929
HMBC of Compound 1HMBC of Compound 1
2828
202033
6655
66 19192929
O
COOH
H
O
OH
12
34 5 6 7
89
10
11
12
13
14
1516
17
18
19
20
21
22
2324
25 26
27
28
29
2323 27272424
COOH
HO
H
OH
MeOOC
H
1
2
34
5
67
89
11
10
12
13
14
1516
17
18
1920 21
22
2324
25 26
27
28
29
30
11HNMR of Compound 2HNMR of Compound 2
OMeOMe
OHOH
2626
25252727
29292323
3030
2424
HSQC of Compound 2HSQC of Compound 2
COOH
HO
H
OH
MeOOC
H
1
2
34
5
67
89
11
10
12
13
14
1516
17
18
1920 21
22
2324
25 26
27
28
29
30
OMeOMe
1818191955
OMeOMe1414 99881010131377 2525
262627272929232330302424 661111
26262525 2727
2929 232330302424
221313
1919
1818
HMBC of Compound 2HMBC of Compound 2
COOH
HO
H
OH
MeOOC
H
1
2
34
5
67
89
11
10
12
13
14
1516
17
18
1920 21
22
2324
25 26
27
28
29
30
HMBC of Compound 2HMBC of Compound 2
2828 33 44
29293030
2323
2424
2626
25,2725,27
2020
OMeOMe
22
18182222
1717
COOH
HO
H
OH
MeOOC
H
1
2
34
5
67
89
11
10
12
13
14
1516
17
18
1920 21
22
2324
25 26
27
28
29
30
Expanded HMBC of Compound 2Expanded HMBC of Compound 2
44 17172020 55 1919
2626
2525 27272929
303024242323
1818
14149988
10101313
NOESY of Compound 2NOESY of Compound 2
COOH
HO
H
OH
MeOOC
H
1
2
34
5
67
89
11
10
12
13
14
1516
17
18
1920 21
22
2324
25 26
27
28
29
30
HO
COOH
11HNMR of Compound 3HNMR of Compound 3
121233 1818
HO
COOH
1313C NMR of Compound 3C NMR of Compound 3
2828 12121313 33 55
Expanded Expanded 1313C NMR of Compound 3C NMR of Compound 3
252524242626
661616 1111
3030
2727
22 1515
2424
xx
2020
22222121
2929 771010
11
4488
181814141919
17179955
HSQC of Compound 3HSQC of Compound 3
HO
COOH
33 18181212
HO
COOH
OH
11HNMR of Compound 4HNMR of Compound 4
1212 OHOH 6633
1818
xx
1313C NMR and DEPT Experiment of Compound 4C NMR and DEPT Experiment of Compound 4
HO
COOH
OH2828 1313 33 66 55
HSQC of Compound 4HSQC of Compound 4
661212 33 1818
HO
COOH
OH
COOH
HO
11HNMR of Compound 5 in (CDClHNMR of Compound 5 in (CDCl33+CD+CD33OD)OD)
331212
xx
COOH
HO
1313C NMR of Compound 5C NMR of Compound 5
2828 13131212 33 55 1818
99
DEPT Experiment of Compound 5DEPT Experiment of Compound 5
1212 33 551818 9919192020
23233030
2626
24242525
2929
2727
6616161111
112222 151522
11HNMR of Compound 6HNMR of Compound 6
COOH
HO
H
HO
COOH
HO
H
HO
11HNMR of Compound 6HNMR of Compound 6
1313C NMR and DEPT Experiment of Compound 6C NMR and DEPT Experiment of Compound 6
2828 332020 1717
55
99 1919 1818 1313
2727242425252626
3030 2323 2929
COOH
HO
H
HO
DEPT Experiment of Compound 6DEPT Experiment of Compound 6
2727
24242525
2626
661111
2929
23233030
22121221211616151577222211
131355
991919
1818
COOH
HO
H
HO
COOH
H
O
OH
11HNMR of Compound 7HNMR of Compound 7
2929 292966
3030
24242525
27272323
2626
COOH
H
O
OH
H HCOSY of Compound 7H HCOSY of Compound 7
2929 66 1919 22
COOH
H
O
OH
DEPT Experiment of Compound 7DEPT Experiment of Compound 7
2929
6655 99
18181919
77 11
1313
2222221616
21211515
1212
2323 2424
1111
3030
25252626
2727
COOH
H
O
OH
HSQC of Compound 7HSQC of Compound 7
2929
66
2020
2828
33
2929 66
11HNMR of Compound 8HNMR of Compound 8
CH2OH
COOH
H
O
OH
2929 663030
262623232727
24242525
CH2OH
COOH
H
O
OH
HSQC of Compound 8HSQC of Compound 8
COOH
HO
H
O
11HNMR of Compound 9HNMR of Compound 9
1313C NMR and DEPT Experiments of Compound 9C NMR and DEPT Experiments of Compound 9
COOH
HO
H
O
Expanded Expanded 1313C NMR of Compound 9C NMR of Compound 9
1717
55
991919
1818
44
141488
11
1313
10102222 77
15153030
1212
161622
23232929
11112424 66
26262525 2727
DEPT Experiments of Compound 9DEPT Experiments of Compound 9
COOH
HO
H
O
COOH
HO
H
O
HSQC of Compound 9HSQC of Compound 9
COOH
HO
H
O
HMBC of Compound 9HMBC of Compound 9