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