Title ナラ枯れ原因菌 Raffaelea quercivora 侵入に応 …...Title ナラ枯れ原因菌...

Title ナラ枯れ原因菌 Raffaelea quercivora 侵入に応答するミズナラの抽出成分に関する研究( 本文(Fulltext) )

Author(s) 今井, 香代子

Report No.(DoctoralDegree) 博士(農学) 甲第622号

Issue Date 2014-03-13

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/49102

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

ナラ枯れ原因菌 Raffaelea quercivora 侵入に応答するミズナラの抽出成分に関する研究

2013年

岐阜大学大学院連合農学研究科

生物資源科学

（岐阜大学）

今井香代子

ナラ枯れ原因菌 Raffaelea quercivora 侵入に応答するミズナラの抽出成分に関する研究

今井香代子

1

700 2000

2

1

2,3,4

5,6,7

- 6,8

1

3

Platypus quercivorus MURAYAMA

Raffaelea quercivora

9,10,11

12,13,14,15 16,17,18,19,20,21

R. quercivora

R. quercivora

R. quercivora

4

1. 1997 46 1

2. 2002 196-201

3. 1996 4-53

4. 2002

307-310

5. Huber, D. P. W. and Borden, J. H. (2001) Angiosperm bark volatiles disrupt response

of Douglas-fir beetle, Dendroctonus pseudotsugae, to attractant-baited traps. J. Chem.

Eco. 27 : 217-233

6. Sadik, G., Islam, R., Rahman, M. M., Khondkar, P., Rashid, M. A. and Sarker, S.D.

(2003) Antimicrobial and cytotoxic constituents of Loranthus globosus. Fitoterapia,

74 : 308–311

7. Yamasaki, T., Saito, M. and Sakoguchi, H. 1997 (-)germacrene D:Masking

Substances of Attractants for the Cerambycid Beetle, Monochamus alternatus (HOPE).

Appl. Entmol. Zool. 32: 423-429

8. Vattem, D.A., Lin, Y.-T., Labbe, R.G. and Shetty, K. (2003) Antimicrobial activity

against select food-borne pathogens by phenolic antioxidants enriched in cranberry

pomace by solid-state bioprocessing using the food grade fungus Rhizopus

oligosporus. Proc. Biochem. in press.

9. Kuroda, K. (2001) Responses of Quercus sapwood to infection with the pathogenic

5

fungus of a new wilt disease vectored by the ambrosia beetle Platypus quercivorus. J.

Wood. Sci. 47: 425-42

10. 2002 11

8-9

11. 1996

78 84-88

12. Igeta, Y., Esaki, K., Kato, K. and Kamata, N. 2003 Influence of light condition on the

stand-level distribution and movement of the ambrosia beetle Platypus quercivorus

(Coleoptera: Platypodidae) Appl. Entomol. Zool. 38:167-175

13. (2002) 35: 26-34

14. Kobayashi, M., Ueda, A. and Takahata, Y. (2001) Inducing Infect ion of Oak logs by a

Pathogenic Fungus Carries by Platypus quercvorus (MURAYAMA) (Coleoptera :

Platypododae). J. For. Res. 6 : 153-136

15. Ueda, A. and Kobayashi, M. (2001) Aggregation of Platypus quercivorus (Murayama)

(Coleoptera : Platyposisae) on Oak Log Bored by Males of the Species. J. For. Res.

6 : 173-179

16. 2000 60-68

17. 2000

10 16-22

18. 1998

6

80 170-175

19. 2003 Raffaelea quercivora

51 199-200

20. Kubono, T. and Ito, S. (2002) Raffaerea quercivora sp. nov. associated with mass

mortality of Japanese oak, and the ambrosia beetle (Platypus quercivorus).

Mycoscience 43 255-260

21. 1999

40 91-96

7

1)

600

Oak wilt 1930

Ceratocystis fagacearm

Pytophthora P. cinnamomi

2, 3)

8

50 Armillaria

spp.

1980

1

4

1 Platypus quercivorus Murayama

5

2

6,7,8

Raffaelea

quercivora Kubono et Shin-Ito sp.nov.9

10 R. quercivora

10

1934

1950 1952 1952

1956 1958 1960 1979

1900

1 1980

1 10

1999

1 15

1 4

17 27 45

Quercus

Castanopsis Pasania Persea Fagaceae

Castanea Fagus

2

11

13,14)

12

0.4 25mm 2 5mm

215 7000

300

Bark beetle

Ambrosia beetle

Mycangia ambrosia fungi

13

1

Frass

3

42

5mm 4

R. quercivora

Swietenia macrophylla

Crossotarsus externe-dentatus

Platypus gerstaeckeri Triplochiton

Sclerozylon Trachyostus ghanaensis 14

14

18

R. quercivora

1941

15

41

R. quercivora

R. quercivora

13,41

16

53,54

Castanea crenata

17

51)

3

2

18

600

Castanopsis Castanea Fagus

Quercus Pasania

F. sylvatica Z

-3-hexenol Sabinen Q. laurifolia Q. rubra

52

Castalagin Vescalagin Castanea sativa

Castanea crenata Q. petraea Q. robur

Q. suber valoneic acid dilactone Castalagin

Vescalagin Castacrenin Castanopsin grandinin roburin pedunculagin

catechin taxifolin hamamelitannin

6,27,52) 3

20

1. (2002)

35: 4-9

2. 2000

10 16-22

3. 1998

80 170-175

4. 1998

47 222-229

5. 2000

10 16-22



Wood. Sci. 47: 425-42

7. 2002 11 8-9

8. 1996

78 84-88




21

10. 1999

40 91-96

22

1

1-1

1

Platypus quercivorus MURAYAMA

Raffaelea quercivora

1,2,3

4,5,6 7,8,9,10,11,12,13

23

1-2

1-2-1

1-2-1-1

40 2007 11

(Operating

Instructions Ultra Centrifugal Mill Type ZM200, Retsch)

580.6g 70% 35.9L

(NS-e) NS-e HPLC

1-2-1-2

40 2007

11 -30

37

1/2 1/2

(Operating Instructions Ultra Centrifugal Mill Type

ZM200, Retsch) 725.6g 70% 11.0L

(DS-e) HPLC

24

1-2-2 HPLC

NS-e DS-e

HPLC

Pump : SHIMADZU LC-10AD (0.05 % TFA in water)

SHIMADZU LC-10AT (Methanol)

Detector : SHIMADZU SPD-M10Avp

Column ocen : SHIMADZU CTO-10A

Communication bus module : SHIMADZU CBM-10A

Degasser : SHIMADZU DGU-12A

Column Deverosil ODS HG-5Φ4.6x250mm

Flow rate 1ml/min

Column temp 40

Detector wave length 200-600nm SHIMAZU SPD-M10A VP

Elution MeOH / 0.01%TFA aq. = 5 / 95 100 / 0 45min

25

1-2-3

NS-e DS-e 14,15)

26

1-2-3-1 Folin- Ciocalteu

16)

40 0.1mg / ml 1ml

24ml 14ml 1

1ml 3

2ml

20ml 10 1

750nm Jasco V-520

+ -

+ -

P = 100 Y / S

P ( % )

Y (mg / ml)

27

S (mg / ml)

28

1-2-3-2

8 6

17)

18) +

- 500nm

40 0.2 0.3mg / ml 1ml

25ml 4 -

6ml 3ml

5 15 500nm

Jasco V-520 + -

+ -

4 -

29

= 100 /

P ( % )

Y (mg / ml)

S (mg / ml)

30

1-2-3-3

19,20)

0.1 *1 1mg / ml BSA 200 l 40

5mg / ml 200 l 1

200 l

100mg 120 10

2ml

pH 5.1 0.1 *2 3ml 108 5

570nm Jasco V-520

BSA

BSA

*1

36g 18.5ml

2ml 200ml

31

*2

0.5 /

32

1-2-3-4

21

5mg 3N 5ml

110 10 N

2.5ml 8ml

100 l

100 l

HPLC

HPLC

Column Cosmosil 5CMS WATERS 4.6mm × 250mm

Flow rate 1ml / min

Column temp 35

Detector wave length SHIMADZU SPD-M10A VP 200 600nm

Solvent Initial MeOH / 0.01%TFA 5 / 95

Final MeOH / 0.01%TFA 100 / 0

33

Gradient time 45min

34

1-3

1-3-1

Table1-1

NS-e (DS-e)

NS-e DS-e Folin-Denis Vanillin-HCl Ninhydrin

NS-e DS-e 45%

1% F / P

0.02

F / P

0.6 0.8 22,23

NS-e 47.3 DS-e 85.6

NS-e 59.3

35

NS-e 59.3 DS-e 71.6 DS-e

BSA

36

Table 1-1 Chemical analysis of the extracts of Quercus crispula

Note :These values were expressed with the percentage based on dry NS-e and DS-ea: NS-e stand for the 70 % aqueous extracts from normal part of Q. crispula sapwood b: DS-e stand for the 70 % aqueous extracts from infected-colored part of Q. crispula sapwood c: Protein precipitation was calculated by the method described at experimental section

37

1-3-2 HPLC

NS-e DS-e HPLC Fig. 1-1 HPLC

NS-e 30

DS-e

31 31

UV

max 252, 365

UV UV

24 UV

38

Fig. 1-1 HPLC chromatograms of extracts from Q. crispula sapwood at 280 nm.NS-e : Extracts from normal sapwood of Q. crispulaDS-e : Extracts from infected-colored sapwood of Q. crispula

39

1-3-3

NS-e HPLC Fig. 1-2

DS-e

40

Fig. 1-2 HPLC chromatograms of hydrolysate of NS-ehydrolyzed with 3N H2SO4 at 110 ºC for 10 hr. at 280 nm

41

1-4

Quercus

2008 Journal of wood science 25)

42

1-5



Wood. Sci. 47: 425-42

2. 2002 11

8-9

3. 1996

78 84-88

4. Igeta, Y., Esaki, K., Kato, K. and Kamata, N. 2003 Influence of light condition on the

stand-level distribution and movement of the ambrosia beetle Platypus quercivorus

(Coleoptera: Platypodidae) Appl. Entomol. Zool. 38:167-175

5. (2002) 35: 26-34

6. Kobayashi, M., Ueda, A. and Takahata, Y. (2001) Inducing Infection of Oak logs by a

Pathogenic Fungus Carries by Platypus quercvorus (MURAYAMA) (Coleoptera :

Platypododae). J. For. Res. 6 : 153-136

7. Ueda, A. and Kobayashi, M. (2001) Aggregation of Platypus quercivorus (Murayama)

(Coleoptera : Platyposisae) on Oak Log Bored by Males of the Species. J. For. Res.

6 : 173-179

8. 2000 60-68

9. 2000

43

10 16-22

10. 1998

80 170-175


51 199-200




13. 1999

40 91-96

14. Takagi K, Shimomura K, Koizumi Y, Mitsunaga T, Abe I (1999) Tyrosinase Inhibitors

from the Pericarp of Jatoba (Hymenaea courbaril L.) (in Japanese with English

summary). Natural Medicines. 53(1): 15-21

15. Takagi K, Mitsunaga T (2002) A Tyrosinase Inhibitors from the Asam ( Mangifera

quadrifida). Natural Medicines. 56(3): 97-103

16. Swain T., and Hillis W.E. 1959. The Phenolic Constitutes of Prunus domestica. II. The

analysis of tissues of the Victoria plum tree. J. Sci. Fd. Agri. 10: 63-68

17. Hagernan A.E. 1989. Chemistry of tannin-protein complexation, pp 323-333, in R.W.

Hemingway and J.J. Karchesy (eds.). Chemistry and Significance of Condensed

Tannins.Plenum Press, New York.

44

18. Mitsunaga T., Doi T., Kondo Y. and Abe I. 1998. Color development of

proanthocyanidins in vanillin-hydrochloric acid reaction. J.Wood. Sci. 44:125-130

19. Harbone, J. B. (1988) The Flavonoids. Chapman and Hall , London, 21-61

20. Takagi, K. and Mitsunaga, T. 2002 A tyrosinase inhibiter from the wood of Asam

(Mangifera quadrifida J.) Nat. Medic. 56 97-103

21. 1997 46 1

22. 1981

27 6 :491-497

23. 1999 Jatoba Hymenaea

courbaril L. Nat. Medic. 53 15-21

24. 2002 196-201

25. K. Imai , T. Mitsunaga , H. Takemoto , T. Yamada , S. Ito , H. Ohashi (2008)

Extractives of Quercus crispula sapwood infected by the pathogenic fungi Raffaelea

quercivora (I) : Comparison of sapwood extractives non-infected and infected. J.Wood.

Sci. 55:126-132

45

2

2-1

Raffaerea quercivora

1)

HPLC

46

2-2

2-2-1

R. quercivora 2

R. quercivora Wa A

2-2-2

2-2-2-1

121 15min PDA

PGA

28 10

50 ml 10 ml

R. quercivora No. 4

2 2 ml

200 mg / ml

0.1 ml / ml 28

3 HPLC

47

2-2-2-2 HPLC

2-2-2-1 2

200 mg / ml 0.1 ml / ml

3

200 l

12,000 rpm 10 min HPLC

200 l 12,000 rpm

10 min. HPLC

48

2-3

2-3-1

3

HPLC Fig. 3-1

10 25

UV

49

Fig. 3-1 HPLC chromatograms of tannic acid added in culture medium of R. quercivora after fungus body was removed (a) and control (b)

50

Fig. 3-2 HPLC chromatograms of tannic acid added in culture medium of R. quercivora after fungus body was removed (a) and control (b) (zoom up)

51

2-3-2

HPLC Fig. 3-3

52

Fig. 3-3 HPLC chromatograms of tannic acid added in culture medium of R. quercivora with fungus body (a), water soluble part of fragmentized fungus body after cultured with tannic acid (b) and control (c) (zoom up)

53

2-4


54

2-5

1. K. -T. Chung, Z. Lu and M. W. Chou. 1998. Mechanism of inhibition of tannic acidnext

term and related compounds on the growth of intestinal bacteria . Food and Chemical

Toxicology. 36: 1053-1060


51 199-200

3. . 1985.

. 39:65-74

4. K. Imai , T. Mitsunaga , H. Takemoto , T. Yamada , S. Ito , H. Ohashi (2008) Extractives of

Quercus crispula sapwood infected by the pathogenic fungi Raffaelea quercivora (I) :

Comparison of sapwood extractives non-infected and infected. J.Wood. Sci. 55:126-132

55

3

3-1

PGG

56

3-2

3-2-1 PGG

HPLC

Pump : SHIMADZU LC-10AD (0.05 % TFA in water), SHIMADZU LC-10AT (Methanol)





Column ; Deverosil ODS HG-5 4.6 250 mm

Flow rate ; 2 ml / min.

Column temp. ; 35

Detector wave length ; 280 nm

Solvent; 0.1 % TFA in acetonitrile % TFA in H2O

Initial 0 / 100 , Final 30 / 70

Gradient time ; 120 min.

HPLC NMR PGG

57

3-2-2 PGG

121 15min PDA

PGA

28 10

50 ml 1) 10 ml

R. quercivora No. 4

200 mg / ml 50 % PGG

20 μl PGG 4 mg / 10 ml 28

1,2,4,6,9,11 2 30 μl

-30 20μl HPLC

HPLC PGG

PGG

PGG

58

3-3

3-3-1 PGG

PGG NMR (Fig. 3-1)

NMR 6.8~7.2ppm 5

5 4.4~6.3ppm 5

2)

PGG NMR PGG

59

Fi

g.

3-

1

1H

N

M

R

chart of PGG isolated from tannic acid

60

3-3-2 PGG

R. quercivora PGG

1,2,4,6,9,11 HPLC

PGG

Fig. 3-2 PGG 11 2

PGG 1 5

PGG R. quercivora

purprogallincarboxylic acid (PGCA)

3) R. quercivora 4) R. quercivora

PGCA

4)

PGG

PGCA

PGCA

PGCA λmax 224 304 398nm3, 4)

61

280nm

62

Fig. 3-2 Change of PGG and gallic acid amount added in the culture medium of R.

quercivora.

μ

μ

μ

μ

63

3-4

PGG

PGCA


64

3-5

1. . 1985.

. 39:65-74

2. Richard T, Vitrac X, Merillon JM, Monti JP (2005) Role of peptide primary sequence in

polyphenol-protein recognition : An example with neurotensin. Biochimica et Biophysica

Acta. 1726:238-243

3. Karl H (1954) Über die Bildung der Purpurogallincarbonsäure durch fermentative

Oxydation der Gallussäure. (zugleich II Mitteilung über gerbstoffartige Substanzen). Archiv

der Pharmazie 287:497-503

4.



quercivora (I) : Comparison of sapwood extractives non-infected and infected. J.Wood. Sci.

55:126-132

65

4

4-1

Raffaerea quercivora

HPLC

66

4-2

4-2-1 HPLC

HPLC

Pump : SHIMADZU LC-10AD (0.05 % TFA in water)

SHIMADZU LC-10AT (Methanol)





Column Deverosil ODS HG-5Φ4.6x250mm

Flow rate 1ml/min

Column temp 40

Detector wave length 200-600nm SHIMAZU SPD-M10A VP


67

4-2-2

HPLC gallic acid

0.05 mol / l

pH 5.5 0.05 mol / l pH 5.5 0.05 mol/l

2 0.05 mol / l NaOH pH 5.5

0.32 % 3 mg / 100 ml

0.05 mol / l pH 5.5 500 l

10 30 100 l

100 l

30 100 l

100

l 50 l 50

l 20 l HPLC

4 ml

1 ml 5 50 200 l

200 l 100 l

50 l 20 l HPLC

69

4-2-3

2006 11

NS-e 3L

3 3L 8 n- 3L 3

HPLC

70

4-2-4 NS-EtOAc-S HPLC

70 % buffer pH 5.5

0.32 % 2 ml

0.3 mg / 10 ml 500 μl

over night 100 μl

200 μl

100 μl 50 μl

20 μl HPLC

71


121 15min PDA

PGA

28 10

50 ml 1) 10 ml

R. quercivora No. 4

200 mg / ml 50 %

NS-EtOAc-S 20 μl NS-EtOAc-S 4 mg / 10 ml

28 6 1000 μl

12,500

rpm, 5 min 500μl 500μl

300μl

50μl

20μl HPLC HPLC 3-2-1

( 100 or 200)

72

4-3

4-3-1

Aspergillus oryzae

30 units / mg

(Fig. 4-1)

1

pH 5.5 30 1

1 mol 1 unit

1 mol

pH 5.5 30

1 1 mol 1 unit

HPLC

60 HPLC

(Fig. 4-2) HPLC R.T. 26 min.

HPLC

73

5 10

(Fig. 4-3)

HPLC

(Fig. 4-4)

Fig. 4-3 1 6.1 10-4 μmol

HPLC 20 l

1

0.3 mg / 10 ml

1000 l 0.03 mg 1mg

762.5 10-4 μmol / 0.03 mg = 2.5 μmol

pH 5.5 30 1 1 mol

1 unit 2.5unit

0.05 M

0.05 M

2 0.05 M NaOH

mol105.762200

5000100200

2050mol101.6 44

74

Fig. 4-2 HPLC

75

Fig. 4-1 Time course of Abs of tannic acid treated by tannase

76

Fig. 5 HPLC chromatograms of tannic acid before treatment by tannase (down side), and after 60 min. treatment by tannase (upper side).

gallotannins

gallic acid

gallic acid

Fig. 5 HPLC chromatograms of tannic acid before treatment by tannase (down side), and after 60 min. treatment by tannase (upper side).

gallotannins

gallic acid

gallic acid

Fig. 4-2 HPLC chromatograms of tannic acid before (a) and after (b) 60min treated by tannase

77

μ

78

μ

79

4-3-2 NS-EtOAc-S

580.0g NS-e

0.9g 2.59g n- 3.17g n- 2.64g

HPLC (Fig. 4-5) NS-e

1-3-4

30

31

UV

30 n-

n-

NS-EtOAc-S

80

Fig. 4-5 HPLC chromatograms of 70% aqueous acetone extracts (a), ether soluble part (b), ethyl acetate soluble part (c), n-BuOH soluble part (d) and n-BuOH insoluble part (e)

81


R. quercivora

A. oryzae

HPLC

NS-EtOAc-S HPLC

(Fig. 4-6)

1-3-4 NS-e

NS-e

NS-EtOAc-S

1-3-3

NS-e

R. quercivora

PGG EtOAc-S

R. quercivora HPLC

83

Retention time (min)Fig. 4-6 HPLC chromatograms of EtOAc-S (a) treated with commercial tannase (b) blank.

a)

b)

84


NS-EtOAc-S

NS-EtOAc-S

HPLC (Fig. 4-7)

30

R.T. 32 min.

UV

R. quercivora

R. quercivora

10

85

a)

b)

ellagic acid

Fig. 4-7 HPLC chromatograms of EtOAc-S (a) treated with crude enzyme of R. quercivora. (b) blank.

Retention time (min)

86

4-3-5 NS-EtOAc-S

NS-EtOAc-S

(Photo)

NS-EtOAc-S 2-3

87

Photo Fungus body of R. quercivora before (a, 100) and after (b, 200) incubation

with NS-EtOAc-S

a)

b)

88

4-4


89

4-5

1. . 1985.

. 39:65-74

2. Shashi Sharma, Lata Agarwal, Rajendra Kumar axena. 2008. Purification, immobilization

and characterization of tannase from Penicillium cariable. Bioresource Technology.

99:2544-2551



quercivora (I) : Comparison of sapwood extractives non-infected and infected. J.Wood. Sci.

55:126-132

90

5

5-1

1-3-3

HPLC

Quercus Q. coccifera Q. suber

1)

91

5-2

5-2-1

1-2-1-2 (DS-e)

DS-e

3500rpm, 10mim, 4 ) DS-WS DS-WI DS-e

DS-WS DS-WI HPLC

HPLC





Column Deverosil ODS HG-5 4.6x250mm

Flow rate 1 ml/min

Column temp 40

Detector wave length 280nm SHIMAZU SPD-M10A VP


Fig. 5-1

92

damaged Q. crispula sapwood (725.6g )

70% aqueous acetoneconcentrated

centrifuged (3500rpm, 10mim, 4 )

extracts (DS-e)

supernatant (DS-WS) precipitate (DS-WI)

Fig. 5-1 Scheme of extraction from damaged Q. crispula sapwood and precipitation

93

5-2-2 DS-WS

DS-WS 10% 30%

2,000rpm, 2min

DS-WS1MS DS-WS3MS 30%

DS-WS3MI 5-2-1 HPLC

94

5-2-3 DS-WS1MS

5-2-3-1 DS-WS1MS

DS-WS1MS GL Science

C18 120A 20/40μm 10%

20% 30% 35% 40% 50%

2L 2L

HPLC

95

5-2-3-2 DS-WS1MS-3 HPLC

DS-WS1MS-3

10% 15% 20% 25% 30% 35% 40% 50% 2L

1L 15 HPLC

Fr. 3 HPLC HPLC





Column : Inartsil® PDS-3 (10×250 mm) (GL Sciences Ins., Japan).

Flow rate : 3 ml / min.

Detector wave length : 280 nm

Solvent : 0.05% TFA in water / acetonitrile = 100 / 0 (initial) to 70 / 30 (finish) (60min.)

HPLC NMR

96


HPLC DS-WS1MS-4

HPLC

Column : Inartsil® PDS-3 (10×250 mm) (GL Sciences Ins., Japan).

Flow rate : 3 ml / min.

Detector wave length : 280 nm

Solvent : 0.05% TFA in water / acetonitrile = 80 / 20 ( isocratic condition)(60min.)

HPLC NMR

97

5-2-4 DS-WS3MS

5-2-4-1 DS-WS3MS

DS-WS3MS GL Science

C18 120A 20/40μm 10%

40% 50% 100%

2L 2L HPLC


DS-WS1MS-4 DS-WS3MS-2 HPLC

NMR FAB-MS NMR

FAB-MS

NBA

98

(-)-lyoniresinol

Yellow, needle crystal ; FAB-MS (positive mode) m/z 420.0 [M]+ (calculated for C22H28O8,

420.0); 1H NMR (CD3OD, 600 MHz) δ 1.60-1.61 (1H, m, H-8’), 1.94-1.96 (1H, m, H-8), 2.55

(1H, dd, J=11.52 and 14.58 H-7’) 2.67 (1H, dd, J=4.8 and 15.12 H-7’), 3.35 (3H, s, H-a),

3.47 (1H, m, H-9’), 3.57 (1H, dd, J= 4.8 and 10.3 H-9’), 3.48 (2H, s, J=2.76, H-a), 3.71 (6H,

s, H-b,b’), 3.82 (3H, s, H-c), 4.29 (1H, d, J=5.52, H-7), 6.36 (2H, s, H-2,6), 6.56 (1H, s,

H-2’); 13C NMR (CD3OD, 150 MHz): δ 32.2(C7’), 39.5(C8’), 41.0(C-7), 47.69(C-8),

55.3(C-c), 55.5(C-b, b’), 58.8(C-a), 62.9(C-9), 65.5(C-9’), 105.6(C-2, 6), 106.5(C-2’),

124.9(C-1’), 128.9(C-6’), 133.2(C-4), 137.5(C-4’), 138.0(C-1), 146.4(C-5’), 147.3(C-3’),

147.7(C-3)

OH

OH

OH

OH

OMe

MeO

OMeMeO

1

2

3

4

56 7

89

1’2’

3’

4’5’

6’

7’

8’

9’

99

5-2-5 DS-WS3MI

DS-WS3MI 40%

40, 50, 60% 5

Fr. 4 5 Amid-80

Column : TSK GEL Amide-80 10μm 21.5mm 30 cm

Solvent : A / Acetonitrile, B / H2O

Gradient program : 100% Solvent A to 85% Solvent A in Solvent B for 30 min

85 % Solvent A in Solvent B isocratic condition for 30 min

Flow rate : 3 ml/min

Pump : Jasco 880-PU Intelligent HPLC Pump

Detector : Jasco 875-UV Intelligent UV-VIS Detector

Degassor : 880-51 2-Line Degassor

Recorder : SHIMADZU C-R6A CHROMATOPAC

Amide-80 ODS

HPLC

HPLC

Column : Inertsil®ODS-3 10 250 mm

Solvent : A / 0.05 % TFA in H2O, B / methanol


100






NMR

DS-WS Fig. 5-2

101

WS1MS

medium-pressure liquid chromatography

WS1MS-1…

WS1MS-4…

WS1MS-6

preparative HPLC

isolated compound

Fig. 5-2 Preparation scheme of DS-WS

DS-WS

10% aqueous methanol

30% aqueous methanol

WS3MIWS3MSmedium-pressure liquid chromatography

WS3MS-1…

WS3MS-3…

WS1MS-4

preparative HPLC

isolated compound

middle pressure column chromatography

eluted with 40, 50 and 60% aqueous methanol and methanol

WS3MI-1 WS3MI-4 WS3MI-5

… …Fr. A Fr. B

WS3MI4-1 WS3MI4-2

P-HPLC(Amide-80 column)

…

…Fr. C Fr. D

…Fr. E Fr. F

(3 compounds)(1 compound)

(3 compounds)(2 compounds)

P-HPLC(ODS column)

102

5-2-6 DS-WI

DS-WI

2,000rpm, 2 min DS-WIES ES-WIEI

HPLC

Column : SHIMADZU Shim-pack VP-ODS 4.6 25 cm


Column temp : 40

Detector wave length : 280nm SHIMAZU SPD-M10A VP

Elution : MeOH / 0.05%TFA aq. = 5 / 95 100 / 0 45min

103

5-2-7 DS-WIES

LH-20 2.4cm 20cm WIES 0.7883g

500ml 50% 200ml 50%

200ml 70% 200ml 100ml

8 HPLC

HPLC

59.5mg Fr. 5 HPLC

HPLC

Column : Inertsil®ODS-3 10 250 mm


Solvent : A / 0.05 % TFA in H2O, B / methanol






NMR

104

5-2-8 WIEI

LH-20 6cm 21cm WIEI 4.4385g 50%

600ml 50% 1000ml 50%

400ml 70% 400ml 100ml

70% 200l

20 HPLC

DS-WI Fig.5-3

105

ethanol solublepart (WIES)

ethanol insoluble part (WIEI)

LH-20 columneluted with ethanol, 50%aqueous ethanol,50% aqueous methanol and 70% aqueous acetone

WIES 1 WIES 2 WIES 3 … WIES 5 WIES 8…

… …WIES 5-1 WIES 5-11

LH-20 column

ellagic acid

isolated compound

DS

DS-WS DS-WI

Fig. 5-3 Preparation scheme of DS-WI

106

4,5-dihydroxy-6-(3,7,8-trihydroxy-5,10-dihydro-chromeno[5,4,3-cde]chromen-2-yloxy)-tetra

hydro- pyran-3-yl ester (1)

Brown oil; 5.5 mg; LC-TOFMS: m/z 584.9888 [M-H]-, (calcd. for C26H18O16); 1H NMR (500

MHz, CD3OD) δ 7.79, 7.49 (2H in total, each s, ellagic acid unit protons), 7 .09 (2H, s,

galloyl-H), 3.62, 3.73, 3.88, 4.22, 5.00, 5.11 (6H in total, sugar protons), see table 2; 13C

NMR (150 MHz, CD3OD) δ166.4 (galloyl C-7), 148.8 (ellagic acid unit C-4’), 146.9 (ellagic

acid unit C-4), 145.2 (galloyl C-3,4), 141.4 (ellagic acid unit C-3), 139.6 (ellagic acid unit

C-3’), 138.7 (galloyl C-6), 136.7 (ellagic acid unit C-2’), 136.4 (ellagic acid unit C-2), 119.7

(galloyl C-1), 115.4 (ellagic acid unit C-1), 112.4 (ellagic acid unit C-5), 112.0 (ellagic acid

unit C-1’), 110.6 (ellagic acid unit C-5’), 109.0 (galloyl C-2,5), 108.8 (ellagic acid unit C-6’),

108.0 (ellagic acid unit C-6), 102.9 (xylose C-1), 73.3 (xylose C-2), 73.1 (xylose C-3), 71.5

(xylose C-4), 62.5 (xylose C-5).

107

5-3

5-3-1 lyoniresinol

DS-WS1MS-4 NMR Tale 5-1

FAB MS 420 13C NMR 1H NMR

C22H28O81H NMR 6.35ppm 2 6.55ppm

1 4.28ppm 1

HMBC 39.55 47.4 62.89 105.55 124.91

128.86 137.98 146.35ppm 3

105.55 146.35ppm

105.55ppm 106.46ppm 2

2

COSY 1.60ppm

2.57ppm 2.67ppm 3.50ppm 1.95ppm 1.95ppm

1.60ppm 3.47ppm .28ppm

3.50ppm 3.47ppm

2.66ppm 2

HMBC 106.46 124.91

108

128.85 3

HMQC 40.97ppm

4 3.35ppm 58.84ppm

3.71ppm 55.46ppm 2 3.82ppm

55.29ppm 146.35 147.65 2 147.33ppm

146.35ppm 2

HMBC COSY

Fig. 5-4 5

109

Table 5-1 1H and 13C NMR spectral data and key COSY and HMBC correlations of

lyoniresinol

Position δC (ppm) δH (ppm) COSY HMBC 1 138.0 2 and 6 105.6 6.36 2H, s C1, C3, C4, C5, C7 3 and 5 147.7 4 133.2 7 41.0 4.29 1H, d, J=5.52 8 C1, C2, C8, C9, C1’,C5’, C6’,C8’ 8 47.7 1.94-1.96 1H, m 7, 9, 8’ C9, C7’, C8’, C9’ 9 62.9 3.48 2H, d, J=2.76 1’ 124.9 2’ 106.5 6.56 1H, s C1’, C3’, C4’, C7’ 3’ 147.3 4’ 137.5 5’ 146.4 6’ 128.9 7’ 32.2 2.55, 2.67 1H, dd, J=11.5, 15.0, 1H,

dd, J=4.8, 15.0 7’,8’ C8, C1’, C2’, C6’, C8’, C9’

8’ 39.5 1.60-1.61 1H, m 8, 7’, 9’ C8, C9, C9’ 9’ 65.5 3.47,3.54 1H, m, 1H, dd, J=4.8 C8, C7’ 5’-OMe 58.8 3.82 3H, s C3’ 3-OMe, 5-OMe 55.5 3.71 3H 2, s C5 3’-OMe 55.3 3.35 3H, s C5’

110

OHOH

MeO

OH

OMe

OMe

HO

MeO

H

H

Fig. 11 Structure of isolated compoound lyoniresinol. Arrows show the diagnostically significant C-H correlation found by HMBC

OHOH

MeO

OH

OMe

OMe

HO

MeO

H

H

Fig. 11 Structure of isolated compoound lyoniresinol. Arrows show the diagnostically significant C-H correlation found by HMBC

Fig. 5-10Fig 5-4 Structure of isolated compound loniresinol.

Arrows show the diagnostically significant C-H

111

OHOH

MeO

OH

OMe

OMe

HO

MeO

H

H

Fig. 12 Structure of isolated compoound lyoniresinol. Arrows show the diagnostically significant H-H correlation found by COSY

Fig. 5-11

Fig 5-5 Structure of isolated compound loniresinol.

Arrows show the diagnostically significant H-H

112

Fig. 5-6 Alangium

premnifolium lyoniresinol lyoniresinol

[ ]23.3 -9.51429 (c=0.21,

MeOH) Mitsuhiko Miyamura (+)-lyoniresinol [ ]D = +58.0

2) lyoniresinol (-)-lyoniresinol

lyoniresinol (+) (-) 3)

4,5) (+) 6) (+)-lyoniresinol

7) (+) (-) 8,9,10)

(-)-lyoniresinol

Kirkia acuminata 11)

DS-WS1MS-4 DS-WS3MS-2 HPLC

NMR (-)-lyoniresinol

113

Fig. 5-6 Structure of (-)-lyoniresinol

OH

OH

OH

OH

OMe

MeO

OMeMeO

1

2

3

4

56 7

89

1’2’

3’

4’5’

6’

7’

8’

9’

114

5-3-2

LC-TOF MS 2

m/z584.9888 m/z 300.9526

LC-TOF MS HHDP

M-galloyle +

2 LC-TOF

MS

2 m/z584.9888 m/z 300.9526

2 C1

2 1H, 13C NMR HMBC Table2

1H NMR 7.09ppm HMBC

1H NMR 3.62 5.11ppm H-1H COSY

3.73ppm 5.11ppm 3.88ppm

5.00ppm 3.88ppm 3.62 4.22ppm

3.62ppm 4.22ppm 5.11ppm

xylose

HMBC xylose 4’’

7’’’

115

7’’’ xylose4’’ =200 HMBC

xylose 1 5.11ppm 4 146.9ppm

xylose1 4

Fig5-7

xylose 4 1 4,5-dihidroxy-6-(3,7,8-trihydroxy-5,10-dihydro

-chromeo[5,4,3-cde]chromen-2-xyloxy)-tetrahydro-pyran-3-yl ester

P.strobilacea pedunculgin

1(β)-O-galloylpedunculagin, casuariin 3’-O-methyl-4,5-dihidroxy-6

-(3,7,8-trihydroxy-5,10-dihydro-chromeo[5,4,3-cde]chromen-2-xyloxy)-tetrahydro-pyran-3-

yl ester 12)

R.

quercivora tannase laccase

116

Table5-2 1H and 13C NMR specral data and key HMBC correlations of new ellagitannin,

4,5-dihydroxy-6-(3,7,8-trihydroxy-5,10-dihydro-chromeno[5,4,3-cde]chromen-2-yloxy)-tetra

hydro- pyran-3-yl ester

Moiety Position δC (ppm) δH (ppm) HMBC 1 115.4 2 136.4 3 141.4 4 146.9 5 112.4 7.79 1H, s C1, C2, C3, C4, C5, C6, C7 6 108.0 Ellagic acid group 7 1’ 112.0 2’ 136.7 3’ 139.6 4’ 148.8 5’ 110.6 7.49 1H, s C1’, C2’, C3’, C4’, C6’, C7’ 6’ 108.8 7’ 1” 102.9 5.11 1H, d, J=6.9 C4 2 “ 73.3 3.73 1H, dd, J=6.85, 8.60 3 “ 73.1 3.88 1H, t , J=9.15 xylose 4” 71.5 5.00 1H, m C7’’ 5” 62.5 3.62

4.22 1H, dd, J=9.75, 11.45 1H, dd, J=5.70, 12.00

1’’’ 119.7 2’’’, 5’’’ 109.0 7.09 2H, s Gallic acid group 3’’’, 4’’’ 145.2 6’’’ 138.7 7’’’ 166.4

C7’’

117

Fig 5-7 Structure of new ellagitannin, 4,5-dihydroxy-6-(3,7,8-trihydroxy-5,10-dihydro-

chromeno[5,4,3-cde]chromen-2-yloxy)-tetrahydro- pyran-3-yl ester

118

5-4

(-)-lyoniresinol

NMR LC-TOF MS 4,5-

dihidroxy-6-(3,7,8-trihydroxy-5,10-dihydro-chromeo[5,4,3-cde]chromen-2-xyloxy)-tetrahyd

ro-pyran-3-yl ester


119

5-5

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