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
9
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
19
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
6. Kuroda, K. (2001) Responses of Quercus sapwood to infection with the pathogenic
fungus of a new wilt disease vectored by the ambrosia beetle Platypus quercivorus. J.
Wood. Sci. 47: 425-42
7. 2002 11 8-9
8. 1996
78 84-88
9. 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
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
1. Kuroda, K. (2001) Responses of Quercus sapwood to infection with the pathogenic
fungus of a new wilt disease vectored by the ambrosia beetle Platypus quercivorus. J.
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
11. 2003 Raffaelea quercivora
51 199-200
12. 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
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
2008 Journal of wood science 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
2. 2003 Raffaelea quercivora
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)
Detector : SHIMADZU SPD-M10Avp
Column ocen : SHIMADZU CTO-10A
Communication bus module : SHIMADZU CBM-10A
Degasser : SHIMADZU DGU-12A
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
2008 Journal of wood science 5)
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.
5. 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
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)
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
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
68
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
4-2-5 NS-EtOAc-S HPLC
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
4-3-3 NS-EtOAc-S HPLC
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
82
83
Retention time (min)Fig. 4-6 HPLC chromatograms of EtOAc-S (a) treated with commercial tannase (b) blank.
a)
b)
84
4-3-4 NS-EtOAc-S HPLC
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
2008 Journal of wood science 2)
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
2. 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
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
Pump : SHIMADZU LC-10AD (0.05 % TFA in water), SHIMADZU LC-10AT (Methanol)
Detector : SHIMADZU SPD-M10Avp
Column ocen : SHIMADZU CTO-10A
Degasser : SHIMADZU DGU-12A
Column Deverosil ODS HG-5 4.6x250mm
Flow rate 1 ml/min
Column temp 40
Detector wave length 280nm SHIMAZU SPD-M10A VP
Elution MeOH / 0.01%TFA aq. = 5 / 95 100 / 0 45min
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
Pump : SHIMADZU LC-10AD (0.05 % TFA in water), SHIMADZU LC-10AT (Methanol)
Detector : SHIMADZU SPD-M10Avp
Column ocen : SHIMADZU CTO-10A
Degasser : SHIMADZU DGU-12A
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
5-2-3-3 DS-WS1MS-4 HPLC
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
5-2-4-2 DS-WS3MS-2 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
Gradient program : 50% Solvent A to 70% Solvent A in Solvent B for 60 min
100
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
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
Flow rate : 1 ml/min
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
Flow rate : 3 ml/min
Solvent : A / 0.05 % TFA in H2O, B / methanol
Gradient program : 50% Solvent A to 70% Solvent A in Solvent B for 60 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
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
2013 Journal of wood science 13)
119
5-5
1. Hideyuki Ito, Koji Yamaguchi, Tae-Hoon Kim, Seddik Khennouf, Kamel Gharzouli and
Takashi Yoshida. 2002. Dimeric acnd Trimeric Hydrolysable Tannins from Quercus coccifera
and Quercus suber. J. Nat. Prod. 65:339-345
2. Mitsuhiko Miyamura, Toshihiro Nohara, Toshiaki Tomimatsu and Itsuo Nishioka. 1983.
Seven aromatic compounds from bark of Cinnamomum cassia. Phytochemistry. 22:215 -218
3. Kaneda N., Dinghorn A.D., Farnsworth N.R., Tuchinda P., Udchachon J. Santisuk T. and
Reutrakul V. 1990. Two diarylheptanoids and a lignan from Casuarina junchuhniana.
Phytochemistry. 29:3366-3368
4. Masateru Ono, Eriko Oda, Takemi Tanaka, Yoshihiko Iida, Toru Yamasaki, Chikako
Masuoka, Tsuyoshi Ikeda and Toshihiro Nohara. 2008. DPPH radical-scavenging effect on
some consitituents from the aerial parts of Lippia triphylla. J. Nat. Med. 62:101 -106
5. Marie-Françosie Nonier, Nicolas Vivas Nathalie Vivas de Gaulejac and Eric Fouquent.
2008. Origin of brown discoloration in the staves of oak used in cooperage-Characterization
of two new lignans in oak wood barrels. C. R. Chimie. Article in Press.
120
6. Samir Kumar Sadhu, Panadda Phattanawasin, M. shahabuddin Kabin Choudhuri, Takashi
Ohtsuki and Masami Ishibashi. 2006. A new lignan from Aphanamixis polystachya. J. Nat.
Med. 60:258-260
7. 1991. (Quercus mongolica FISCHER var
grosseserrata REHD. et WILS.) .
37:82-87
8. Kaori Yuasa, Toshinori Ide, Hideaki Totsuka, Choei Ogimi, Eiji Hirata, Anki Takushi and
Yoshio Takeda. 1997. Lignan and neolignan glycosides from Stems of Alangium
Premnifolium. Phytochemistry. 45:611-615
9. Klaus Peter Latté, Maki Kaloga, Andreas Shäfer and Herbert Kolodziej. 2008. An
ellagitannin, n-butyl gallate, two aryltetralin lignans, and an unprecedented diterpene ester
from Pelargonium reniforme. Phytochemistry. 69:820-826
10. Nilubon Jong-Anurakkun, Megh Raj Bhandari, Jun Kawabata. 2007. α-Glucosidase
inhibitors from Devil tree (Alastronia scholaris). Food Chemistry. 103:1319-1323
11. Mulfolland D.A., Cheplogoi P., and Crouch N.R. 2003. Secondary metabolites from
Kirkia acuminate and Kirkia wilmisii (Kirkiaceae). Biochemical Systematic and Ecology.
121
31:793-797
12. Maeda H, Kakoki, Ayabe M, Koga Y, Oribe T, Matsuo Y, Tanaka T, Kouno I (2011)
ent-Eudesmane sesquiterpenoids, galloyl esters of the oak lactone precursor, and a
3-O-methylellagic acid glycoside from the wood of Platycarya strobilacea. Phytochemistry
72: 796-803
13. Kayoko Imai, Kosei Yamauchi, Tohru Mitsunaga (2013) Extractives of Quercus crispula
sapwood infected by the pathogenic fungus Raffaelea quercivora (II): Isolation and
identification of phenolic compounds from infected sapwood . J.Wood. Sci. article not
assigned to an issue. (Article in press)
122