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약학석사학위논문
Diospyros burmanica 의 잎과 수피에서
분리한 성분
Chemical Constituents from Diospyros burmanica
leaves and barks
2013 년 8 월
서울대학교 대학원
약학과 생약학전공
이 준 철
i
Abstract
Diospyros burmanica Kurz is the evergreen broad-leaved tree distributed in Mandalay
of Myanmar, which belongs to the family of Ebenaceae. In Myanmar, it is used to treat
diarrhea, diabetes, and shigellosis. More than 350 plants belonging to the genus Diospyros are
known worldwide. However, not many on D. burmanica are studied yet except for the
leishmaniasis inhibiting properties studied by Japanese researchers. Thus, the objective of this
study is to isolate and identify the chemical constituents from the leaves and the barks of
Diospyros burmanica.
Dried leaves and barks of Diospyros burmanica are extracted with 100% methanol,
suspended in distilled water and fractionated with methylene chloride, ethyl acetate, and n-
buthanol. Then through silica gel column chromatography, counter current chromatography,
HP column chromatography, ODS-A gel column chromatography, MPLC, HPLC, seven
flavonoids, a methyl gallate, and five triterpenes were isolated.
Isolated compounds were identified as (-)-catechin 3-O-α-L-rhamnopyranoside (1), (-)-
catechin (2), methyl gallate (3), (-)-2,3-trans-dihydrokaempferol 3-O-rhamnopyranoside (4), (-)-
epicatechin 3-O-gallate (5), (+)-catechin 3-O-gallate (6), (-)-epicatechin (7), (+)-afzelechin 3-O-
α-L-rhamnopyranoside (8), lupeol (9), methyl lup-20(29)-en-3-one-28-oic acid (10), 3β-
hydroxy-D:B-friedo-olean-5-ene (11), β-amyrin (12), urs-12-ene-3β-ol (13) from UV, Q-TOF
LC/MS, FAB-LRMS, 1H-NMR,
13C-NMR,
1H-
1H COSY, HSQC, and HMBC spectrum.
Compounds 1, 3, 4, and 8 (catechins and methyl gallate) showed significant inhibitory
effect on NO production induced by lipopolysaccharide on the BV2 microglia cell line. Also
ii
catechin type compounds showed significant difference in their activity depending on the
substitution of the glycosides on C-3 position.
Keywords : Diospyros burmanica, catechin, epicatechin, afzelechin, dihydrokaempferol,
triterpene, lupeol, ursenol, amyrin, friedo-oleanene
Student Number : 2011-21760
iii
Contents
List of Abbreviations ..................................................................................................................... v
List of Figures ............................................................................................................................. vii
List of Tables ........................................................................................................................... viiiiii
List of Schemes ............................................................................................................................ ix
I. Introduction ................................................................................................................................ 1
II. Materials and Methods ............................................................................................................. 2
1. Isolation of chemical constituents from bark of D. burmanica .............................................. 2
1.1. Plant material ........................................................................................................... 2
1.2. Reagents and equipments......................................................................................... 2
1.3. Methods ................................................................................................................... 5
2. Evaluation of Inhibitory effect on NO production in BV2 microglia cell line ................... 29
2.1. BV2 microglia cell culture and reagents ................................................................ 29
2.2. BV2 microglia cell culture ..................................................................................... 29
2.3. NO production induced by LPS ............................................................................. 29
2.4. Griess assay ........................................................................................................... 30
2.5. Statistical analysis .................................................................................................. 30
III. Result and discussion ............................................................................................................ 31
1. Structure elucidation of isolated compounds from D. burmanica ....................................... 31
1.1. Compounds 1 and 2 ............................................................................................... 31
iv
1.2. Compounds 3 and 4 ............................................................................................. 344
1.3. Compounds 5 and 6 ............................................................................................... 37
1.4. Compounds 7 and 8 ............................................................................................... 40
1.5. Compound 9........................................................................................................... 43
1.6. Compound 10 ......................................................................................................... 45
1.7. Compound 11 ......................................................................................................... 47
1.8. Compound 12 ......................................................................................................... 49
1.9. Compound 13 ......................................................................................................... 50
2. Inhibitory effect against NO production by the compounds isolated from D.burmanica . 53
IV. Conclusions ......................................................................................................................... 566
V. References ............................................................................................................................. 577
v
List of Abbreviations
AcCN: acetonitrile
n-BuOH: n-butanol
c.c.: column chromatography
ccc.: counter current chromatography
CH2Cl2: methylene chloride
COSY: correlation spectroscopy
d: doublet
dd: doublet of doublet
DMEM: Dulbecco’s modified eagle’s medium
DMSO: dimethylsulfoxide
dt: doublet of triplet
ESIMS: electron spray impact mass spectroscopy
EtOAc: ethyl acetate
fr.: fraction
HMBC: heteronuclear multi-bond correlation
Hz: hertz
m: multiplet
M: methanol
MeOH: methanol
vi
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NMR: nuclear magnetic resonance
RP: reverse phase
s: singlet
t: triplet
UV: ultraviolet absorption spectroscopy
W: water
vii
List of Figures
Figure 1. 1H and
13C NMR spectra of compound 1………………………………………………32
Figure 2. 1H and
13C NMR spectra of compound 2………………………………………………33
Figure 3. 1H and
13C NMR spectra of compound 3………………………………………………35
Figure 4. 1H and
13C NMR spectra of compound 4………………………………………………36
Figure 5. 1H and
13C NMR spectra of compound 5………………………………………………38
Figure 6. 1H and
13C NMR spectra of compound 6………………………………………………39
Figure 7. 1H and
13C NMR spectra of compound 7………………………………………………41
Figure 8. 1H and
13C NMR spectra of compound 8………………………………………………42
Figure 9. HSQC spectrum of compound 8……………………………………………………...…43
Figure 10 . 1H and
13C NMR spectra of compound 9………………………………….…………44
Figure 11. 1H and
13C NMR spectra of compound 10……………………………………………46
Figure 12. HSQC spectrum of compound 10……………………………………………..………46
Figure 13. 1H and
13C NMR spectra of compound 11…………………………………………...48
Figure 14. 1H and
13C NMR spectra of compound 12………………………………...…………50
Figure 15 . 1H and
13C NMR spectra of compound 13………………………………………..…51
viii
List of Tables
Table 1. 1H NMR and
13C NMR spectral data of compound 1…………………………………16
Table 2. 1H NMR and
13C NMR spectral data of compound 2…………………………………17
Table 3. 1H NMR and
13C NMR spectral data of compound 3…………………………………18
Table 4. 1H NMR and
13C NMR spectral data of compound 4…………………………………19
Table 5. 1H NMR and
13C NMR spectral data of compound 5…………………………………20
Table 6. 1H NMR and
13C NMR spectral data of compound 6…………………………………21
Table 7. 1H NMR and
13C NMR spectral data of compound 7…………………………………22
Table 8. 1H and
13C NMR spectral data of compound 8……………………………………...…23
Table 9. 1H and
13C NMR spectral data of compound 9……………………………………...…24
Table 10. 1H and
13C NMR spectral data of compound 10…………………………………...…25
Table 11. 1H NMR and
13C NMR spectral data of compound 11………………………………26
Table 12. 1H NMR and
13C NMR spectral data of compound 12………………………………27
Table 12. 1H NMR and
13C NMR spectral data of compound 13………………………………28
ix
List of Schemes
Scheme 1. Extraction and fractionation of the barks of D. burmanica…………………………..5
Scheme 2. Extraction and fractionation of the leaves of D. burmanica………………………....6
Scheme 3. Isolation of compounds from EtOAc fraction of D. burmanica (barks) …….….….8
Scheme 4. Isolation of compounds from CH2Cl2 fraction of D. burmanica (barks) ……….…10
Scheme 5. Isolation of compounds from CH2Cl2 fraction of D. burmanica (leaves) …………11
1
I. Introduction
The family Ebenaceae contains only three genera and the genus Diospyros is of the largest
(Willis, 1966). Thus the chemical constituents of Ebenaceae are generally confined to the
genus Diospyros. More than 350 plants of genus Diospyros are known worldwide and many
are used as traditional medicine in Ayurveda, the African folklore and Chinese medicine
(Tangmouo et al., 2006; Chen et al., 2008). The family Ebenaceae and the genus Diospyros
show the characteristics of producing naphthoquinones, flavonoids, and triterpenes, especially
of the lupine series (Zhong et al., 1984). The stems and leaves of this genus generally contain
triterpenoids, when the roots are generally known to contain naphthols and naphtoquinones
(Bhakuni et al., 1971). The antibacterial, antifungal, and termite-resistant properties of the
genus Diospyros showed attribution to the chemical composition of naphthoquinones
(Waterman and Mbi, 1979). Recent studies also showed that many of these species contain
antitumor, antidiabetic, antioxidant, and hypocholesterolemic effects (Chen et al., 2008).
Diospyros burmanica Kurz is a tree distributed throughout certain regions of Myanmar,
such as Mandalay, which belongs to the family of Ebenaceae. No studies have been done on
such plant and no medicinal effects are known yet except for the activity against leishmaniasis
studied by the Japanese research center. According to the study, the methanol extract of the D.
burmanica woods showed some potent inhibition against Leishmania while no other Diospyros
plants showed activity against Leishmania spp. (K. Mori-Yasumoto et al., 2012). Furthermore,
not many chemical constituents of D. burmanica are reported yet.
2
II. Materials and Methods
1. Isolation of chemical constituents from the barks and leaves
of D. burmanica
1.1. Plant material
Barks and leaves of Diospyros burmanica Kurz (Ebenaceae) were collected from
Mandalay, Myanmar in February 2012.
1.2. Reagents and equipments
1.2.1. Reagents
Analytical TLC: Silicagel 60 F254, Art. 5715, Merck, Germany
First grade solvent for extraction, fractionation and isolation: Dae Jung Pure chemical Eng. Co.
Ltd., Korea
HPLC grade solvent: Fisher Scientific, Pittsburgh, PA, USA
3
ODS gel: YMC-Pack ODS-A, 12nm S-5 m, AA12S05-252OWT, YMC, Japan
Silica gel: Kiesgel 60, 40-63 mm, 230-400 mesh, Art. 9385, Merck, Germany
1.2.2. Equipments
Analytical balance: Shimadzu AUX220, Japan
Drying oven: HB-5025, Han Baek Scientific Co. Korea
HPLC system (Preparative):
- Gilson 321 pump, USA
- Gilson UV/Vis-155 detector, USA
- Gilson GX-271 Liquid Handler and GX-271 ASPECTM
, USA
-Agilent Technologies 1200 Infinity Series, 1260 Infinity, Germany
HPLC-DAD-ESIMS system:
- Finnigan Surveyor MS pump plus
- Finnigan Surveyor PDA detector
- Finnigan LCQ advantage Max
- Column: Ascentis Express C18 HPLC Column (4.6 x 150 mm, 2.7 μm)
MPLC: IOTA S300, PN PYC00000, ECOM, Czech Republic
NMR: Bruker Avance III 500 Spectrometer (500 MHz), Germany
Polarimeter: JASCO, DIP-1000, Japan
Rotary evaporator: EYELA, Tokyo Rikakikai Co., Japan
Sonicator: Branson 5510, UK
4
UV lamp: CN-6 Cedex 1, France
UV spectrometer: Shimadzu UV-1800 Spectrophotometer, Japan
HPCCC:
-DE Spectrum Centrifuge, Spectrum 11020304, Dynamic Extractions Ltd., United Kingdom
-DE Midi Centrifuge, Midi 11021101, Dynamic Extractions Ltd., United Kingdom
Nitrogen gas generator:
Nitrogen Generator for Evaporation, Evan-0100, Goo Jung Engineering, Pressured Gas Blowing
Concentrator, MG-2200, EYELA, Japan
5
1.3. Methods
1.3.1. Extraction and fractionation of D. burmanica
1.3.1.1. Barks of D. burmanica
Dried barks (1.3 kg) of D. burmanica were extracted with 100% MeOH in a sonicator.
After evaporating the solvent in vacuo, the 100% MeOH extract (221.5 g) was suspended in
H2O and fractionated with CH2Cl2 (8.1 g), EtOAc (68.9 g), and n-BuOH (93.9 g) (Scheme 1).
1.3.1.2. The leaves of D. burmanica
Scheme 1. Extraction and fractionation D. burmanica barks
6
Dried leaves (630.0 g) of D. burmanica were extracted with 100% MeOH using a
sonicator. The solvents were evaporated in vacuo and suspended 100% MeOH extract (103.0
g) in H2O and fractionated with CH2Cl2 fraction (3.6 g), EtOAc fraction (15.5 g), and n-BuOH
fraction (16.6 g) (Scheme 2).
Scheme 2. Extraction and fractionation D. burmanica leaves
7
1.3.2. Isolation of compounds from D. burmanica barks
1.3.2.1. Isolation of compounds from EtOAc fraction
The EtOAc fraction was subjected to silica gel column chromatography and operated
solid phase extraction (CHCl3:MeOH 10:1→1:1→MeOH; v/v ratio) to yield five fractions
(E1~E5) (Scheme 3). Fraction E1 (7.5 g) was subjected to counter current chromatography
with solvent composition of Hexane-EtOAC-MeOH-water (HEMW) (2:8:2:8) to yield five
fractions (E1-1~E1-5). Compound 1 (282.9 mg) was obtained by subjecting E1-2 to silica gel
c.c. with mixtures of CMW (20:5:1) and then subjecting E1-2-3 to ODS gel c.c. with mixtures
of MeOH-water (MW) (1:9). Compound 2 (243.7 mg) was obtained from E1-1 by operating
ccc under different solvent composition of HEMW (1:9:2:8) and purifying the E1-1-7 fraction
with silica gel c.c. with mixtures of CHCl3-MeOH-water (CMW) (12:5:1). Compound 3 (27.0
mg) and 5 (99.7 mg) were obtained from E1-3-4 through silica gel c.c. (CM 5:1) and preparative
HPLC (ODS-A, 250 x 20 mm, MeOH-water, 30:70, 4 mL/min, UV 254, 210 nm). Compound 4
(1.7 mg)and 6 (7.6 mg) were obtained from E2 through silica gel c.c. (CMW 15:5:1) and
preparative HPLC (Phenyl-hexyl, MeOH-water, 33:67). Compound 7 (27.3mg) was obtained
from E1-3 through silica gel c.c. (CM 5:1) and preparative HPLC (Phenyl-hexyl, 250 x 21.20
mm, AcCN-water, 25:75, 5 mL/min, UV 254, 210 nm). Compound 8 (56.5mg) was also
obtained from E1-3 as compound 7 did but under different HPLC condition (Phenyl-hexyl,
AcCN-water, 0:100→30:70). Scheme 3 summarizes the overall procedures of isolation of
compounds from EtOAc fraction.
8
Scheme 3. Isolation of compounds from EtOAc fraction of D. burmanica barks
A: MPLC Silica gel C.C. B: MPLC ODS gel C.C. C: HPLC ODS-A gel C.C. D: HPLC Phenyl-hexyl gel C.C. E: Silica gel C.C. F: Counter Current C.
9
1.3.2.2. Isolation of compounds from CH2Cl2 fraction
The CH2Cl2 fraction was subjected to silica gel column chromatography and yielded
seven fractions (M1~M7) using solid phase extraction (Hexane-EtOAc 10:1→1:1→CM
10:1→5:1; v/v ratio) (Scheme 4). Then M1 was subjected to silica gel c.c. and eluted with
solvent, Hexane-EtOAc (HE) (10:1) and two fractions (M1-1, M1-2) were yielded. Compound
10 was obtained from M1-1 using preparative HPLC (ODS-A, 250 x 20 mm, MeOH 100%, 4
mL/min, UV 254, 210 nm). Compound 13 was also obtained from M1-2 by preparative HPLC
(ODS-A, MeOH 100%). Compound 9 was obtained from M2 fraction using silica gel c.c. twice
(HE 15:1, HE 20:1) and preparative HPLC twice (Phenyl-hexyl, 250 x 21.20 mm, AcCN 100, 5
mL/min, UV 254, 210 nm). Scheme 4 summarizes the overall procedures of isolation of
compounds from CH2Cl2 fraction.
10
Scheme 4. Isolation of compounds from CH2Cl2 fraction of D. burmanica barks
A: Silica gel C.C. B: MPLC silica gel C.C. C: MPLC ODS gel C.C. D: HPLC ODS-A gel C.C. E: HPLC Phenyl-hexyl gel C.C. F: Counter Current C.
11
1.3.3. Isolation of compounds from the leaves of D. burmanica
1.3.3.1. Isolation of compounds from CH2Cl2 fraction
The CH2Cl2 fraction was subjected to silica gel c.c. and eluted with solvent, hexane-
EtOAc (8:1; v/v ratio) to yield 4 fractions (Fr. 1~ Fr. 4) (Scheme 5). Then Fr. 2 was subjected to
silica gel c.c. again with different solvent composition of HE (10:1). Compound 11 and 12 were
obtained from Fr. 2-1 through preparative HPLC (ODS-A, 250 x 20 mm, MeOH 100%, 4
mL/min, UV 254, 210 nm). Scheme 5 summarizes the overall procedures of isolation of
compounds from CH2Cl2 fraction of the leaves of D. burmanica.
Scheme 5. Isolation of compounds from CH2Cl2 fraction of D. burmanica leaves
A: Silica gel C.C. B: MPLC silica gel C.C. C: MPLC ODS gel C.C. D: HPLC ODS-A gel C.C. E: HPLC Phenyl-hexyl gel C.C.
F: Counter Current C.
12
1.3.4. Compounds isolated from D. burmanica
Compound 1
light pink-orange amorphous powder
C15H14O6
ESI-Q-TOF MS: m/z 291.0866 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 1
13C NMR (MeOD-d4, 500 MHz): see Table 1
Compound 2
dark brown amorphous powder
C21H24O10
ESI-Q-TOF MS: m/z 437.1454 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 2
13C NMR (MeOD-d4, 500 MHz): see Table 2
Compound 3
dark purple amorphous powder
C22H18O10
ESI-Q-TOF MS: m/z 443.2216 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 3
13C NMR (MeOD-d4, 500 MHz): see Table 3
13
Compound 4
brown amorphous powder
C15H14O6
ESI-Q-TOF MS: m/z 291.1953 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 4
13C NMR (MeOD-d4, 500 MHz): see Table 4
Compound 5
dark purple amorphous powder
C22H18O10
ESI-Q-TOF MS: m/z 443.2216 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 5
13C NMR (MeOD-d4, 500 MHz): see Table 5
Compound 6
brown amorphous powder
C21H24O9
ESI-Q-TOF MS: m/z 421.2333 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 6
13C NMR (MeOD-d4, 500 MHz): see Table 6
14
Compound 7
yellowish amorphous powder
C21H22O10
ESI-Q-TOF MS: m/z 435.2365 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 7
13C NMR (MeOD-d4, 500 MHz): see Table 7
Compound 8
dark purple amorphous powder
C8H8O5
ESI-Q-TOF MS: m/z 185.0443 [M+H]+
1H NMR (MeOD-d4, 500 MHz): see Table 8
13C NMR (MeOD-d4, 500 MHz): see Table 8
Compound 9
white powder
C30H50O
HRFAB MS: m/z 426.3862 [M+H]+
1H NMR (CDCl3-d1, 500 MHz): see Table 9
13C NMR (CDCl3-d1, 500 MHz): see Table 9
15
Compound 10
white powder
C31H48O3
LRFAB MS: m/z 469 [M+H]+
1H NMR (CDCl3-d1, 500 MHz): Table 10
13C NMR (CDCl3-d1, 500 MHz): Table 10
Compound 11
white powder
C30H50O
HRFAB MS: m/z 426.3855 [M+H]+
1H NMR (CDCl3-d1, 500 MHz): see Table 11
13C NMR (CDCl3-d1, 500 MHz): see Table 11
Compound 12
white powder
C30H50O
HRFAB MS: m/z 426.3856 [M+H]+
1H NMR (CDCl3-d1, 500 MHz): see Table 12
13C NMR (CDCl3-d1, 500 MHz): see Table 12
16
Compound 13
white powder
C30H50O0
HRFAB MS: m/z 426.3853 [M+H]+
1H NMR (CDCl3-d1, 500 MHz): see Table 13
13C NMR (CDCl3-d1, 500 MHz): see Table 13
Table 1. 1H NMR and
13C NMR spectral data of compound 1 and (-)-catechin
1H Position
1 (-)-catechin 13C Position 1 (-)-catechin
δH (J in Hz)
2 4.56, d(7.5) 4.55, d (7.4) 2 83.0 82.8
3 3.96, m 3.95, m 3 69.0 68.8
4eq 2.85, dd(16.3,
5.3)
2.86, dd (16.3,
5.3) 4 28.7 28.5
4ax 2.50, dd(16.2,
8.0)
2.50, dd (16.3,
8.9) 5 157.7 157.6
6 5.85, d(2.2) 5.84, d (2.3) 6 96.4 96.3
8 5.92, d(2.2) 5.90, d (2.3) 7 158.0 157.8
2’ 6.84, d(2.0) 6.82, d (1.8) 8 95.6 95.5
5’ 6.76, d(8.0)
6.68-6.77, m
9 157.1 156.9
6’ 6.72, dd(8.3,
2.0) 10 100.9 100.8
1’ 132.4 132.2
2’ 115.4 115.2
3’ 146.4 146.2
4’ 146.4 146.2
5’ 116.2 116.1
6’ 120.2 120.0
17
Table 2. 1H NMR and
13C NMR spectral data of compound 2 and (-)-catechin 3-O-α-L-
rhamnopyranoside
1H Position
2
(-)-catechin
3-O-α-L-
rhamnopyranoside 13
C Position 2
(-)-catechin
3-O-α-L-
rhamnopyranoside δH (J in Hz)
2 4.62, d(7.7) 4.58, d (7.4) 2 81.3 81.1
3 3.93, m 3.92, m 3 76.1 75.9
4eq 2.88, dd(16.2,
5.6)
2.82, dd (16.3,
5.3) 4 28.1 27.9
4ax 2.64, dd(16.1,
8.3)
2.57, dd (16.3,
7.9) 5 157.7 157.4
6 5.85, d(2.2) 5.83, d (2.3) 6 95.7 95.5
8 5.93, d(2.4) 5.91, d (2.3) 7 158.1 157.7
2’ 6.84, d(1.9) 6.82, d (1.8) 8 96.6 96.4
5’ 6.76, d(8.0)
6.68-6.72, m
9 157.0 156.9
6’ 6.72, dd(8.2,
1.9) 10 102.3 102.1
1’’ 4.29, d(1.2) 4.26, d (1.2) 1’ 132.1 131.9
2’’ 3.51, m
3.49-3.72
overlapping
2’ 115.2 115.0
3’’ 3.57, dd(9.6,
3.3) 3’ 146.4 146.2
4’’ Under the
MeOH 4’ 146.5 146.2
5’’ 3.68, m 5’ 116.3 116.1
6’’ 1.25, d 1.22, d (6.1) 6’ 120.0 119.8
1’’ 100.8 100.6
2’’ 72.2 72.0
3’’ 72.4 72.2
4’’ 74.1 73.9
5’’ 70.5 70.3
6’’ 18.1 17.9
18
Table 3. 1H NMR and
13C NMR spectral data of compound 3 and (+)-catechin 3-O-gallate
1H Position
3 (+)-catechin 3-
O-gallate 13C Position 3
(+)-catechin
3-O-gallate δH (J in Hz)
2 5.06, d(6.1) 5.05, d (5.9) 2 79.5 79.3
3 5.37, m 5.36, dt (5.9,
5.1) 3 71.3 71.1
4eq 2.82, dd(16.5,
5.1)
2.80, dd (16.6,
5.1) 4 24.5 24.3
4ax 2.71, dd(16.5,
6.0)
2.70, dd (16.6,
5.9) 5 156.6 156.5
6 5.94, d(2.2) 5.93, d (2.2) 6 96.6 96.4
8 5.96, d(2.2) 5.95, d (2.2) 7 157.7 157.6
2’ 6.84, s 6.82, s 8 95.8 95.6
5’, 6’ 6.72, s 6.71, s 9 158.2 158.1
2’’, 6’’ 6.96, s 6.95, s 10 99.8 99.6
1’ 131.6 131.5
2’ 114.6 114.4
3’ 146.3 146.2
4’ 146.4 146.3
5’ 116.4 116.2
6’ 119.4 119.2
1’’ 121.5 121.4
2’’, 6’’ 110.3 110.1
3’’, 5’’ 146.5 146.4
4’’ 140.0 139.8
COO 167.7 167.5
19
Table 4. 1H NMR and
13C NMR spectral data of compound 4 and (-)-epicatechin
1H Position
4 (-)-epicatechin 13C Position 4 (-)-epicatechin
δH (J in Hz)
2 4.82, s 4.88, s 2 80.0 79.8
3 4.17, m 4.16, m 3 67.7 67.4
4eq 2.73, dd(16.8,
2.8)
2.72, dd (16.8,
2.8) 4 29.4 29.2
4ax 2.86, dd(16.5,
4.9)
2.85, dd (16.7,
4.5) 5 157.9 157.6
6 5.91, d(2.2) 5.90, d (2.3) 6 96.0 95.8
8 5.94, d(2.3) 5.93, d (2.3) 7 158.2 158.0
2’ 6.97, d(1.8) 6.96, d (1.9) 8 96.5 96.3
5’ 6.75, d(8.2) 6.74, d (8.1) 9 157.5 157.3
6’ 6.79, dd(8.4,
1.7)
6.79, dd (8.1,
1.8) 10 100.2 100.8
1’ 132.5 132.3
2’ 115.5 115.3
3’ 146.1 145.9
4’ 146.0 145.7
5’ 116.0 115.8
6’ 119.5 119.3
20
Table 5. 1H NMR and
13C NMR spectral data of compound 5 and (-)-epicatechin 3-O-gallate
1H Position
5 (-)-epicatechin
3-O-gallate 13C Position 5
(-)-epicatechin
3-O-gallate δH (J in Hz)
2 5.03, s 4.97, s 2 78.8 78.6
3 5.52, m 5.47, m 3 70.1 69.9
4eq 2.99, dd(17.3,
4.7)
2.95, dd (17.4,
4.6) 4 27.0 26.8
4ax 2.85, dd(17.3,
2.1)
2.80, dd (17.5,
2.2) 5 157.4 157.2
6 Under the
H2O shift
5.91, d (2.9) 6 96.7 96.5
8 5.92, d (2.4) 7 158.0 157.8
2’ 6.93, d(1.9) 6.88, d (2.0) 8 96.0 95.8
5’ 6.69, d(8.3) 6.64, d (8.2) 9 158.0 157.8
6’ 6.81, dd(8.2,
1.7)
6.76, dd (8.4,
2.0) 10 99.6 99.3
2’’, 6’’ 6.95, s 6.9, s 1’ 131.6 131.4
2’ 115.3 115.0
3’ 146.1 145.9
4’ 146.1 145.9
5’ 116.2 115.9
6’ 119.5 119.3
1’’ 121.6 121.4
2’’, 6’’ 110.4 110.1
3’’, 5’’ 146.5 146.3
4’’ 139.9 139.8
COO 167.8 167.5
21
Table 6. 1H NMR and
13C NMR spectral data of compound 6 and (+)-afzelechin 3-O-α-L-
rhamnopyranoside
1H Position
6
(+)-afzelechin
3-O-α-L-
rhamnopyranoside 13
C Position 6
(+)-afzelechin
3-O-α-L-
rhamnopyranoside δH (J in Hz)
2 4.66, d(7.9) 4.74, d (7.9) 2 81.3 80.5
3 3.94, m 4.02, ddd
(8.03, 7.9, 5.7) 3 76.4 74.8
4eq 2.65, dd(16.3,
8.9)
2.67, d (16.3,
8.3) 4 28.4 27.9
4ax 2.91, dd(15.9,
5.7)
2.92, dd (16.3,
5.8) 5 157.1 156.9
6 5.85, d(2.2) 5.94, d (2.3) 6 95.6 95.5
8 5.94, d(2.1) 6.09, d (2.3) 7 157.7 157.4
2’, 6’ 7.23, d(8.5) 7.28, d (8.6) 8 96.6 96.4
3’, 5’ 6.79, d(8.8) 6.88, d (8.6) 9 158.1 158.0
1’’ 4.25, br. s 4.33, d (1.6) 10 100.8 100.4
2’’ 3.47, m 3.56, dd (3.3,
1.6) 1’ 131.4 131.1
3’’ 3.56, dd(9.4,
3.4)
3.65, dd (9.4,
3.4) 2’, 6’ 129.5 129.4
4’’ Under the
MeOH
3.42, dd (9.4,
9.4) 3’, 5’ 116.2 116.0
5’’ 3.69, m 3.73, dq (9.4,
6.2) 4’ 158.7 158.3
6’’ 1.25, d(6.3) 1.26, d (6.2) 1’’ 102.4 101.5
2’’ 72.1 71.5
3’’ 72.4 72.3
4’’ 74.1 73.7
5’’ 70.5 69.7
6’’ 18.1 17.9
22
Table 7. 1H NMR and
13C NMR spectral data of compound 7 and (-)-2,3-trans-dihydrokaempferol
3-O-α-L-rhamnopyranoside
1H Position
7
(-)-2,3-trans-dihydrokaempferol
3-O-α-L-
rhamnopyranoside
13C Position 7
(-)-2,3-trans-dihydrokaempferol 3-
O-α-L-
rhamnopyranoside δH (J in Hz)
2 5.14, d(11.4) 5.14, d (10.4) 2 84.0 83.9
3 4.62, d(11.2) 4.62, d (10.4) 3 78.8 78.7
6 5.92, d(2.2) 5.91, d (2.2) 4 196.2 196.0
8 5.89, d(1.9) 5.89, d (2.2) 5 165.7 165.4
2’ 7.36, d(8.8) 7.35, d (8.6) 6 97.6 97.4
3’ 6.84, d(8.8) 6.83, d (8.6) 7 164.3 164.1
5’ 6.84, d(8.8) 6.83, d (8.6) 8 96.4 96.3
6’ 7.36, d(8.8) 7.35, d (8.6) 9 168.9 168.7
1’’ 4.00, d(1.4) 4.0, d (1.6) 10 102.4 102.2
2’’ 3.50, dd(3.1,
1.6)
3.49, dd (3.2,
1.6) 1’ 128.8 128.6
3’’ 3.65, dd(9.7,
3.5)
3.64, dd (9.6,
3.2) 2’, 6’ 130.2 130.0
4’’ Under the
MeOH
3.30 (under the
MeOH) 3’, 5’ 116.6 116.5
5’’ 4.26, (m) 4.25, dq (9.7,
6.4) 4’ 159.6
6’’ 1.18, d(6.2) 1.18, d (6.4) 1’’ 102.6 102.5
2’’ 71.9 71.8
3’’ 72.3 72.2
4’’ 73.9 73.8
5’’ 70.7 70.5
6’’ 18.0 17.8
23
Table 8. 1H NMR and
13C NMR spectral data of compound 8 and Methyl gallate
1H Position
8 Methyl gallate 13C Position 8 Methyl gallate
δH (J in Hz)
2 7.04, s 7.02, s 1 121.6 121.4
6 7.04, s 7.02, s 2, 6 110.2 110.0
OCH3 3.81, s 3.80, s 3, 5 146.6 146.5
4 139.9 139.7
C=O 169.2 169.0
OCH3 52.4 52.3
24
Table 9. 1H NMR and
13C NMR spectral data of compound 9 and Lupeol
1H Position
9 Lupeol 13C Position 9 Lupeol
δH (J in Hz)
3 3.16, dd (11.5,
5.2)
3.18, dd (9.6,
6.2) 1 39.0 38.7
19 2.35, m 2.39, m 2 27.6 27.4
21 1.90, m 1.90, m 3 79.2 79.0
Me-23 0.77, s 0.78, s 4 39.1 38.9
Me-24 0.81, s 0.81, s 5 55.5 55.3
Me-25 0.92, s 0.92, s 6 18.6 18.3
Me-26 0.94,s 0.94, s 7 34.5 34.3
Me-27 1.01, s 1.02, s 8 41.1 40.8
Me-28 0.74, s 0.75, s 9 50.7 50.4
29a 4.54, m 4.56, m 10 37.4 37.2
29b 4.66, d (2.4) 4.69, d (2.4) 11 21.2 20.9
Me-30 1.66, s 1.67, s 12 25.4 25.2
13 38.3 38.1
14 43.1 42.9
15 27.7 27.5
16 35.8 35.6
17 43.2 43.0
18 48.6 48.3
19 48.2 48.0
20 151.2 151.0
21 30.1 29.9
22 40.2 40.0
23 28.2 28.0
24 15.6 15.4
25 16.3 16.1
26 16.2 16.0
27 14.8 14.6
28 18.2 18.0
29 109.5 109.3
30 19.5 19.3
25
Table 10. 1H NMR and
13C NMR spectral data of compound 10 and Methyl lup-20(29)-en-3-
one-28-oic acid
1H Position
10 Methyl lup-
20(29)-en-3-one-
28-oic acid 13
C Position 10
δH (J in Hz)
3 2.98, m 3.06-3.03, m 1 38.4
26.6
218.2
39.7
56.6
19.7
33.7
42.5
55.0
36.9
21.4
25.6
37.0
47.0
29.7
34.2
47.4
49.9
49.4
150.5
32.1
40.6
30.6
15.8
19.4
16.0
14.7
176.7
109.7
21.1
51.3
19 2.47, m 2.48, m 2
Me-23 0.90, s 0.91, s 3
Me-24 0.93, s 0.95, s 4
Me-25 0.95, s 0.97, s 5
Me-26 0.99,s 1.01, s 6
Me-27 1.04, s 1.06, s 7
29a 4.58, m 4.60, s 8
29b 4.71, m 4.73, s 9
Me-30 1.66, s 1.68, s 10
Me-31 3.65, s 3.67, s 11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
26
Table 11. 1H NMR and
13C NMR spectral data of compound 11 and β-amyrin
1H Position
11 β-amyrin 13C Position 11 β-amyrin
δH (J in Hz)
3 3.20, dd (10.2,
4.5)
3.21, dd (11.0,
5.0) 1 38.8 38.7
12 5.16, t (3.6) 5.18, t (3.5) 2 27.2 26.9
Me-23 0.98, s 1.00, s 3 79.3 79.0
Me-24 0.77, s 0.79, s 4 39.0 38.8
Me-25 0.92, s 0.94, s 5 55.4 55.2
Me-26 0.95, s 0.97, s 6 18.6 18.4
Me-27 1.11, s 1.13, s 7 32.9 31.0
Me-28 0.81, s 0.83, s 8 41.9 41.7
Me-29, 30 0.85, s 0.87, s 9 47.9 47.6
10 37.2 36.9
11 23.9 23.7
12 121.9 121.7
13 145.4 145.2
14 40.0 41.7
15 28.3 28.1
16 26.4 26.1
17 32.9 32.6
18 47.5 47.2
19 47.1 46.8
20 32.7 31.0
21 35.0 34.7
22 37.4 37.1
23 28.6 28.4
24 15.7 15.4
25 15.8 15.5
26 17.0 16.8
27 26.2 25.9
28 27.5 27.2
29 33.6 33.3
30 23.8 23.5
27
Table 12. 1H NMR and
13C NMR spectral data of compound 12 and urs-12-ene-3β-ol
1H Position
12 Urs-12-ene-3β-ol 13
C Position 12 Urs-12-ene-3β-ol δH (J in Hz)
3 3.21, dd(10.4,
5.1)
3.23, dd(9.9,
5.1) 1 39.0 38.7
12 5.10, t(3.6) 5.13, dd(3.6,
3.6) 2 27.5 27.2
Me-23 0.98, s 1.00, s 3 79.3 78.3
Me-24 0.76-0.78
overlapped 0.79, s 4 39.0 38.7
Me-25 0.93, s 0.96, s 5 55.4 55.2
Me-26 0.99, s 1.01, s 6 18.6 18.3
Me-27 1.05, s 1.07, s 7 33.2 32.9
Me-28 0.76-0.78
overlapped 0.80, s 8 40.2 40.0
Me-29 0.76-0.78
overlapped 0.79, d(5.6) 9 47.9 47.7
Me-30 0.89, s 0.92, d(5.9) 10 37.1 36.9
11 23.6 23.3
12 124.6 124.3
13 139.8 139.3
14 42.3 42.0
15 26.8 26.6
16 28.4 28.1
17 34.0 33.7
18 59.3 58.9
19 39.9 39.6
20 39.8 39.6
21 31.5 31.2
22 41.7 41.5
23 28.3 28.1
24 15.9 15.6
25 15.8 15.6
26 17.1 16.8
27 23.5 23.3
28 29.0 28.7
29 17.7 17.4
30 21.6 21.3
28
Table 13. 1H NMR and
13C NMR spectral data of compound 13 and 3β-Hydroxy-D:B-friedo-
olean-5-ene
1H Position
13 3β-Hydroxy-D:B-
friedo-olean-5-ene 13C Position 13
3β-Hydroxy-D:B-
friedo-olean-5-ene δH (J in Hz)
3 3.5, m 3.47, dd(3.1,
2.6) 1 18.4
18.2
6 5.66, m 5.63, m 2 28.0 27.8
Me-23 1.08, s 1.04, s 3 76.6 76.4
Me-24 1.17, s 1.14, s 4 41.1 40.8
Me-25 0.88, s 0.85, s 5 141.8 141.7
Me-26 1.13, s 1.09, s 6 122.3 122.1
Me-27 1.04, s 1.01, s 7 23.9 23.7
Me-28 1.19, s 1.16, s 8 47.7 47.4
Me-29 1.02, s 0.99, s 9 35.1 34.9
Me-30 0.98, s 0.95, s 10 49.9 49.7
11 34.8 34.6
12 30.6 30.4
13 38.1 37.9
14 39.5 39.3
15 32.3 32.1
16 36.2 36.1
17 30.3 30.0
18 43.3 43.1
19 35.3 35.1
20 28.5 28.3
21 33.3 33.2
22 39.1 39.0
23 29.2 29.0
24 25.7 25.5
25 16.4 16.2
26 18.6 19.6
27 19.8 18.4
28 32.2 32.1
29 32.6 32.4
30 34.7 34.5
29
2. Evaluation of Inhibitory effect on NO production in BV2
microglia cell line
2.1. BV2 microglia cell culture and reagents
DMEM, HBSS, sodium bicarbonate, penicillin/streptomycin, and trypsin were
purchased from Sigma (St. Louis, USA), and fetal calf serum was purchased from Hyclone
(Utah, USA). Reagents (sulfanilamide, N-1-naphtylethylenediammine dihydrochloride and
phosphoric acid) required for Griess assay were purchased from Sigma (St. Louis, USA).
2.2. BV2 microglia cell culture
BV2 microglia cell line was provided from Kyung Hee University and used 10% FBS,
100 IU/mL penicillin and DMEM containing 100g/mL streptomycin as a culture media and
cultivated in 37℃ incubator providing gas mixture of air (95%) and CO2 (5%) consistently
(Kim et al., 2013).
2.3. NO production induced by LPS
BV2 microglia cells were transplanted in a culture dish, substituted the culture media to
phenol red free/FBS free DMEM after 24 hours, and treated the samples. After one hour,
100ng/mL of LPS were treated to induce the NO production and used Griess assay and MTT
assy after 24 hours, to measure the inhibitory effect of NO production and cell viability.
30
2.4.Griess assay
100L of BV2 microglia cell culture media were treated with 100L of Griess reagent
(1% sulfanilamide, 0.1% naphtylethylenediamine dihydrochloride, 2% phosphoric acid) in a 96
well plate and exposed to room temperature for 15 minutes. NO production was measured by
measuring absorbance of sodium nitrite at 540nm (Kim et al., 2013).
2.5. Statistical analysis
All data were expressed as means ± S.D. The evaluation of statistical significance was
determined by ANOVA test using computerized statistical package, with p < 0.05 *, p < 0.01
**
and p < 0.001 ***
considered to be statistically significant.
31
III. Result and discussion
1. Structure elucidation of isolated compounds from D.
burmanica
1.1. Compounds 1 and 2
Compound 1 was gained as light pink-orange amorphous powder and the molecular
formula, C15H14O6, was identified using the positive mode HRESI-Q-TOFMS [m/z 291.0866
[M+H]+
(calcd. for 291.0869)]. The signals for 1,3,4-substituted aromatic protons [δH 6.84 (1H,
d, J = 2.0, H-2'), 6.76 (1H, d, J = 8.0, H-5'), 6.72 (1H, dd, J = 8.3, 2.0, H-6')], two meta coupling
protons [δH 5.92 (1H, d, J = 2.2, H-8), 5.85 (1H, d, J = 2.2, H-6)] and equatorial H-4 and axial
H-4 protons [δH 2.85 (1H, dd, J = 16.3, 5.3, H-4eq), 2.50 (1H, dd, J = 16.2, 8.0, H-4ax)] were
shown in the 1H NMR spectrum. Thus compound 1 was identified as (-)-catechin comparing
the spectral data above to literature values (H.L. Zhang et al., 1998).
Compound 2 was obtained as dark brown amorphous powder and the ion peak at m/z
437.1454 [M+H]+ (calcd. for 437.1448) of the positive mode HRESI-Q-TOFMS established the
molecular formula, C21H24O10. Similar patterns were found in both 1H and
13C NMR spectra
with the spectra of compound 1 except for the region of hexose moiety. The coupling constant
of anomeric proton [δH 4.29 (1H, d, J = 1.2, H-1'')], the chemical shift of methyl group [δH 1.25
(3H, d, J = 6.2, H-6'')] and the chemical shift of anomeric carbon [δc 100.8 (C-1'')] suggested α-
L-rhamnopyranoside. By comparing these spectral data with literature values, the structure of
compound 2 was identified as (-)-catechin 3-O-α-L-rhamnopyranoside (M.S. Zheng et al., 2011).
32
Figure 1.
1H and
13C NMR spectra of compound 1
33
Figure 2. 1H and
13C NMR spectra of compound 2
34
1.2. Compounds 3 and 4
Compound 3 was isolated as dark purple amorphous powder and its molecular
formula was figured out as C22H18O10 by the positive mode HRESI-Q-TOFMS [m/z 443.2216
[M+H]+ (calcd. for 443.0979)] and the
13C
NMR spectrum. The signals for 1,3,4-substituted
aromatic protons [δH 6.84 (1H, s, H-2'), 6.72 (2H, s, H-5', 6’)] and two meta coupling protons
[δH 5.96 (1H, d, J = 2.2, H-8), 5.94 (1H, d, J = 2.2, H-6)] and equatorial H-4 and axial H-4
protons [δH 2.82 (1H, dd, J = 16.5, 5.1, H-4eq), 2.71 (1H, dd, J = 16.5, 6.0, H-4ax)] existed in
the 1H NMR spectrum. Also the region of sugar moiety existed and the moiety was identified
as gallate by the 1H NMR spectrum showing the signal of symmetrical proton at H-2” and H-6”
[δH 6.96 (2H, s, H-2”,6”)] and the 1C NMR spectrum showing COO group [δC 167.7, COO)].
From these observed spectral data and by comparing to literature values, compound 3 was
identified as (+)-catechin 3-O-gallate (A. Saito, 2004).
Compound 4 was obtained as brown amorphous powder and its molecular formula
turned out to be C15H14O6 using the positive mode HRESI-Q-TOFMS [m/z 291.1953 [M+H]+
calcd. for 291.0869)] and the spectral data. The 1H and
13C NMR spectral data of compound 4
showed similar patterns with the spectral data of compound 3 except for the 1,3,4-substitued
aromatic proton signal pattern [δH 6.97 (1H, d, J = 1.8, H-2'), 6.75 (1H, d, J = 8.2, H-5'), 6.79
(1H, dd, J = 8.4, 1.7, H-6')]. From the comparison of above data with literature values,
compound 4 was identified to be (-)-epicatechin (Santos-Buelga, 2012).
35
Figure 3. 1H and
13C NMR spectra of compound 3
36
Figure 4.
1H and
13C NMR spectra of compound 4
37
1.3. Compounds 5 and 6
Molecular formula of compound 5 was determined as C22H18O10 by ion peak at m/z
443.2216 [M+H]+ (calcd. for 443.0979) of the positive mode HRESI-Q-TOFMS and dark
purple amorphous powder was obtained. The 1H and
13C NMR spectrum displayed similar
patterns with compound 4 except for the existence of a gallate group signal [δH 6.95 (2H, s, H-
2”,6”)/δc 167.8 (COO)]. From these spectral data with comparison of literature values,
compound 5 was assigned as (-)-epicatechin 3-O-gallate (Tommasi, 2003).
Compound 6 was isolated as brown amorphous powder and the molecular formula was
figured out to be C21H24O9 from the ion peak at m/z 421.2333 [M+H]+ (calcd. for 421.1499) of
the positive mode HRESI-Q-TOFMS and the NMR spectral data. The 1H and
13C NMR
spectrum displayed similar patterns with the spectral data of compound 2 except for the signals
of 1,4-substituted aromatic protons [δH 7.23 (2H, d, J = 8.5, H-2', 6'), 6.79 (2H, d, J = 8.8, H-3',
5')] Using these spectral data and by comparing with literature values, compound 6 was
assigned as (+)-afzelechin 3-O-α-L-rhamnopyranoside (Drewes et al., 1992).
38
Figure 5. 1H and
13C NMR spectra of compound 5
39
Figure 6. 1H and
13C NMR spectra of compound 6
40
1.4. Compounds 7 and 8
Yellowish amorphous powder was obtained for compound 7 and the molecular formula
was determined as C21H22O10 by ion peak at m/z 435.2365 [M+H]+ (calcd. for 435.1292) of the
positive mode HRESI-Q-TOFMS. The 1H and
13C NMR spectrum showed similar patterns
with those of compound 6 except for the existence of C=O [δc 196.2 (C-4)] at C-4 instead of the
signals of equatorial and axial H-4 signals. From these spectral data, compound 7 was
assigned as (-)-2,3-trans-dihydrokaempferol 3-O-α-L-rhamnopyranoside with comparison to the
literature values (M. Fujiwara et al., 2011).
Compound 8 was dark purple amorphous powder and its molecular formula was
established as C8H8O5 by the negative mode HRESI-Q-TOFMS [m/z 185.0451 [M+H]+ (calcd.
for 291.0869)] and the NMR spectra. The
1H NMR spectrum showed the signals for
symmetrical protons at H-2 and H-6 [δH 7.04 (2H, s, H-2, 6)], and -OCH3 signal [δH 3.81 (3H, s,
-OCH3)]. The 13
C NMR spectrum also showed the signal of –OCH3 [δc 52.4 (-OCH3)] and
C=O [δc 169.2 (C=O)]. Based on above data and with comparison to the literature values,
compound 8 was identified as methyl gallate (M.T. Ekaprasada et al., 2009).
41
Figure7. 1H and
13C NMR spectra of compound 7
42
Figure8. 1H and
13C NMR spectra of compound 8
43
1.5. Compound 9
Compound 9 was obtained as white powder, and displayed ion peaks at m/z 426.3862
[M+H]+ (calcd. for 427.3941)on the positive mode HRFABMS showing that the molecular
formula was C30H50O. The 13
C NMR spectrum showed thirty carbons, one hydroxyl group [δc
79.2 (C-3)], and one double bond between two carbons [δc 151.2 (C-20) and 109.5 (C-29)].
Thus, the structure of compound 9 was defined as lupeol by comparison with literature values of
both 1H NMR and
13C NMR spectrum (D.A. da Silva et al., 2012).
Figure9. HSQC spectrum of compound 8
44
Figure10. 1H and
13C NMR spectra of compound 9
45
1.6. Compound 10
Compound 10 was white powder, showed ion peaks at m/z 469 [M+H]+ (calcd. for 470)
on the positive mode LRFABMS and the molecular formula was C31H48O3. The 13
C NMR
spectrum counted out thirty one carbons, one ketone group [δc 218.4 (C-3)], one double bond
between two carbons [δc 150.7 (C-20) and 109.9 (C-29)] and one –COOCH3 group [δc 176.9 (C-
28)]. Through above spectral data and comparison with the literature values for both 1H NMR
and 13
C NMR spectrum, the structure of compound 10 was defined as methyl lup-20(29)-en-3-
one-28-oic acid (Qiu, 2012).
46
Figure 11. 1H and
13C NMR spectra of compound 10
Figure 12. HSQC spectrum of compound 10
47
1.7. Compound 11
Compound 11 was isolated as white powder and the molecular formula was determined
as C30H50O by ion peak at m/z 426.3855 [M+H]+ (calcd. for 427.3941) of the positive mode
HRFABMS and the NMR spectral data. The 13
C NMR spectrum showed that there are thirty
carbons, one hydroxyl group [δc 79.3 (C-3)], and one double bond between two carbons [δc
145.3 (C-13) and 121.9 (C-12)]. Through above spectral data and comparison with the
literature values for both 1H NMR and
13C NMR spectrum, the structure of compound 11 was
defined as β-amyrin (Woo, 2012).
48
Figure 13. 1H and
13C NMR spectra of compound 11
49
1.8. Compound 12
Compound 12 was isolated as white powder and the molecular formula was identified
as C30H50O by ion peak at m/z 426.3856 [M+H]+ (calcd. for 427.3941) of the positive mode
HRFABMS and the NMR spectral data. The 13
C NMR spectrum showed similar patterns with
those of compound 11 except for the chemical shifts of the double bonded carbons, C-12 and C-
13 [δc 139.8 (C-13) and 124.6 (C-12)]. With this unique chemical shifts at C-12 and C-13 and
through comparison with the literature values for both 1H NMR and
13C NMR spectrum, the
structure of compound 12 was defined as urs-12-ene-3β-ol (Y. Wang et al., 2012).
50
1.9. Compound 13
Compound 13 was obtained as white powder and the molecular formula was figured
out to be C30H50O by ion peak at m/z 426.3853 [M+H]+ (calcd. for 427.3941) of the positive
mode HRFABMS and NMR spectral data. Similar patterns of 13
C NMR spectrum with those of
compound 11 were exposed and the difference was the location of the double bonded carbons,
C-5 and C-6 [δc 141.8 (C-5) and 122.3 (C-6)] and the chemical shift value of hydroxyl group at
C-3[δc 76.6 (C-3)]. With these specific chemical shifts and with comparison to the literature
values of both 1H NMR and
13C NMR spectrum, the structure of compound 13 was defined as
3β-Hydroxy-D:B-friedo-olean-5-ene (A.G. Gonzalez et al., 1987).
Figure 14. 1H and
13C NMR spectra of compound 12
51
Figure15.
1H and
13C NMR spectra of compound 13
52
R1 R1
1 OH 4 OH
2 O-Rha 5 O-gallate
R1 R2 R3
3 OH H2 OH
6 O-Rha H2 H
7 O-Rha =O H
Figure15. The structures of compounds isolated from D.burmanica barks and leaves
53
2. Inhibitory effect against NO production by the compounds
isolated from D. burmanica
Compounds isolated from D. burmanica were screened about their inhibitory effect
against NO production induced by treating LPS on BV2 microglia cell line. When all
compounds were injected to the cell by 10 and 100 M, compounds 1, 3, 4, and 8 significantly
inhibited the NO production. From these results, catechins and methyl gallate revealed to be
effective in inhibiting NO production. Also all four compounds showed no toxicity in MTT
assay when injected as either 10 or 100 M. Furthermore, catechins showed lower inhibitory
effect when sugar moiety was substituted at C-3. Compound 1, (-)-catechin, showed relative
NO production of 73.90% at 10μM and 30.54% at 100μM compared to compound 2, (-)-
catechin 3-O-rhamnopyranoside, showing 112.94% at 10μM and 97.30% at 100μM.
Compound 4, (-)-epicatechin, showed 72.35% at 10μM and 15.62% at 100μM, while
compound 5, (-)-epicatechin 3-O-gallate showed 116.62% at 10μM and 54.44% at 100μM.
Thus, catechins without sugar moiety substituted at C-3 showed more effective inhibition of NO
production.
54
Figure16. The effect of the isolated compounds from D. burmanica on LPS-induced NO production in BV2 microglia cells
BV2 cells were washed with DMEM and incubated with compounds for 1 hr. Then the cultures were stimulated by 100 ng/ml of LPS for 24hrs. After the
incubation, NO production (NP) was measured by the Griess assay and sodium nitrite was used as a standard. Respectively, NP of control and LPS-treated
cultures were 2.78 + 0.4 and 40.5 + 2.7μM. Relative production (%) was calculated by (NP of sample treated – NP of control) / (NP of LPS-treated – NP of
control) x 100. Mean value is significantly different (*p<0.05, **p<0.01, ***p<0.001) compared with the LPS-treated.
55
0
20
40
60
80
100
120
140
cell
via
bil
ity
(%
of
con
tro
l)
MTT assay
10ug/ml
100ug/ml
Figure17. The cell viability of the compounds isolated from D. burmanica on LPS-induced NO production in BV2 microglia cells
56
IV. Conclusions
Eleven compounds including seven flavonoids (1-7), a methyl gallate (8), and three
triterpenes (9, 10, 13) were isolated from EtOAC and MC fraction of the barks and two more
triterpenes (11, 12) were isolated from MC fraction of the leaves. Isolated compounds were
identified as (-)-catechin (1), (-)-catechin 3-O-α-L-rhamnopyranoside (2), (+)-catechin 3-O-
gallate (3), (-)-epicatechin (4), (-)-epicatechin 3-O-gallate (5), (+)-afzelechin 3-O-α-L-
rhamnopyranoside (6), (-)-2,3-trans-dihydrokaempferol 3-O-α-L-rhamnopyranoside (7), methyl gallate
(8), lupeol (9), methyl lup-20(29)-en-3-one-28-oic acid (10), β-amyrin (11), urs-12-ene-3β-ol (12),
3β-Hydroxy-D:B-friedo-olean-5-ene (13) using various spectral data. Among these isolated
compounds, compounds 1, 3, 4, and 8 (flavonoids and methyl gallate) showed significant
inhibitory effect against NO production. Furthermore, in the study of flavonoid structure and
activity relationship, glycosylation at C-3 showed less inhibitory effect against NO production.
57
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Chen, X.N., Fan, J.F., Yue, X., Wu, X.R., Li, L.T., 2008. Radical scavenging activity and phenolic
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C. Santos-Buelga, 2012. Characterization of sulfated quercetin and epicatechin metabolites. J. Agric.
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G.Y. Kim, C.H. Kang, R.G.P.T.Jayasooriya, Y.H. Choi, S.K. Moon, W.J. Kim, 2013. β-ionone attenuates
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H.L. Zhang, A. Nagatsu, H. Okuyama, H. Mizukami and J. Sakakibara, 1998. Sesquiterpene glycosides
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M.S. Zheng, Y. Li, Y.K. Lee, H.W. Chang and J.K. Son, 2011. Protective constituents against sepsis in
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M.T. Ekaprasada, H. Nurdin, S. Ibrahim, and D., 2009. Antioxidant activity of methyl gallate isolated
from the leaves of Toona sureni. Indo. J. Chem., 9(3): 457-460.
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S.E. Drewes, C.W. Taylor and A.B. Cunningham, 1992. (+)-Afzelechin 3-rhamnoside from Cassipourea
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Tangmouo, J.G., Meli, A.L., Komguem, J., Kuete, V., Ngounou, F.N., Lontsi, D., Beng, V.p., Choudhary,
M.I., Sondengam, B.L., 2006. Crassiflorone, a new naphthoquinone from Diospyros crassiflora (Hien).
Tetrahedron Lett. 47: 3067-3070.
Waterman, P.G., Mbi, C.N., 1979. The sterols and dimeric naphthoquinones of the barks of three West
African Diospyros species. Planta Med. 37: 241-246.
59
Willis, J.C., (rev. K.A. Airy Shaw), 1966. A dictionary of flowering plants and ferns. seventh ed.
Cambridge University Press, London, p.360.
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60
국 문 초 록
Diospyros burmanica 는 Ebenaceae (감나무과)에 속하는 상록활엽수로서 미얀마
Mandalay 지역에 자생하는 식물이다. 미얀마 현지에서는 민간의약학 적으로 이질,
설사, 당뇨의 치료에 사용하고 있으며 Burmese ebony 라 하여 목재의 용도로
이용되고 있다. Diospyros 속 식물은 전 세계적으로 350 종 이상이 알려져 있는데 그
중 D. kaki, D. lotus, D. melanoxylon 등이 식용할 수 있는 과실로 인해 가장 잘 알려져
있고 그 외 다수 Diospyros 속 식물은 심재의 아름다운 색으로 인하여 목재로서
건축자재의 용도로 이용되고 있다. 하지만 D. burmanica 의 경우 현지 토착민의 사용
예가 있음에도 전 세계적으로 일본 연구진에 의한 리슈만편모촌충증(Leishmaniasis)
활성 이외의 과학적인 연구가 되어 있지 않으므로 본 연구는 D. burmanica 의
수피에서 식물화학 성분을 분리, 규명하고자 하였다.
건조된 D. burmanica 의 수피 1.1 kg 을 100% 메탄올로 추출하여 얻은 추출물을
증류수에 현탁한 후 메틸렌클로라이드, 에틸아세테이트, 부탄올, 수용액 층으로
각각 분획하였다. 이후 이 분획을 silica gel column chromatography, counter current
chromatography, HP column chromatography, ODS-A gel column chromatography, MPLC,
HPLC 등을 이용하여 8 종의 flavonoid 와 5 종의 triterpene 을 분리하였다.
분리한 화합물들은 UV, Q-TOF LC/MS, FAB-LRMS, 1H-NMR,
13C-NMR,
1H-
1H COSY,
HSQC, HMBC spectrum 등의 분광학적 데이터를 종합하여 보고된 자료와 비교한 후
그 구조를 각각 (-)-catechin 3-O-α-L-rhamnopyranoside (1), (-)-catechin(2), methyl gallate
(3), (-)-2,3-trans-dihydrokaempferol 3-O-rhamnopyranoside (4), (-)-epicatechin 3-O-gallate (5),
(+)-catechin 3-O-gallate (6), (-)-epicatechin (7), (+)-afzelechin 3-O-α-L-rhamnopyranoside(8),
lupeol(9), methyl lup-20(29)-en-3-one-28-oic acid(10), 3β-hydroxy-D:B-friedo-olean-5-ene (11),
β-amyrin (12), urs-12-ene-3β-ol(13) 으로 동정하였다. 이 모든 화합물들은 Diospyros
burmanica 에서 처음 분리 보고되는 물질이다.
61
13 종의 분리한 화합물에 대해 lipopolysaccharide (LPS)를 처리하여 NO 생성을
유도한 BV2 microglia 세포주를 검색계로 하여 NO 생성 억제율을 측정하였다. 이 중
화합물 1, 3, 4, 8 (catechins and methyl gallate)이 유의성 있는 저해 활성을 나타내었다.
또한 catechin 계열 화합물의 경우, 3 번 탄소에 당의 결합 여부가 활성에 영향을
미치는 것으로 나타났다.
주요어 : Diospyros burmanica, catechin, epicatechin, afzelechin, dihydrokaempferol,
triterpene, lupeol, ursenol, amyrin, friedo-oleanene
학번 : 2011-21760