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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


Figure 3. 1H and


Figure 4. 1H and


Figure 5. 1H and


Figure 6. 1H and


Figure 7. 1H and


Figure 8. 1H and


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


Table 3. 1H NMR and


Table 4. 1H NMR and


Table 5. 1H NMR and


Table 6. 1H NMR and


Table 7. 1H NMR and


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





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]+



Compound 3

dark purple amorphous powder

C22H18O10

ESI-Q-TOF MS: m/z 443.2216 [M+H]+



13

Compound 4

brown amorphous powder

C15H14O6

ESI-Q-TOF MS: m/z 291.1953 [M+H]+



Compound 5


C22H18O10

ESI-Q-TOF MS: m/z 443.2216 [M+H]+



Compound 6

brown amorphous powder

C21H24O9

ESI-Q-TOF MS: m/z 421.2333 [M+H]+



14

Compound 7

yellowish amorphous powder

C21H22O10

ESI-Q-TOF MS: m/z 435.2365 [M+H]+



Compound 8


C8H8O5

ESI-Q-TOF MS: m/z 185.0443 [M+H]+



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]+



Compound 12

white powder

C30H50O

HRFAB MS: m/z 426.3856 [M+H]+



16

Compound 13

white powder

C30H50O0

HRFAB MS: m/z 426.3853 [M+H]+



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-


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-


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


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


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


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


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


34


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


36

Figure 4.

1H and


37


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


39

Figure 6. 1H and


40


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


42

Figure8. 1H and


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


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


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


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


51

Figure15.

1H and


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

V. References

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26(10): 2785-2788.

Akiko Saito, 2004. Stereoselevtive synthesis of procyanidin B3-3-O-gallate and 3,3”-di-O-gallate, and

their abilities as antioxidant and DNA polymerase inhibitor. Tetrahedron, 60: 12043-12049.

Bhakuni, D.S., Satish, S., Shukla, Y.N., Tandon, J.S., 1971. Ebenaceae, chemical constituents of

Diospyros buxifolia, D.tomentosa, D. ferrea, D. lotus, Rhus parviflora, Polygonus recumbens, Balintes

aegyptiacea and Pyrus pachia. Phytochemistry, 10: 2829-2831.

Chen, X.N., Fan, J.F., Yue, X., Wu, X.R., Li, L.T., 2008. Radical scavenging activity and phenolic

compounds in Persimmon (Diospyros kaki L. cv. Mopan). J. Food Sci. 73: C24-C28.

C.C. da Silva and D.A. da Silva, 2012. Antiproliferative activity of Luehea candicans Mart. et Zucc.

(Tiliaceae). Natural Product Research, 26(4): 364-369.

C. Santos-Buelga, 2012. Characterization of sulfated quercetin and epicatechin metabolites. J. Agric.

Food Chem., 60: 3592-3598.

E.R. Woo, Q. Jin, H.G. Jin, and A. R. Kim, 2012. A new megastigmane palmitate and a new oleanane

triterpenoid from Aster yomena Makino. Helvetica Chimica Acta, 95.

G.Y. Kim, C.H. Kang, R.G.P.T.Jayasooriya, Y.H. Choi, S.K. Moon, W.J. Kim, 2013. β-ionone attenuates

LPS-induced pro-inflammatory mediators such as NO, PGE2 and TNF-αin BV2 microglial cells via

suppression of the NF-kB and MAPK pathway. Toxicology in Vitro 27: 782-787.

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H.L. Zhang, A. Nagatsu, H. Okuyama, H. Mizukami and J. Sakakibara, 1998. Sesquiterpene glycosides

from cotton oil cake. Phytochemistry, 48: 665-668.

K. Mori-Yasumoto, 2012. Leishmanicidal activities and cytotoxicities of bisnaphthoquinone analogues

and naphthol derivatives from Burman Diospyros burmanica. Bioorg. Med. Chem. 20: 5215-5219.

M. Fujiwara, N. Yagi and M. Miyazawa, 2011. Tyrosinase inhibitory constituents from the bark of

Peltophorum dasyrachis (yellow batai). Natural Product Research, 25(16): 1540-1548.

M. Liu, W.W. Qiu, and J. Tang, 2012. Synthesis and biological evaluation of heterocyclic ring-fused

betulinic acid derivatives as novel inhibitors of osteoclast differentiation and bone resorption. J. Med.

Chem., 55: 3122-3134.

M.S. Zheng, Y. Li, Y.K. Lee, H.W. Chang and J.K. Son, 2011. Protective constituents against sepsis in

mice from the root barks of Ulmus davidiana var. japonica. Arch Pharm Res, 34: 1443-1450.

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.

N. de Tommasi, 2003. Chemical composition and antioxidant activity of phenolic compounds from wild

and cultivated Sclerocarya birrea (Anacardiaceae) leaves. J. Agric. Food Chem., 52: 6689-6695.

S.E. Drewes, C.W. Taylor and A.B. Cunningham, 1992. (+)-Afzelechin 3-rhamnoside from Cassipourea

gerrardii. Phytochemistry, 31(3): 1073-1075.

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.

X. He, Y. Wang, L. Xiang, M. Chen, and Z. Zhang, 2012. Substrate specificity for the 2α-hydroxylation

of ursolic acid by Alternaria alternata and the antitumor activities of those metabolites. J. Molecular

Catalysis B: Enzymatic, 83: 51-56.

Zhong, S.M., Walterman, P.G., Jeffreys, J.A.D., 1984. Naphthoquinones and triterpenes from African

Diospyros species. Phytochemistry 23: 1067-1072.

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

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