Botanical Studies (2006) 47: 37-43.

*

Corresponding author: E-mail: wchou@tmu.edu.tw; Fax:

886(2) 2378-0134.

Structure-activity relationships of five myricetin

galloylglycosides from leaves of

Acacia confusa

Tzong-Huei LEE

1

, Der-Zen LIU

2

, Feng-Lin HSU

1

, Wen-Chung WU

1

, and Wen-Chi HoU

1*

1

Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan

2

Graduate Institute of Biomedical Materials, Taipei Medical University, Taipei, Taiwan

(Received June 30, 2005; Accepted September 7, 2005)

ABSTRACT.

Five structure-related myricetin galloylglycosides isolated from leaves of Acacia confusa

were previously reported (Lee et al., 2000, J. Nat. Prod., 63, 710-712). However, the structure-activity

relationships were not reported. In this research, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

activity, and inhibitory activities against semicarbazide-sensitive amine oxidase (SSAo) and angiotensin

converting enzyme (ACE) were compared among five compounds, namely, myricetin 3-O-(3"-O-galloyl)-α-

rhamnopyranoside 7-methyl ether (compound 1, 630 Da), myricetin 3-O-(2"-O-galloyl)-α-rhamnopyranoside

7-methyl ether (compound 2, 630 Da), myricetin 3-O-(2"-O-galloyl)-α-rhamnopyranoside (compound 3,

616 Da), myricetin 3-O-(3"-O-galloyl)-α-rhamnopyranoside (compound 4, 616 Da), myricetin 3-O-(2", 3"

-di-O-galloyl)-α-rhamnopyranoside (compound 5, 768 Da). For DPPH scavenging activity, the IC

50

for five

compounds was 591, 1522, 3210, 1389, and 867 μM, respectively. For SSAo inhibitory activity, the IC

50

for

five compounds was 36.16, 93.20, 119.50, 88.20, and 39.35 μM, respectively. The IC

5 0

of positive control

of semicarbazide was 34.21 μM. The five compounds have the same orders of compound 1> compound 5>

compound 4> compound 2> compound 3 for DPPH scavenging activity and SSAO inhibitiony. It was found

that gallic acid in the R

3

position was the key role for both biological activities. For ACE inhibitory activity,

compound 1, compound 2, and compound 5 showed dose-dependent inhibitory modes and the IC

50

was 60.32,

151.90, and 19.82 μM, respectively.

Keywords: Acacia confusa; Angiotensin converting enzyme (ACE); 1, 1-diphenyl-2-picrylhydrazyl (DPPH);

Myricetin galloylglycoside; Semicarbazide-sensitive amine oxidase (SSAo); Structure-activity relationships

(SAR).

INTRODUCTION

Free radical-mediated reactions are involved in

degenerative or pathological processes such as aging

(Harman, 1995), cancer, coronary heart disease, and

Alzheimer ’s disease (Ames, 1983; Smith et al., 1996; Diaz

et al., 1997). Meanwhile, there are many epidemiological

results revealing an association between a diet rich in

fresh fruit and vegetable and a decrease in the risk of

cardiovascular diseases and certain forms of cancer in

humans (Salah et al., 1995). Several reports concern

natural compounds in fruits and vegetables for their

antioxidant activities, such as phenolic compounds (Rice-

Evans et al., 1997), anthocyanin (Espin et al., 2000),

echinacoside in Echinaceae root (Hu and Kitts, 2000),

methanolic and hot-water extracts of Liriope spicata L.

(Hou et al., 2004), the storage proteins of sweet potato

root (Hou et al., 2001a), yam tuber (Hou et al., 2001b),

potato tuber (Liu et al., 2003b), and yam mucilages (Hou

et al., 2002; Lin et al., 2005).

The semicarbazide-sensitive amine oxidase (SSAo,

EC 1.4.3.6), which contains a cofactor of one or

more topaquinone, is a common name for a group of

heterogenous enzymes widely distributed in nature, in

plants, microorganisms, and the organs of mammals

(vasculature, dental pulp, eye and plasma) (Boomsma

et al., 2000). SSAo converts primary amines into the

corresponding aldehydes, generating hydrogen peroxide

and ammonia. It was found that the endogenous

compounds aminoacetone and methylamine are good

substrates for most SSAos (Precious et al., 1988). In

recent research, plasma SSAo was found raised in

diabetes mellitus and heart failure and implicated in

roles in atherosclerosis, endothelial damage, and glucose

transport into adipocytes (Yu and Zuo, 1993, 1996;

Boomsma et al., 1997, 2003).

Several classes of pharmacological agents have been

used in the treatment of hypertension (Mark and Davis,

2000). one class of anti-hypertensive drugs known as

angiotensin I converting enzyme (ACE) inhibitors (i.e.

peptidase inhibitors) has a low incidence of adverse side-

effects and is the preferred class of anti-hypertensive

agents when treating patients with concurrent secondary

BIOCHEMISTRY

38

Botanical Studies, Vol. 47, 2006

diseases (Fotherby and Panayiotou, 1999). ACE (EC

3.4.15.1) is a dipeptide-liberating exopeptidase which

has been classically associated with the renin-angiotensin

system regulating peripheral blood pressure (Mullally

et al., 1996). The potent ACE inhibitors were frequently

derived from food proteins (Ariyoshi, 1993; Hsu et

al., 2002). However, pomegranate juice (Aviram and

Dornfeld, 2001), flavan-3-ols and procyanidins (Actis-

Goretta et al., 2003), and tannins (Liu et al., 2003a) were

also reported to have ACE inhibitory activity.

Acacia confusa (Leguminosae) is widely distributed on

the hills and lowlands of Taiwan. Five structure-related

myricetin galloylglycosides (Figure 1)—namely, myricetin

3-O-(3"-O-galloyl)-α-rhamnopyranoside 7-methyl ether

(compound 1, 630 Da), myricetin 3-O-(2"-O-galloyl)-

α-rhamnopyranoside 7-methyl ether (compound 2, 630

Da), myricetin 3-O-(2"-O -galloyl)-α-rhamnopyranoside

(compound 3, 616 Da), myricetin 3-O-(3"-O-galloyl)-

α -rhamnopyranoside (compound 4, 616 Da), and

myricetin 3-O-(2", 3"-di-O-galloyl)-α-rhamnopyranoside

(compound 5, 768 Da)—isolated from leaves of Acacia

confusa were previously reported (Lee et al., 2000).

However, the structure-activity relationships (SAR)

were not reported. In this research, the 1,1-diphenyl-

2-picrylhydrazyl (DPPH) radical scavenging activity,

and inhibitory activities against SSAo and ACE were

compared among five compounds.

MATERIALS AND METHODS

Materials

ACE (I unit, rabbit lung) was purchased from Fluka

Chemie GmbH (Switzerland); DPPH, benzylamine,

2’, 2-azinodi(3-ethylbenzthiazoline-6-sulfonic acid,

ABTS), bovine plasma (P-4639, reconstituted with 10 ml

deionized water), horseradish peroxidase (148 units/mg

solid), N-(3-[2-furyl] acryloyl)-Phe-Gly-Gly (FAPGG),

semicarbazide, and other chemicals and reagents were

from Sigma Chemical Co. (St. Louis, Mo, USA).

Extraction and purificaction of five structure-

related myricetin galloylglycosides from

Acacia

confusa

Five structure-related flavonol galloylglycosides

isolated from leaves of Acacia confusa were previously

reported (Lee et al., 2000). The methanolic extracts were

isolated in series by Sephadex LH-20 column, Si-flash

column, and reverse-phase HPLC. Each structure was

identified by

13

C and

1

H NMR, CoSY, HMQC and HMBC

spectra. The purity of each compound was higher than

99%, determined by reverse-phase HPLC.

Scavenging activity against DPPH radical

analyzed by spectrophotometry

The scavenging activity of five structure-related

flavonol galloylglycosides against DPPH radical was

measured according to the method of Hou et al. (2001a,

b). Each sample was dissolved in DMSo to a final

concentration of 2 mg/ml as stock solutions. Each 0.3 ml

sample solution (compound 1, 158.73, 317.46, 476.19,

634.92 μM; compound 2, 317.46, 555.56, 793.65, 1587.3

μM; compound 3, 811.69, 1623.38, 2435.06, 3246.75

μM; compound 4, 487.01, 811.69, 1217.53, 1623.38 μM;

compound 5, 390.63, 520.83, 651.04, 976.56 μM) was

added to 0.1 ml of 1 M Tris-HCl buffer (pH 7.9) and

then mixed with 0.6 ml of 100 μM DPPH in methanol

for 20 min under light protection at room temperature.

The decrease of absorbance at 517 nm was measured and

expressed as .A517. Means of triplicates were measured.

DMSo was used as a blank experiment. The scavenging

activity of DPPH radicals (%) was calculated with the

equation: (.A517

blank

. .A517

sample

) ÷ .A517

blank

× 100%.

The IC

50

stands for the concentration of 50% scavenging

activity.

SSAO inhibitory activities of five structure-

related myricetin galloylglycosides

SSAo inhibitory activity was determined by

spectrophotometric method according to Szutowicz et al.

(1984) with some modifications. The total 200 μl reaction

solution [containing 50 μl of 200 mM phosphate buffer,

pH 7.4, 50 μl of 8 mM benzylamine, bovine plasma

(containing SSAo, 2.53 units) and different amounts

of sample solution (compound 1, 15.87, 31.75, 47.62,

63.49 μM; compound 2, 47.62, 63.49, 95.24, 111.11

μM; compound 3, 81.17, 113.64, 129.87, 194.81 μM;

compound 4, 64.94, 97.40, 129.87, 162.34 μM; compound

5, 26.04, 39.06, 52.08, 65.10 μM) and semicarbazide

(6.25, 12.5, 25, and 50 μM)] was placed at 37°C for

one h and then heated at 100°C to stop reaction. After

cooling and a brief centrifugation, the 90 μl reaction

solution was isolated and added to the 710 μl solution

containing 200 μl of 200 mM phosphate buffer (pH 7.4),

100 μl of 2 mM ABTS solution, and 25 μl of horseradish

peroxidase (10 μg/ml). The changes of absorbance at 420

nm were recorded during 1 min and expressed as .A420

nm/min. Means of triplicates were measured. DMSo was

used as a blank experiment. The SSAo inhibition (%)

was calculated with the equation: (.A420 nm/min

blank

-

.A420 nm/min

sample

) ÷ .A420 nm/min

blank

× 100%. The

IC

50

stands for the concentration of 50% inhibitions.

Determination of ACE inhibitory activity of

structure-related myricetin galloylglycosides by

spectrophotometry.

The ACE inhibitory activity was measured according

to the method of Holmquist et al. (1979) and Lee et al.

(2003) with some modifications. The 15 μl (15 mU)

commercial ACE (1 U/mL, rabbit lung) were mixed with

50 μl of sample solution (compound 1, 14.76, 44.60,

79.21, 148.90 μM; compound 2, 44.60, 74.44, 148.90,

178.73 μM; compound 5, 12.11, 24.35, 36.59, 48.83 μM)

and then 1 mL of 0.5 mM FAPGG [dissolved in 50 mM

Lee et al. — SAR of five myricetin galloylglycosides

39

Tris-HCl buffer (pH 7.5) containing 0.3 M NaCl] was

added. The decreased absorbance at 345 nm (.A

inhibitor

)

was recorded during 5 min at room temperature. DMSo

was used instead of sample solution for blank experiments

(.A

blank

). The ACE activity was expressed as .A345 nm,

and the ACE inhibition (%) was calculated as follows: [1-

(.A

inhibitor

÷ .A

control

)

] × 100%. Means of triplicates were

determined.

RESULTS AND DISCUSSION

Flavonoids are one type of polyphenol—a group which

includes isoflavones, flavones, and flavanones—and

have been reported to exhibit many biological activities

(Hodnick et al., 1994; Hoult et al., 1994; Siess et al., 1995;

Naasani et al., 1998; Lee et al., 2001). Lee et al. (2000)

isolated five structure-related myricetin galloylglycosides

(Figure 1), and the IC

50

of anti-hatch activity against brine

shrimp from four of them was 50 μg/ml (79.37 μM), 89

μg/ml (141.27 μM), 75 μg/ml (121.75 μM), and 64 μg/ml

(83.33 μM), respectively, for compound 1, compound

2, compound 4, and compound 5. Compound 3 was not

measured. The four compounds ranked for their anti-hatch

activity against brine shrimp take the order: compound

1> compound 5> compound 4> compound 2. Chang et al.

(2001) reported the antioxidant activity of 70% ethanolic

extracts from Acacia confusa bark and heartwood. In

this research, the DPPH radical scavenging activity

and inhibitory activities against SSAo and ACE were

compared among five myricetin galloylglycosides.

Scavenging activitiy against DPPH radical

analyzed by spectrophotometry

DPPH radicals were widely used in the model system

to investigate the scavenging activities of several natural

compounds. When DPPH radical was scavenged, the

color of the reaction mixture changed from purple to

yellow with decreasing of absorbance at wavelength

517 nm. Figure 2 shows the scavenging activity against

DPPH radicals from five structure-related myricetin

galloylglycosides. The dose-dependent DPPH radical

scavenging activities from five pure compounds were

found. For compound 1, the scavenging activities against

DPPH were 13.86, 24.71, 43.74, and 52.59%, respectively,

for 158.73, 317.46, 476.19, 634.92 μM; for compound 2,

the scavenging activities against DPPH were 15.36, 23.71,

33.39, and 51.86%, respectively, for 317.46, 555.56,

793.65, 1587.3 μM; for compound 3, the scavenging

activities against DPPH were 20.87, 39.40, 47.58, and

50.25%, respectively, for 811.69, 1623.38, 2435.06,

3246.75 μM; for compound 4, the scavenging activities

against DPPH were 23.21, 38.06, 48.25, and 52.42%,

respectively, for 487.01, 811.69, 1217.53, 1623.38 μM; for

compound 5, the scavenging activities against DPPH were

24.37, 36.56, 42.57, and 53.42%, respectively, for 390.63,

520.83, 651.04, 976.56 μM. The IC

50

of each compound

against DPPH radial was shown at Table 1. The IC

50

for

five compounds were 591, 1522, 3210, 1389, and 867

μM, respectively. The five compounds had the order of

compound 1> compound 5> compound 4> compound 2>

compound 3 for DPPH scavenging activity, which was the

same order as for anti-hatch activity against brine shrimp

(Lee et al., 2000). For anti-DPPH activity, with the same

methyl group in the R

1

position, the gallic acid in the R

3

position (compound 1) was more effective (2.5 fold) than

in the R

2

position (compound 2), and this efficiency was

Figure 1. Structures of five myricetin galloyoglycosides

isolated from leaves of Acacia confusa.

Figure 2. The scavenging activity of five myricetin

galloylglycosides against DPPH radicals. Means of triplicates

were measured. The scavenging activity of DPPH radical (%)

was calculated according to the following equation: (A517

blank

.

A517

sample

) ÷ A517

blank

× 100%.

40

Botanical Studies, Vol. 47, 2006

also found in compound 3 and compound 4. With the

same hydrogen atom in the R

1

position, the gallic acid

in the R

3

position (compound 4) was more effective (2.3

fold) than in the R

2

position (compound 3). With the same

gallic acid in the R

3

position and hydrogen atom in the R

2

position, the methyl group in the R

1

position (compound

1) was more effective (2.35 fold) than the hydrogen atom

in the same position (compound 4), and this efficiency

was also found in compound 2 and compound 3. With the

same gallic acid in the R

2

position and the hydrogen atom

in the R

3

position, the methyl group in the R

1

position

(compound 2) was more effective (2.11 fold) than the

hydrogen atom in the same position (compound 3). While,

with two gallic acid groups in the R

2

and R

3

positions

(compound 5) were more effective than one in the R

2

position (compound 3) or in the R

3

position (compound

4). It was concluded that the gallic acid in the R

3

position

was the key for anti-DPPH activity among five structure-

related compounds.

SSAO inhibitory activities of five structure-

related myricetin galloylglycosides

Figure 3 shows the SSAo inhibitory activity from

five structure-related myricetin galloylglycosides. The

dose-dependent SSAo inhibitory activities from five

pure compounds were found. For compound 1, the

SSAo inhibitory activities were 12.17, 41.44, 67.05, and

81.44%, respectively, for 15.87, 31.75, 47.62, 63.49 μM;

for compound 2, the SSAo inhibitory activities were

20.83, 33.47, 50.65, and 74.43 %, respectively, for 47.62,

63.49, 95.24, 111.11 μM; for compound 3, the SSAO

inhibitory activities were 25.36, 44.55, 60.85, and 72.58

%, respectively, for 81.17, 113.64, 129.87, 194.81 μM;

for compound 4, the SSAo inhibitory activities were

33.45, 56.72, 71.64, and 88.44%, respectively, for 64.94,

97.40, 129.87, 162.34 μM; for compound 5, the SSAO

inhibitory activities were 31.99, 48.93, 76.48, and 88.44

%, respectively, for 26.04, 39.06, 52.08, 65.10 μM. The

IC

50

of SSAo inhibitory activity of each compound was

shown at Table 1. For SSAo inhibitory activity, the IC

50

for five compounds was 36.16, 93.20, 119.50, 88.20,

and 39.35 μM, respectively. The IC

50

of semicarbazide

(positive control) was 34.21 μM which was close to that

of compound 1. The five compounds had the order of

compound 1> compound 5> compound 4> compound

2> compound 3, which was the same as for DPPH

scavenging activities (above-mentioned) and anti-hatch

activity against brine shrimp (Lee et al., 2000). For SSAo

inhibitory activity, with the same methyl group in the R

1

position, the gallic acid in the R

3

position (compound

1) was more effective (2.6 fold) than in the R

2

position

(compound 2), and this efficiency was also found in

compound 3 and compound 4. With the same hydrogen

atom in the R

1

position, the gallic acid in the R

3

position

(compound 4) was more effective (1.35 fold) than in the

R

2

position (compound 3). With the same gallic acid in the

R

3

position and the hydrogen atom in the R

2

position, the

methyl group in the R

1

position (compound 1) was more

effective (2.44 fold) than the hydrogen atom in the same

position (compound 4), and this efficiency was also found

in compound 2 and compound 3. With the same gallic acid

Table 1. Comparisons of the DPPH scavenging activity and inhibitory activities against SSAO and ACE among five myricetin

galloyoglycosides isolated from leaves of Acacia confusa.

Myricetin galloylglycoside Scavenging activity of DPPH radical

(μM)

a

SSAo inhibitory activity

(μM)

a

ACE inhibitory activity

(μM)

a

Compound 1

591

36.16

60.32

Compound 2

1522

93.20

151.90

Compound 3

3210

119.50

ND

b

Compound 4

1389

88.20

ND

Compound 5

867

39.35

19.82

a

Expressed as the IC

50

value.

b

Not detectable.

Figure 3. The effects of five myricetin galloylglycosides on the

activities of semicarbazide-sensitive amine oxidase (SSAo, 2.53

units) from bovine plasma. The semicarbazide (6.25, 12.5, 25,

and 50 μM) was used as a positive control. Deionized water was

used as a blank experiment. The changes of absorbance at 420

nm were recorded during 1 min and expressed as .A420 nm/

min. The SSAo inhibition (%) was calculated with the equation:

(.A420 nm/min

blank

. .A420 nm/min

sample

) ÷ .A420 nm/min

blank

× 100%.

Lee et al. — SAR of five myricetin galloylglycosides

41

in the R

2

position and hydrogen atom in the R

3

position,

the methyl group in the R

1

position (compound 2) was

more effective (1.28 fold) than the hydrogen atom in the

same position (compound 3). Two gallic acid groups in the

R

2

and R

3

positions (compound 5) were more effective than

one in the R

2

position (compound 3) or in the R

3

position

(compound 4). It was concluded that the gallic acid in the

R

3

position was the key role for SSAo inhibitory activity

among five structure-related compounds.

ACE inhibitory activity of myricetin

galloylglycosides by spectrophotometry

Figure 4 shows the ACE inhibitory activity from

five structure-related myricetin galloylglycosides. It

was found that three out of five compounds had dose-

dependent ACE inhibitory activities. For compound 1, the

ACE inhibitory activities were 34.33, 46.27, 54.48, and

64.90 %, respectively, for 14.76, 44.60, 79.21, 148.90

μM; for compound 2, the ACE inhibitory activities were

35.80, 47.02, 49.25, and 56.71 %, respectively, for 44.60,

74.44, 148.90, 178.73 μM; for compound 5, the ACE

inhibitory activities were 37.31, 57.46, 67.91, and 71.64

%, respectively, for 12.11, 24.35, 36.59, 48.83 μM. The

IC

50

of ACE inhibitory activity of each compound was

shown at Table 1. The IC

50

of compound 1, compound

2, and compound 5 was 60.32, 151.90, and 19.82 μM,

respectively. For ACE inhibitory activity, with the same

methyl group in the R

1

position, the gallic acid in the R

3

position (compound 1) was more effective (2.52 folds)

than in the R

2

position (compound 2). Two gallic acid

groups in the R

2

and R

3

positions (compound 5) were more

effective than one in the R

2

position (7.66 folds, compound

2) or in the R

3

position (3.04 folds, compound 1). Liu et

al. (2003) reported that gallotannins of five gallic acid

groups of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (IC

50

,

73.1 μM) was more effective in ACE inhibitory activity

than four gallic acid groups of 1,2,3,6-tetra-O-galloyl-β-

D-glucose (IC

50

, 101.4 μM). The IC

50

of two or three gallic

acid groups in gallotannin were higher than 200 μM.

In conclusion, the SAR of DPPH radical scavenging

activity and inhibitory activities of SSAo and ACE in

five structure-related myricetin galloylglycosides were

reported. The five compounds have the same orders of

compound 1> compound 5> compound 4> compound 2>

compound 3 for DPPH scavenging activity and SSAo

inhibition. It was postulated that gallic acid in the R

3

position was the key role for anti-DPPH radicals and

SSAo inhibitory activity. For ACE inhibitory activity, the

orders were compound 5> compound 1> compound 2. It

was postulated that more gallic acid groups were the key

to ACE inhibitory activity.

Acknowledgments. The authors want to thank the

financial support (NSC93-2313-B038-001) from the

National Science Council, Republic of China.

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43

相思樹葉子五種楊梅樹皮素配糖體之結構－活性關係

李宗徽

1

　劉得任

2

　徐鳳麟

1

　吳紋君

1

　侯文琪

1

1

臺北醫學大學生藥學研究所

2

臺北醫學大學生物醫學材料研究所

　　從相思樹葉子分離五種楊梅樹皮素配糖體在之前就已經有報告 (Lee et al., 2000, J. Nat. Prod., 63,

710-712)，但是結構-活性之間的關係並沒有報告。本½研究將比較五種分離的化合物，包括 myricetin

3-O-(3"-O-galloyl)-α-rhamnopyranoside 7-methyl ether (化合物 1，分子量 630 Da), myricetin 3-O-(2"

-O-galloyl)-α-rhamnopyranoside 7-methyl ether (化合物 2，分子量 630 Da), myricetin 3-O-(2"-O-galloyl)-

α-rhamnopyranoside (化合物 3，分子量 616 Da), myricetin 3-O-(3"-O-galloyl)-α-rhamnopyranoside (化

合物 4，分子量 616 Da), 及myricetin 3-O-(2", 3"-di-O-galloyl)-α-rhamnopyranoside (化合物 5，分子量

768 Da)，對於清除 DPPH 自由基與抑制 Semicarbazide-sensitive 胺.（SSAo）及血管收縮素轉化.

（ACE）活性的關係。就清除 DPPH 自由基方面，五種化合物 50% 抑制所需濃度分別為 591, 1522,

3210, 1389, 及 867 μM。就抑制 Semicarbazide-sensitive 胺.方面，五種化合物 50% 抑制所需濃度分別

為 36.16, 93.20, 119.50, 88.20, 及 39.35 μM。就上面清除 DPPH 自由基與抑制 Semicarbazide-sensitive 胺

.的結果可以發現，五種結構相關化合物顯現出以下相同次序：化合物 1> 化合物 5> 化合物 4> 化合物

2> 化合物 3，而 gallic acid 位在 R3 的位置，與上述兩種活性有密切的關係。就抑制血管收縮素轉化.

（ACE）方面，只有化合物 1，化合物 2，及化合物 5 具有濃度相關抑制活性, 三種化合物 50% 抑制所

需濃度分別為 60.32, 151.90, 及 19.82 μM。

關鍵詞：相思樹；DPPH 自由基；Semicarbazide-sensitive 胺.；血管收縮素轉化.；楊梅樹皮素配糖

體；結構-活性關係。