Anti-complement activity of constituents from the stem-bark of Juglans mandshurica

Article abstract of DOI:10.1248/bpb.26.1042

Four known flavonoids and two galloyl glucoses isolated from the stem-bark of Juglans mandshurica (Juglandaceae), namely taxifolin (1), afzelin (2), quercitrin (3), myricitrin (4), 1,2,6-trigalloylglucose (5), and 1,2,3,6-tetragalloylglucose (6), were evaluated for their anti-complement activity against complement system. Afzelin (2) and quercitrin (3) showed inhibitory activity against complement system with 50% inhibitory concentrations (IC₅₀) values of 258 and 440 μM. 1,2,6-Trigalloylglucose (5) and 1,2,3,6-tetragalloylglucose (6) exhibited anti-complement activity with IC ₅₀ values of 136 and 34 μM. In terms of the evaluation of the structure-activity relationship of 3,5,7-trihydroxyflavone, compounds 2, 3, and 4 were hydrolyzed with naringinase to give kaempferol (2a), quercetin (3a), and myricetin (4a) as their aglycones, and these were also tested for their anti-complement activity. Of the three aglycones, kaempferol (2a) exhibited weak anti-complement activity with an IC₅₀ value of 730 μM, while quercetin (3a) and myricetin (4a) were inactive in this assay system. Among the compounds tested, 1,2,3,6-tetragalloylglucose (6) showed the most potent anticomplement activity (IC₅₀, 34 μM).

Full text of DOI:10.1248/bpb.26.1042

1
042
Notes
Biol. Pharm. Bull. 26(7) 1042—1044 (2003)
Vol. 26, No. 7
Anti-complement Activity of Constituents from the Stem-Bark of
Juglans mandshurica
a
a
a
a
a
b
Byung-Sun MIN, Sun-Young LEE, Jung-Hee KIM, Joong-Ku LEE, Tae-Jin KIM, Dong-Hee KIM,
c
a
,a
d
d
Young-Ho KIM, Hyouk JOUNG, Hyeong-Kyu LEE,* Norio NAKAMURA, Hirotsugu MIYASHIRO, and
d
Masao HATTORI
a
Laboratory of Immunomodulator, Korea Research Institute of Bioscience and Biotechnology; Daejeon 305–333, Korea:
b
c
Laboratory of Pathology, College of Oriental Medicine, Deajeon University; Daejeon 300–176, Korea: College of
d
Pharmacy, Chungnam National University; Daejeon 305–764, Korea: and Institute of Natural Medicine, Toyama Medical
and Pharmaceutical University; 2630 Sugitani, Toyama 930–0194, Japan.
Received January 8, 2003; accepted April 4, 2003
Four known flavonoids and two galloyl glucoses isolated from the stem-bark of Juglans mandshurica (Jug-
landaceae), namely taxifolin (1), afzelin (2), quercitrin (3), myricitrin (4), 1,2,6-trigalloylglucose (5), and 1,2,3,6-
tetragalloylglucose (6), were evaluated for their anti-complement activity against complement system. Afzelin (2)
and quercitrin (3) showed inhibitory activity against complement system with 50% inhibitory concentrations
(
IC ) values of 258 and 440 mM. 1,2,6-Trigalloylglucose (5) and 1,2,3,6-tetragalloylglucose (6) exhibited anti-com-
50
plement activity with IC values of 136 and 34 mM. In terms of the evaluation of the structure–activity relation-
5
0
ship of 3,5,7-trihydroxyflavone, compounds 2, 3, and 4 were hydrolyzed with naringinase to give kaempferol (2a),
quercetin (3a), and myricetin (4a) as their aglycones, and these were also tested for their anti-complement activ-
ity. Of the three aglycones, kaempferol (2a) exhibited weak anti-complement activity with an IC50 value of
7
1
30 mM, while quercetin (3a) and myricetin (4a) were inactive in this assay system. Among the compounds tested,
,2,3,6-tetragalloylglucose (6) showed the most potent anticomplement activity (IC , 34 mM).
50
Key words Juglans mandshurica; anti-complement activity; 1,2,3,6-tetragalloylglucose; 1,2,6-trigalloylglucose; 3,5,7-trihy-
droxyflavone
The invertebrate complement system can be activated by a totoxic activity against human cancer cell lines, inhibitory ef-
cascade mechanism of the classical pathway (CP), an alterna- fects on DNA polymerase and on the RNase H activities of
tive pathway (AP), or the MBL/MASP (mannan binding HIV-1 reverse transcriptase, as well as anti-HIV-1 activity in
1
)
5—10)
lectin/MBL-associated serine protease) pathway. The thirty MT-4 cells.
In this paper we describe the anti-comple-
odd complement fragments that make up the system include ment activity of compounds from this plant.
proteolytic pro-enzymes, non-enzymatic components that
form functional complexes, co-factors, regulators, and recep- MATERIALS AND METHODS
tors. The pro-enzymes are the complements, which become
sequentially activated in the cascade of events leading to the
Plant Material The stem-bark of J. mandshurica was
2
)
activation of the complement system. The proteolytic cas- collected during September 1998 at a mountain area of Kim-
cade allows for tremendous amplification, since each pro- chun, Kyungbook, Korea, and dried at room temperature for
teinase molecule activated at a single step can generate mul- 3 weeks. A voucher specimen is deposited at the herbarium
tiple copies of an activated enzyme later in the cascade, of the College of Pharmacy, Chungnam National University,
which in turn cleaves non-enzymatic components, such as Korea.
C3, C4, and C5. The larger fragments derived from C3, C4,
Chemicals Sheep red blood cell (SRBC) was obtained
and C5 (i.e. C3b, C4b, and C5b) are involved in biologic ef- from College of Agriculture, Chungnam National University
fector functions, such as opsonization, phagocytosis, and im- (Daejeon, Korea). Normal human serum was collected from
munomodulation. However, the smaller molecules, C3a, C4a, a healthy volunteer (Man). Hemolysin, gelatin, MgCl2,
and C5a, called anaphylatoxins, induce the release of media- CaCl , sodium barbital, barbituric acid, and naringinase were
2
tors from mast cells and lymphocytes, which cause a variety purchased from Sigma Chemical (St. Louis, U.S.A.). Ros-
of inflammatory diseases, and may be fatal if they occur after marinic acid and tiliroside were isolated from Agastache ru-
2
—4)
organ transplantation.
plement activity should be useful in the therapy of inflamma- tained from Merck Co. (Darmstadt, Germany). Sephadex
tory diseases. LH-20 was purchased from Amersham Pharmacia Biotech
Therefore, the modulation of com- gosa and Magnolia fargesii, respectively. Silica gel was ob-
During the course of our research on anti-inflammatory (Uppsala, Sweden). ODS was obtained from Fuji Silysia
agents from natural sources, flavonoids and galloyl glucoses Chemical (Aichi, Japan).
from the stem-bark of Juglans mandshurica MAXIMOWICZ
Juglandaceae) were tested for their anti-complement activ- (3.0 kg) was extracted with MeOH at room temperature for
ity. J. mandshurica is used as a folk medicine for the treat- 24 h to give a dark-brown extract (390 g). The MeOH extract
Extraction and Isolation Stem-bark of J. mandshurica
(
5
)
ment of cancer in Korea. Several naphthoquinones, naph- (300 g) was suspended in H O (2500 ml) and extracted with
2
thalenyl glucoses, a-tetralonyl glucoses, diarylheptanoyl glu- hexane (2500ϫ3) to give a hexane-soluble fraction (48 g).
coses, and flavonoids have been isolated from this plant, and The resulting H O layer was extracted with CH Cl
2
2
2
furthermore, these compounds have been shown to have, cy- (3000 mlϫ3), EtOAc (3000 mlϫ3), and BuOH (3000 mlϫ3),
∗
To whom correspondence should be addressed. e-mail: hykylee@kribb.re.kr
© 2003 Pharmaceutical Society of Japan

July 2003
1043
2
ϩ
successively. The EtOAc-soluble fraction (90 g) was chro-
matographed on a column of silica gel (1 kg). The column
gelatin veronal buffer (GVB , 80 ml) with or without sam-
ple. Each sample was dissolved in DMSO, and it was used as
negative control. The mixture was pre-incubated at 37° for
was eluted using a gradient of CHCl , MeOH, and H O to
3
2
give six fractions (Fr. A—F: 2.7 g, 15.9 g, 23.9 g, 7.5 g, 5.4 g,
and 10.2 g, respectively). Repeated column chromatography
3
0 min, after which sensitized erythrocyte (sheep red blood
cells, 40 ml) was added. After incubation under the same con-
ditions, the mixture was centrifuged (4 °C, 1500 rpm) and the
optical density of the supernatant (100 ml) was measured at
of Fr. B on silica gel (CHCl –MeOH, 9 : 1), Sephadex LH-20
3
(
CHCl –MeOH, 1 : 9), and ODS column (50% aqueous
3
1
1)
MeOH), followed by MPLC on RP-18 (50% aq. MeOH and
0% aq. CH CN) afforded compounds 1 (165 mg) and 2
4
50 nm. Tiliroside and rosmarinic acid were used as posi-
1
2)
7
3
tive controls. Anti-complement activity was determined as
a mean of triplicate measurements and expressed as the 50%
inhibitory concentrations (IC ) values from complement-de-
(
189 mg). Repeated column chromatography of Fr. C on
Sephadex LH-20 (MeOH and CHCl –MeOH, 1 : 9), silica gel
3
5
0
1
3)
(
CHCl –MeOH, 8 : 2), and ODS column (40% aq. MeOH),
3
pendent hemolysis of the control.
followed by MPLC on RP-18 (40% aq. MeOH and 80% aq.
CH CN) furnished compounds 3 (168 mg), 4 (345 mg), 5
3
RESULTS AND DISCUSSION
8
)
(
340 mg), and 6 (294 mg).
Taxifolin (5,7,3Ј,4Ј-Tetrahyrdoxyflavanol, 1): White amor-
The human complement system plays an important role in
the host defense system against foreign invasive organisms,
i.e. viruses, bacteria, and fungi, as well as an external wound.
Its effects are normally beneficial to the host, but it can also
cause adverse effects depending on the site, extent, and dura-
tion of complement activation. Activation of the system
may lead to pathologic reactions in a variety of inflammatory
and degenerative diseases such as multiple sclerosis, sys-
temic lupus erythematosus, Sjogren syndrome, dermatologi-
Ϫ1
phous powder, [a] ϩ20° (cϭ0.1, MeOH). IR n
3
cm :
D
max
420, 1620, 1520, 1470, 1360, 1265, 1165. UV l
nm
max
(
log e): 288 (4.2), 328 (sh).
Afzelin (Kaempferol 3-O-a-L-Rhamnopyranoside, 2): Yel-
low amorphous powder, [a] Ϫ184° (cϭ0.1, MeOH). IR
1
4)
D
Ϫ1
n
(
cm : 3280, 1655, 1615, 1500, 1450, 1365. UV lmax nm
max
log e): 264 (4.3), 342 (4.1).
Quercitrin (Quercetin 3-O-a-L-Rhamnopyranoside, 3):
1
5)
Yellow amorphous powder, [a] Ϫ178° (cϭ0.1, MeOH). IR
D
cal disease, rheumatoid arthritis, and gout.
Ϫ1
n
cm : 3320, 1660, 1610, 1500, 1450, 1360. UV l nm
max
max
As part of an ongoing program of research in our labora-
tory to detect natural products with anti-complement activity,
the inhibitory properties of compounds from the stem-bark
of J. mandshurica were investigated. Repeated column chro-
matography of the EtOAc-soluble fraction of the MeOH ex-
tract of J. mandshurica on silica gel, ODS, and Sephadex
LH-20 led to the isolation of four flavonoids (1—4) and two
galloyl glucoses (5, 6). These compounds were identified as
taxifolin (1), afzelin (2), quercitrin (3), myricitrin (4), 1,2,6-
trigalloylglucose (5), and 1,2,3,6-trigalloylglucose (6) by
comparing their spectral data to those previously reported
(
log e): 254 (4.2), 314 (sh), 350 (4.1).
Myricitrin (Myricetin 3-O-a-L-Rhamnopyranoside, 4):
Yellow amorphous powder, [a] Ϫ141° (cϭ0.1, MeOH). IR
D
Ϫ1
n
cm : 3270, 1655, 1610, 1500, 1455, 1340. UV l nm
max
max
(
log e): 254 (4.2), 312 (sh), 354 (4.1).
1
,2,6-Trigalloylglucose (5): White amorphous powder,
Ϫ1
[
1
a] Ϫ94° (cϭ0.1, MeOH). IR n cm : 3420, 1710, 1610,
D
max
540, 1525, 1450, 1355. UV l
4.4).
nm (log e): 216 (4.6), 278
max
(
1
,2,3,6-Tetragalloylglucose (6): White amorphous powder,
Ϫ1
8
)
[
1
a] ϩ39° (cϭ0.1, MeOH). IR n cm : 3400, 1700, 1610,
D
max
(Fig. 1). Furthermore, kaempferol (2a), quercetin (3a), and
myricetin (4a) were obtained from afzelin (2), quercitrin (3),
and myricitrin (4), respectively, by enzymatic hydrolysis.
The compounds 1—6 isolated were tested for their anti-
complement activity and the results obtained are summarized
in Table 1 (IC50 values). Afzelin (2), quercitrin (3), 1,2,6-tri-
540, 1455, 1355. UV l nm (log e): 216 (4.9), 278 (4.5).
max
Enzymatic Hydrolysis of 2, 3, and 4 Naringinase
(
1
100 mg) was added to a suspension of 2, 3, or 4 (each
0 mg) in 50 mM acetate buffer (pH, 5.5) and the mixture was
stirred at room temperature for 3 h. The reaction mixture was
extracted with EtOAc (20 ml) and evaporated to dryness. The
each residue was chromatographed on silica gel eluting
with CHCl –MeOH (1 : 0→8 : 2) to give kaempferol (2a),
3
quercetin (3a), and myricetin (4a).
Kaempferol (2a): Yellow amorphous powder. [a] : ϩ5.0°
D
1
(
(
(
cϭ0.2, MeOH). H-NMR (DMSO-d , 300 MHz): d 6.16
6
1H, d, Jϭ1.1 Hz, H-6), 6.40 (1H, d, Jϭ1.1 Hz, H-8), 6.92
2H, d, Jϭ8.7 Hz, H-3Ј, 5Ј), 8.03 (2H, d, Jϭ9.0 Hz, H-2Ј, 6Ј).
Quercetin (3a): Yellow amorphous powder. [a] : ϩ5.6°
D
1
(
(
(
cϭ0.2, MeOH). H-NMR (DMSO-d , 300 MHz): d 6.14
6
1H, d, Jϭ1.8 Hz, H-6), 6.35 (1H, d, Jϭ1.8 Hz, H-8), 6.87
1H, d, Jϭ8.4 Hz, H-5Ј), 7.53 (1H, dd, Jϭ8.4, 2.4 Hz, H-6Ј),
7
.65 (1H, d, Jϭ1.8 Hz, H-2Ј).
Myricetin (4a): Yellow amorphous powder. [a] : Ϫ4.0°
D
1
(
(
(
cϭ0.3, MeOH). H-NMR (DMSO-d , 300 MHz): d 6.18
6
1H, d, Jϭ1.2 Hz, H-6), 6.37 (1H, d, Jϭ1.2 Hz, H-8), 6.93
2H, s, H-2Ј, 6Ј).
Anti-complement Assay A diluted solution of normal
Fig. 1. Chemical Structures of Compounds from the Stem-Bark of
human serum (complement serum, 80 ml) was mixed with a Juglans mandshurica

1
044
Vol. 26, No. 7
Table 1. Inhibitory Effects of Compounds on the Complement System of tive in this assay system. These results demonstrate the role
Classical Pathway in Vitro
which the number of hydroxyl groups on the B-ring of 3,5,7-
trihydroxyflavone may play in structure–activity relation-
a)
Compound
Taxifolin (1)
Afzelin (2)
Quercitrin (3)
Myricitrin (4)
IC50 (mM)
ships: it seems that the inhibitory potencies of 2 (2a), 3 (3a),
and 4 (4a) against anti-complement activity increased in in-
verse proportion to number of free hydroxyl groups on the
B-ring of 3,5,7-trihydroxyflavone. These data agreed with the
anti-complement properties of apigenin and its glycosides
c)
NE
258
440
NE
136
34
1
,2,6-Trigalloylglucose (5)
(apigenin 7-O-glucoside, apigenin 7-O-rutinoside), and lute-
1
,2,3,6-Tetragalloylglucose (6)
Kaempferol (2a)
Quercetin (3a)
Myricetin (4a)
730
NE
NE
180
101.2
olin and its glycosides (luteolin 7-O-glucoside, luteolin 4Ј-O-
glycoside) isolated from Ligustrum vulgare and Phillyrea lat-
ifolia.
Of the compounds tested, 1,2,3,6-tetragalloylglucose (6)
was found to be the most active compound with an IC50 of
16)
b)
Rosmarinic acid
b)
Tiliroside
a) Results are the mean (nϭ3). b) This compound was used as a positive con- 34 mM in this assay system. Comparing the galloylglucoses
1
2)
trol.
c) NE; no effect.
(5, 6) with flavonoids (1—4) shown that the galloyl group
might increase anti-complement properties. This result is
supported by the anti-complement activity of 1,2,3,6-tetra-
galloylglucose (6; IC , 34 mM), which was found to be more
5
0
potent than 1,2,6-trigalloylglucose (5; IC , 136 mM) against
5
0
the complement system.
Acknowledgments This research was supported by a
grant (PF0050213) from Plant Diversity Research Center of
2
1st Century Frontier Research Program funded by Ministry
of Science and Technology of Korea government. We are
1
13
grateful to KBSI for H- and C-NMR spectral measure-
ments.
Fig. 2. Inhibitory Effects of 1,2,6-Trigalloylglucose (5) on Classical Path-
way of Complement System (MeanϮS.D., nϭ3)
REFERENCES
1
2
3
)
)
)
Kirschfink M., Immunopharmacology, 38, 51—62 (1997).
Ember J. A., Hugli T. E., Immunopharmacology, 38, 3—15 (1997).
Abbas D., Lichtman A. K., Pober J. S., “Cellular and Molecular Im-
munology,” W. B. Saunders Company, Philadelphia, 1997, pp. 315—
3
38.
Min B. S., Gao J. J., Hattori M., Lee H. K., Kim Y. H., Planta Med.,
7, 811—814 (2001).
4
)
6
5
6
7
)
)
)
Son J. K., Arch. Pharm. Res., 18, 203—205 (1995).
Joe Y. K., Son J. K., J. Nat. Prod., 59, 159—160 (1996).
Kim S. H., Lee K. S., Son J. K., Je G. H., Lee J. S., Lee C. H., Cheong
C. J., J. Nat. Prod., 61, 643—645 (1998).
8
)
Min B. S., Nakamura N., Miyashiro H., Kim Y. H., Hattori M., Chem.
Pharm. Bull., 48, 194—200 (2000).
9
)
Lee S. W., Lee K. S., Son J. K., Planta Med., 66, 184—186 (2000).
Fig. 3. Inhibitory Effects of 1,2,3,6-Tetragalloylglucose (6) on Classical
Pathway of Complement System (MeanϮS.D., nϭ3)
1
0) Min B. S., Lee H. Y., Lee S. M., Kim Y. H., Bae K. H., Otake T.,
Nakamura N., Hattori M., Arch. Pharm. Res., 25, 441—445 (2002).
1) Yamada H., Ohtani K., Kiyohara H., Cyong J. C., Otsuka Y., Ueno Y.,
Omura S., Planta Med., 51, 121—125 (1985).
1
galloylglucose (5), and 1,2,3,6-tetragalloylglucose (6) 12) Jung K. Y., Oh S. R., Park S. H., Lee I. S., Ahn K. S., Lee J. J., Lee H.
K., Biol. Pharm. Bull., 21, 1077—1078 (1998).
3) Oh S. R., Kinjo J., Shii Y., Ikeda T., Ahn K. S., Kim J. H., Lee H. K.,
Planta Med., 66, 506—510 (2000).
4) Cimanga K., De B. T., Lasure A., Van P. B., Pieters L., Vanden B. D.,
Vlietinck A., Kambuk K., Tona L., J. Nat. Prod., 58, 372—378
(1995).
5) Park S. H., Oh S. R., Jung K. Y., Lee I. S., Ahn K. S., Kim J. H., Kim
Y. S., Lee J. J., Lee H. K., Chem. Pharm. Bull., 47, 1484—1486
showed anti-complement activity with IC50 values of 258,
1
1
4
40, 136, and 34 mM, respectively (Figs. 2, 3). On the other
hand, taxifolin (1) and myricitrin (4) were completely inca-
pable of inhibiting complement activity. Furthermore,
kaempferol (2a), quercetin (3a), and myricetin (4a), which
were obtained from compounds 2, 3, and 4, were also evalu-
ated for their anti-complement activity. Kaempferol (2a) ex-
hibited weak anti-complement activity with an IC50 value of
1
(
1999).
16)
Pieroni A., Pachaly P., Huang Y., Van Poel B., Vlietinck A. J., J.
Ethnopharmacol., 70, 213—217 (2000).
7
30 mM, while quercetin (3a) and myricetin (4a) were inac-