Flavonol glycosides from muehlenbeckia platyclada and their anti-inflammatory activity

Article abstract of DOI:10.1248/cpb.57.280

A new flavonol, morin-3-O-α-rhamnopyranoside (1), along with four known flavonols, kaempferol 3-O-α-rhamnopyranoside (2), kaempferol 3-O-β-glucopyranoside (3), quercetin 3-O-α-rhamnopyranoside (4) and (+)-catechin (5), were isolated from the methanolic ex

Full text of DOI:10.1248/cpb.57.280

280
Notes
Chem. Pharm. Bull. 57(3) 280—282 (2009)
Vol. 57, No. 3
Flavonol Glycosides from Muehlenbeckia platyclada and Their
Anti-inflammatory Activity
Chiao-Ting YEN,a Pei-Wen HSIEH,b Tsong-Long HWANG,b Yu-Hsuan LAN,c Fang-Rong CHANG,a,d and
,a,d,e
*
Yang-Chang WU
a Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University; Kaohsiung 807, Taiwan:
c
b Graduate Institute of Natural Products, Chang Gung University; Tao-Yuan 333, Taiwan: College of Pharmacy, China
Medical University; Taichung 404, Taiwan: dCenter of Excellence for Environmental Medicine, Kaohsiung Medical
e
University; Kaohsiung 804, Taiwan: and National Sun Yat-Sen University-Kaohsiung Medical University Joint Research
Center; Kaohsiung 804, Taiwan.
Received September 25, 2008; accepted November 18, 2008; published online December 3, 2008
A new flavonol, morin-3-O-a-rhamnopyranoside (1), along with four known flavonols, kaempferol 3-O-a-
rhamnopyranoside (2), kaempferol 3-O-b-glucopyranoside (3), quercetin 3-O-a-rhamnopyranoside (4) and (ꢀ)-
catechin (5), were isolated from the methanolic extract of Muehlenbeckia platyclada. The structures of these com-
pounds were determined on the basis of chemical and spectroscopic evidence, as well as acid hydrolysis of the
original glycoside. Isolates were evaluated for inhibition of generation of superoxide anion, and inhibition of re-
lease of neutrophil elastase. Compound 2 showed moderate inhibition of superoxide anion generation with an
IC50 value of 6.11ꢁ0.86 mg/ml; 1, 3 and 5 inhibited neutrophil elastase release with IC50 values of 3.82ꢁ0.80,
8.61ꢁ1.38 and 4.37ꢁ0.72 mg/ml, respectively, and were 15-fold more potent than phenylmethylsulfonyl fluoride
(PMSF), the positive control, in this anti-inflammatory assay.
Key words Muehlenbeckia platyclada; Polygonaceae; flavonoid; superoxide anion generation; neutrophil elastase
Four species of Muehlenbeckia belong to the plant family chronic obstructive pulmonary disease (COPD), rheumatoid
Polygonaceae. Only one species, Muehlenbeckia platyclada arthritis and asthma.8,9) Activated neutrophils can secrete a
(F.V. MUELL) Meisn, is found in Taiwan.1) In previous studies, series of cytotoxins, including superoxide (O˙2ꢀ), granule pro-
the genus Muehlenbeckia exhibited oxytoxic, analgesic and teases, and bioactive lipids.9—12) Regulating neutrophil acti-
wound-healing activities.2,3) Several chemical constituents vation by using chemical agents has been proposed as a way
have been identified from this genus, including flavonoids, to ameliorate inflammatory diseases.9)
acetophenones, lignans, and anthraquinone.2,4—6)
Bioassay-directed fractionation led to isolation of a new
M. platyclada, commonly known as “ribbon-bush” in Tai- flavonoid glycoside, along with four known flavonoids
wan and China, is an herbal medicine used for treatment of [kaempferol 3-O-a-rhamnopyranoside (2), kaempferol 3-O-
poisonous snake bites and fracture injuries, as well as for al- b-glucopyranoside (3), quercetin 3-O-a-rhamnopyranoside
leviating fever and detoxification.7) However, a phytochemi- (4) and (ꢁ)-catechin (5)] from the extract of M. platyclada
cal investigation has never been done. In a continuing search (Figs. 1, 2). Known compounds were identified by compari-
for biologically active constituents from natural sources, the son of their physical and spectroscopic data with literature
methanolic extract of this species was found to have in- values.13—16) The structural elucidation of the new compound
hibitory effects on the generation of human neutrophil super- and the anti-inflammatory effects of 1—5 are reported
oxide anion and the release of neutrophil elastase induced by herein.
formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; Table
1).
Compound 1 was isolated as a brown powder with the mo-
lecular formula, C22H24O10, deduced from the HR-ESI-MS
Neutrophils play an important role in the defense against (m/z 471.1270 [MꢁNa]ꢁ, Calcd 471.1267). Its IR spectrum
invasion by microorganisms and in the pathogenesis of revealed the carbonyl group of a 4-pyrone (1652 cmꢀ1) and a
hydroxyl group (3352 cmꢀ1). UV data, as well as a violet col-
oration with ferric chloride and pink color in the Shinoda
Table 1. Inhibitory Effects of Crude Extracts on Superoxide Anion Gener-
test,17) indicated that 1 was a flavonoid. On acid hydrolysis of
ation and Elastase Release by Human Neutrophils in Response to fMLP/CB
1, morin and rhamnose were liberated and identified by
HPLC. The ESI-MS (negative mode) spectrum of compound
1 showed a deprotonated ion peak at m/z 447.2 and product
ions were observed at m/z 301.1 ([MꢀHꢀ146]ꢀ), suggesting
a rhamnosyl residue in the molecule. This assignment was
confirmed by the 1H-NMR spectrum, which showed the typi-
cal flavonol a-L-rhamnopyranoside proton pattern at dH 0.94
(3H, d, H-6ꢂ)/dC 17.7, 3.57—3.60 (2H, m, H-4ꢂ, 5ꢂ)/dC 73.3
and 72.0, 3.78 (1H, dd, H-3ꢂ)/dC 72.2, 4.22 (1H, dd, H-2ꢂ)/dC
Superoxide anion
Elastase
Crude extracts
IC50 (mg/ml)a) or (Inh %)
IC50 (mg/ml)a) or (Inh %)
CHCl3 extract
BuOH extract
H2O extract
DPIb)
(25.66ꢄ4.98)**
7.04ꢄ1.49**
(23.94ꢄ3.56)***
0.7ꢄ0.4
(23.04ꢄ4.46)***
16.11ꢄ0.37***
(18.29ꢄ5.94)*
PMSFb)
130.9ꢄ29.1
1
71.9, and 5.35 (1H, d, H-1ꢂ)/dC 103.5.15) In addition, the H-
Percentage of inhibition (Inh %) at 10 mg/ml. Results are presented as meanꢄS.E.M.
(nꢅ3). ∗ pꢆ0.05, ∗∗ pꢆ0.01, ∗∗∗ pꢆ0.001 compared with the control value. a) Con-
centration necessary for 50% inhibition (IC50). b) Diphenyleneiodonium (DPI) and
phenylmethylsulfonyl fluoride (PMSF) were used as positive control in anti-inflamma-
tory.
NMR spectrum showed three aromatic proton signals at d
7.32 (H-3ꢃ), d 7.29 (H-5ꢃ) and d 6.91 (H-6ꢃ), indicative of
the presence of ring B of a 2ꢃ,4ꢃ-substituted flavonoid (Table
∗ To whom correspondence should be addressed. e-mail: yachwu@kmu.edu.tw
© 2009 Pharmaceutical Society of Japan

March 2009
281
Table 2. Inhibitory Effects of Compounds on Superoxide Anion Genera-
tion and Elastase Release by Human Neutrophils in Response to fMLP/CB
Superoxide anion
Elastase
Compound
IC50 (mg/ml)a) or (Inh %)
IC50 (mg/ml)a) or (Inh %)
1
2
3
4
(33.13ꢄ4.37)***
6.11ꢄ0.86***
(25.65ꢄ2.35)***
(34.35ꢄ5.85)**
Nb)
3.82ꢄ0.8***
(16.47ꢄ5.27)*
8.61ꢄ1.38***
(16.58ꢄ4.08)*
4.37ꢄ0.72***
5
DPIc)
PMSFc)
0.7ꢄ0.4
130.9ꢄ29.1
Fig. 1. Flavonol Glycosides Isolated from M. platyclada
Percentage of inhibition (Inh %) at 10 mg/ml. Results are presented as meanꢄS.E.M.
(nꢅ3—4). ∗ pꢆ0.05, ∗∗ pꢆ0.01, ∗∗∗ pꢆ0.001 compared with the control value. a)
Concentration necessary for 50% inhibition (IC50). b) 5 reacts with cytochrome C.
c) Diphenyleneiodonium (DPI) and phenylmethylsulfonyl fluoride (PMSF) were used
as positive control in anti-inflammatory.
1, morin-3-O-a-rhamnopyranoside; 2, kaempferol 3-O-a-rhamnopyranoside; 3,
kaempferol 3-O-b-glucopyranoside; 4, quercetin 3-O-a-rhamnopyranoside.
Table 3. 1H- and 13C-NMR Spectroscopic Data of Morin-3-O-a-rham-
nopyranoside Isolated from M. platyclada (CD3OD)
Position
d
H (multiplicity) JH–H (Hz)
dC
HMBC correlation
Fig. 2. The Structure of (ꢁ)-Catechin
2
3
4
5
6
7
8
9
159.3
136.2
179.6
163.2
3), and two meta-coupled protons at d 6.21 (Jꢅ2.0 Hz) and
6.38 (Jꢅ2.0 Hz), characteristic of flavonoids with 5,7-oxy-
genated substitutions. An anomeric methine at dH 5.35 (1H,
d, 1.4 Hz)/dC 103.5 confirmed a monosaccharide moiety on
the flavonol. Moreover, cross peaks [H-6ꢃ (d 6.91) to C-2 (d
159.3)/C-1ꢃ (d 122.8)/C-4ꢃ (d 149.8)/C-5ꢃ (d 116.4) and H-
1ꢂ (d 5.35) to C-3 (d 136.2)/C-2ꢂ (d 71.9)] in the HMBC
spectrum confirmed the presence of a morin structure and 3-
O-linkage of the rhamnosyl moiety, respectively. Therefore,
the structure was established as shown in Fig. 1 and named
morin-3-O-a-rhamnopyranoside.
The morin structure occurs commonly in plants of Psidium
guajava (Myrtaceae), and bioactivities associated with this
structure include antioxidant and antimicrobial effects.18,19)
While flavonoids are widespread in Muehlenbeckia plants,
the morin structure (2ꢃ,4ꢃ,3,5,7-oxygenated flavonoid) has
been found for the first time in this genus, specifically in M.
platyclada.
6.21 (1H, d)
6.38 (1H, d)
2.0
2.0
99.9 C-5, C-7, C-8, C-10
165.9
94.7 C-7, C-9, C-6, C-10
158.5
10
1ꢃ
2ꢃ
105.9
122.8
146.4a)
3ꢃ 7.32 (1H, d)
4ꢃ
2.2
117.0 C-2ꢃ, C-4ꢃ, C-1ꢃ, C-5ꢃ
149.8a)
5ꢃ 7.29 (1H, dd)
6ꢃ 6.91 (1H, d)
1ꢂ 5.35 (1H, d)
2ꢂ 4.22 (1H, dd)
3ꢂ 3.78 (1H, dd)
4ꢂ 3.57—3.60 (1H, m)
5ꢂ 3.57—3.60 (1H, m)
6ꢂ 0.94 (3H, d)
2.2, 7.6
7.6
1.4
1.4, 3.3
3.2
116.4 C-4ꢃ, C-6ꢃ, C-1ꢃ, C-3ꢃ
123.0 C-1ꢃ, C-5ꢃ, C-4ꢃ, C-2
103.5 C-3, C-2ꢂ
71.9
72.2
73.3
72.0
5.8
17.7 C-4ꢂ, C-5ꢂ
a) Interchangeable.
Furthermore, the isolates were subjected to two anti-in- against human neutrophil elastase release. On the basis of
flammatory assays, superoxide anion generation and elastase these findings, the preliminary structure–activity relation-
release of human neutrophils induced by fMLP (Table 2). ships for anti-inflammatory effects were proposed. There-
Compounds 1, 3 and 5 showed inhibitory effects on the re- fore, the inhibition of neutrophil elastase release by the
lease of neutrophil elastase, with IC50 values of 3.82, 8.61 methanolic extract of M. platyclada was mainly due to the
and 4.37 mg/ml, respectively, and were 15-fold more potent presence of morin 3-rhamnopyranoside, kaempferol 3-glu-
than PMSF as a positive control in this anti-inflammatory copyranoside, and (ꢁ)-catechin.
assay (pꢆ0.001). Compound 2 exhibited a selective in-
hibitory effect on generation of neutrophil superoxide anion,
Experimental
General Experimental Procedures Compound purity was checked by
with an IC50 value of 6.11 mg/ml. According to previous
preparative TLC on precoated silica gel plates (Merck, Kieselgel 60 F254
,
studies,20,21) flavonoids are direct inhibitors of human neu-
trophil elastase. In the present study, comparison of the data
of compounds 1, 2 and 4 showed that when hydroxyl group
was changed to hydrogen group at R1 position, the activity of
inhibition of elastase release reduced, but at the same time,
while hydroxyl group was substituted by hydrogen group at
R2 position, the activity increased (Fig. 1, Table 2). Addition-
ally, compound 3 (kaempferol 3-O-b-glucopyranoside) was
more potent than 2 (kaempferol 3-O-a-rhamnopyranoside),
layer thickness 0.25 mm). Melting points were determined on a Melt-Temp
II apparatus, optical rotations were measured with a JASCO P-1020 digital
polarimeter, and UV spectra were obtained on a Hitachi 200-20 spectropho-
tometer in CD3OD. NMR spectra, both 1D (1H, 13C, DEPT) and 2D (COSY,
TOCSY, HSQC, HMBC and NOESY), were performed at 200 and 50 MHz
1
for H and 13C, respectively, and obtained on a Varian NMR spectrometer.
1H-NMR: CD3OD as solvent, dꢅ3.31 ppm and 13C-NMR: CD3OD as sol-
vent, dꢅ49.0 ppm. Low resolution ESI-MS spectra were obtained on an API
3000TM (Applied Biosystems) in positive or negative mode (solvent:
CH3OH), and high resolution ESI-MS spectra on a Bruker Daltonics APEX
II 30e spectrometer in positive or negative mode (solvent: CH3OH). Shi-
suggesting that 3-O-glucosyl substitution was effective madzu LC-6AD pumps, a SPD-M10A UV–Vis detector, and Develosil,

282
Vol. 57, No. 3
University-Kaohsiung Medical University Joint Research Center, and Center
of Excellence for Environmental Medicine (KMU-EM-97-2.1a). We also
thank Mr. Yun-Zhao Chen for providing plant materials.
ODS 5 mm (250ꢇ4.6 mm i.d.) and preparative ODS 5 mm (250ꢇ21.2 mm
i.d.) columns were employed for HPLC.
Plant Material M. platyclada was collected from Yongkang, Tainan,
Taiwan, in September 2006. Samples were authenticated and deposited at
the Graduate Institute of Natural Products, Kaohsiung Medical University,
Taiwan.
References
1) Boufford D. E., Hsieh C. F., Huang T. C., Chashi H., Yang Y. P., Lu S.
Y., “Flora of Taiwan,” 2nd ed., Vol. II, ed. by Editorial Committee of
the Flora of Taiwan, Taipei, 1996, p. 294.
2) Erazo S., Muñoz O., García R., Lemus I., Backhouse N., Negrete R.,
San Feliciano A., Delporte C., Z. Naturforsch. C, 57, 801—804
(2002).
3) Villegas L. F., Fernández I. D., Maldonado H., Torres R., Zavaleta A.,
Vaisberg A. J., Hammond G. B., J. Ethnopharmacol., 55, 193—200
(1997).
4) Autore G., De Tommasi N., Aquina R., Borrelli F., Tartaglia D., Mas-
colo N., Phytother. Res., 10, 202—206 (1996).
5) Pressland A. J., Keenan F. J., Plant Protect. Quart., 1, 62—66 (1985).
6) Pablo M., Lilia G. G., Cienciay Nat., 14, 2—10 (1973).
7) Je-Chian F., Hsiang-Pei Y., Jen-Chen Wu., ”Quanzhou Herbal Medi-
cine,” ed. by Quanzhou Committee on Science and Technology
Quanzhor City Bureau Quanzhou Institute of Medicine Science, ROC,
1961.
8) Nathan C., Nat. Rev. Immunol., 6, 173—182 (2006).
9) Hwang T. L., Hung H. W., Kao S. H., Teng C. M., Wu C. C., Cheng S.
J., Mol. Pharmacol., 64, 1419—1427 (2003).
Extraction and Isolation The air-dried plant (1.2 kg) of M. platyclada
was extracted four times at room temperature with CH3OH. Combined
CH3OH extracts were evaporated under vacuum and distilled to yield a dried
green residue (62.84 g). The latter was dissolved in CHCl3 and extracted
with H2O. The H2O solution was partitioned with BuOH to give a H2O layer
(37.74 g) and a BuOH layer (15.5 g). The BuOH extract was further sepa-
rated on RP-18 column (VersaPakTM, 40ꢇ150 mm, Supelco) and MPLC
with a gradient of 80% H2O/MeOH, 60% H2O/CH3OH, 40% H2O/CH3OH,
20% H2O/CH3OH and CH3OH (each 1000 ml) to yield five fractions (A—
E). Fraction C (740 mg) was separated on Sephadex LH-20 with CH3OH to
give five subfractions (C1—C5). Subfraction C4 was subjected to column
chromatography on silica gel. Elution started with CHCl3/CH3OH 20/1 to
yield 1 (6.08 mg, CHCl3/CH3OH 5/1), kaempferol 3-O-a-L-rhamnopyra-
noside (2) (14.1 mg, CHCl3/CH3OH 10/1) and kaempferol 3-O-b-glucopyra-
noside (3) (3.84 mg). Fraction D (1.12 g) was separated on Sephadex LH-20
with CH3OH to give five subfractions (D1—D5). Subfraction D4 was fur-
ther purified by RP-18 column (LiChroprep, 40—63 mm, Merck) and
preparative HPLC (CH3OH/H2O 65/35, flow rate 3 ml/min, detection at
280 nm) to yield quercitrin (4) (8.7 mg) and (ꢁ)-catechin (5) (14.5 mg).
Morin-3-O-a-rhamnopyranoside (1): Brown amorphous powder. UV
(CH3OH) lmax nm (log e): 260 (4.41), 351 (4.26); mp: 270—280 °C; [a]D
ꢀ20.8° (cꢅ0.003, CH3OH); IR, n (KBr) cmꢀ1 3352, 1652, 1606, 1510,
1355, 1301, 1109, 1088; 1H-NMR (CD3OD, 200 Hz): see Table 3; 13C-NMR
(CD3OD, 50 Hz): see Table 3; HR-ESI-MS m/z: 471.1270 [MꢁNa]ꢁ, Calcd
471.1267 for C22H24O10Na.
10) Ross D., Van Bruggen R., Meischl C., Microb. Infect., 5, 1307—1315
(2003).
11) Witko-Sarsat V., Rieu P., Descamps-Latscha B., Lesavre P., Halb-
wachs-Mecarelli L., Lab. Invest., 80, 617—653 (2000).
12) Borregaard N., Curr. Opin. Hematol., 3, 8—11 (1996).
13) Nørbæk R., Nielsen J. K., Kondo T., Phytochemistry, 51, 1133—1138
(1999).
14) Kazuma K., Noda N., Suzuki M., Phytochemistry, 62, 229—237
(2003).
15) Zhong X. N., Otsuka H., Ide T., Hirata E., Takushi A., Takeda Y., Phy-
tochemistry, 46, 943—946 (1997).
Acid Hydrolysis The mixture of compound 1 and 1% aqueous HCl
(1 ml) was heated in a capped tube on water bath at 85 °C for 2 h. Then,
methanol was added to the resulting solution and sonicated for 10 min. The
solution was re-filtered prior to HPLC injection. The water eluate was col-
lected and submitted to HPLC analysis of the sugar. A further eluate with
MeOH was collected and submitted to HPLC analysis of the flavonoid agly-
cone.
16) Bilia A. R., Morelli I., Hamburger M., Hostettmann K., Phytochem-
istry, 43, 887—892 (1996).
17) Shinoda J., J. Pharm. Soc. Jpn., 48, 214—220 (1928).
18) Bourama N., Annelise L., Byung H. U., Robert A., Mamadou E. K.
Kone, Biochem. Syst. Ecol., 33, 309—312 (2005).
19) Arima H., Danno G.-i., Biosci. Biotechnol. Biochem., 66, 1727—1730
(2002).
Assays of Superoxide Anion Generation and Elastase Release of
Human Neutrophils Induced by fMLP All isolates from the entirety of
M. platyclada were purified before bioassay (purity ꢈ99%). The assays were
carried out according to established protocols.14)
20) Siedle B., Hrenn A., Merfort I., Planta Med., 73, 401—420 (2007).
21) Melzig M. F., Loser B., Ciesielski S., Pharmazie, 56, 967—970
(2001).
Acknowledgement We gratefully acknowledge financial support for
this project from the National Science Council, National Science Technol-
ogy Program/Biotechnology and Pharmaceuticals, National Sun Yat-Sen