The species Dianthus superbus var. longicalysinus is an
Oriental drug for treating diuretic, carcinoma, and
inflamma-tory.
1)In previous studies, nine new triterpene saponins were
isolated, and showed anti-hepatotoxic and anti-inflammatory
activities.
1—4)As part of our research for bioactive
con-stituents of Dianthus sp., we previously investigated the
ex-tract of Dianthus superbus, a Chinese medicine, and isolated
four new cyclic peptides dianthins C—F, along with a new
dianthramide.
5)Among them, a cyclic peptide, dianthin E,
and dianthramide, 4-methoxydianthramide B, showed
selec-tively cytotoxicity to Hep G2 cancer cell line. In a continuing
research of this genus plants, a new cyclic peptide,
longica-lycinin A (1), together with six known compounds,
vaccaro-side A,
6)dianoside A,
1)dianoside G,
3)3-(4-hydroxy-3-methoxy-phenyl)propionic acid methyl ester,
7)p-hydroxy-benzoic acid, and p-hydroxybenzaldehyde, were isolated
from D. superbus var. longicalysinus. The isolation and
structural elucidation of the new compound are reported
herein.
The methanolic extracts of D. superbus var. longicalysinus
were partitioned between n-hexane and 80% aqueous MeOH.
The latter extract was further partitioned between H
2O and
n-BuOH. Among them, the n-BuOH layer showed the peptide
signals in its NMR spectrum. Therefore, this layer was
fur-ther separated, and gave a new compound (Fig. 1),
longica-lycinin A (1), together with six known compounds,
vaccaro-side A, dianosdie A, dianovaccaro-side G,
3-(4-hydroxy-3-methoxy-phenyl)propionic acid methyl ester, p-hydroxybenzoic acid,
and p-hydroxybenzaldehyde. The known compounds were
identified by comparisons of spectral data with those
re-ported.
1,3,6,7)Longicalycinin A (1) was obtained as pale yellow powder.
Absorptions at 3407, 1685, and 1518 cm
1in the IR were
characteristic of amide, carbonyl, and aromatic functions,
re-spectviely. The NMR spectra of 1 (Table 1) showed four
amide N–H signals and five carbonyls, which indicated that 1
might belong to a peptide class of compound. A negative
ninhydrin test indicated its cyclic nature. The molecular
weight 611 Da was obtained from ESI mass spectrum, which
showed the pseudomolecular ion [M
H]
at m/z 612, and
the sodium adduct ion at m/z 634, respectively. Analysis of
2D NMR data (TOCSY and ROESY) and ESI-MS
3data
demonstrated that the amino acid residues are Gly, Pro, Try,
and Phe
2. The sequence of the amino acid residues was
deduced from ESI-MS
3analysis (Fig. 2). As shown, the
colli-sional induced decomposition (CID) experiment on the
[M
H]
ion of 1 gave preferential ring opening at the Phe
2-Tyr
3amide bond, and gave relative B ions (a peptide
frag-mented at a single peptide bond retaining the positive charge
at the N-terminus) of peptide fragments. The fragment ion at
m/z 465 could be attributed to Tyr
3-Pro
4-Phe
5-Gly
1-, and was
followed by the subsequent losses of Gly
1and Phe
2.
Further-more, a series of A ion (a peptide fragmented at a C–C
O
bond retaining the positive charge at the N-terminus)
frag-ments also obtained at m/z 584, 437, 380, 233, and 136,
which were assigned to Phe
2-Gly
1-Phe
5-Pro
4-Tyr
3. Thus the
structure of 1 was established as cyclo(Gly
1-Phe
2-Tyr
3-Pro
4-Phe
5). The difference of
13C-NMR chemical shifts of Pro
4(Dd
Cb-Cg7.6 ppm) provided evidence that the amide bond in
the Pro residue is cis.
8)The configuration of each amino acid
residue was assigned as L, which was deduced by acid
hy-drolysis and Marfey’s analysis of the individual amino
acids.
9,10)The secondary structure of 1 was not included
helix, turn, and b-sheet, and was confirmed by CD spectrum
which showed three negative Cotton effect at 217, 212, and
196 nm.
11—13)However, it still needs more evidence to
estab-lish the conformation of 1.
The cytotoxicities of isolates were evaluated against the
cell lines of human hepatocellular carcinoma Hep G2 and
Hep 3B, human breast carcinoma MCF-7 and
MDA-MB-231, and human lung carcinoma A-549, respectively.
How-ever, only compound 1 showed activity against Hep G2
can-cer cell line with IC
50value 13.52
mg/ml.
∗ To whom correspondence should be addressed. e-mail: [email protected] © 2005 Pharmaceutical Society of Japan
Longicalycinin A, a New Cytotoxic Cyclic Peptide from Dianthus superbus
var. longicalycinus (M
AXIM
.) W
ILL
.
Pei-Wen H
SIEH,
aFang-Rong C
HANG,
aChing-Chung W
U,
aChien-Ming L
I,
bKuen-Yuh W
U,
bSu-Li C
HEN,
aHsin-Fu Y
EN,
cand Yang-Chang W
U*
,aaGraduate Institute of Natural Products, Kaohsiung Medical University; Kaohsiung 807, Taiwan: bDivisions of
Environmental Health and Occupational Medicine, National Health Research Institute; Kaohsiung 807, Taiwan: and
cNational Museum of Natural Science; Taichung, 404, Taiwan. Received October 23, 2004; accepted January 5, 2005
A new cyclic peptide, longicalycinin A (1), and six known compounds, vaccaroside A, dianoside A, dianoside G, 3-(4-hydroxy-3-methoxy-phenyl)propionic acid methyl ester, p-hydroxybenzoic acid, and p-hydroxybenzalde-hyde were isolated from the MeOH extract of Dianthus superbus var. longicalycinus. The amino acid sequences of 1 was elucidated as cyclo(Gly1-Phe2-Tyr3-Pro4-Phe5-) on the basis of ESI tandem mass fragmentation analysis, chemical evidence, and extensive 2D NMR methods. Furthermore, compound 1 showed cytotoxicity to Hep G2 cancer cell line.
Key words Dianthus superbus var. longicalycinus; cyclic peptide; cytotoxicity
336 Notes Chem. Pharm. Bull. 53(3) 336—338 (2005) Vol. 53, No. 3
Experimental
Optical rotations were measured with a JASCO P-1020 digital polarime-ter. The UV spectra were obtained on a Hitachi 200-20 spectrophotometer, and IR spectra were measured on a Hitachi 260-30 spectrophotometer. CD spectra were measured on a Jasco J-810 circular dichroism spectrometer (using 0.5 cm length cell). NMR (using C5D5N as solvents) spectra were
ob-tained on a Varian Unity Plus 400 FT-NMR. ESI-MSnwas recorded on an
API 3000TM(Applied Biosystems). Low-resolution EI-MS were collected on
a Quattro GC/MS spectrometer having a direct inlet system. High-resolution FAB-MS were collected on a Finnigan/Thermo Quest MAT 95XL spectrom-eter. High-resolution EI-MS were collected on a JEOL JMS SX/SX 102A spectrometer. Shimadzu LC-10AT pumps, SPD-10A UV–VIS detector, and Hypersil ODS 5mm (2504.6 mm i.d.) and preparative ODS 5 mm (25021.2 mm i.d.) columns were employed in a HPLC system.
Plant Material D. superbus var. longicalysinus was collected from Nan-Tao (Lu-Guo) and identified by Dr. Hsin-Fu Yen (National Museum of Natural Science, Taichung, Taiwan). The samples were authenticated and deposited in the Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan (KMU-DS-002).
Extraction and Isolation The air-dried plant (180 g) of D. superbus var. longicalysinus was extracted with MeOH at room temperature. The methanol extract (16 g) was partitioned between n-hexane–80% MeOH/H2O
to yield n-hexane and MeOH extracts. The MeOH extract (10 g) was further partitioned between H2O and n-BuOH to yield H2O and n-BuOH extracts.
The n-BuOH extract (2 g) was separated on Sephadex LH-20 with 80% MeOH/H2O to give six fractions (A—F). Fraction C (300 mg) was further
March 2005 337
Table 1. 1H- (400 MHz) and 13C- (100 MHz) NMR Data of 1 in C 5D5N
dH, mult. (J in Hz) dC, mult. ROESY (dH)
Gly1 CO 170.7 (s) NH 8.29 (br d, 8.8) a 4.84 (dd, 16.0, 8.8) 43.6 (t) 3.62 3.60 (dd, 16.0, 8.8) 4.84 Phe2 CO 171.2 (s) NH 10.90 (d, 7.2) 5.05 a 4.37 (m) 57.7 (d) b 3.62 (m) 40.0 (t) 3.85 3.85 (m) 3.62 Ar 7.00—7.41 (m) 140.2 (s) 130.2 (d) 128.9 (d) 126.8 (d) Tyr3 CO 171.3 (s) NH 8.43(br d, 9.6) a 5.34 (ddd, 14.4, 9.6, 9.6) 54.3 (d) 7.56, 7.34, 4.37, 3.95, 3.43 b 3.43 (m) 35.5 (t) 7.56, 7.34, 5.34, 3.95 3.95 (m) 7.56, 7.34, 3.43 Ar 127.5 (s) 7.56 (d, 8.4) 130.7 (d) 7.34 7.34 (d, 8.4) 116.4 (d) 7.56 157.9 (s) Pro4 CO 170.7 (s) a 4.40 (t, 8.0) 60.3 (d) b 1.81 (m) 30.5 (t) g 1.90 (m) 22.9 (t) 1.65 (m) d 3.80 (m) 48.8 (t) 3.42 (m) Phe5 CO 171.2 (s) NH 8.53 (br s) a 5.05 (m) 57.0 (d) 10.90, 3.26, 3.20 b 3.20 (m) 36.7 (t) 5.05, 3.26 3.26 (m) 5.05, 3.20 Ar 7.00—7.41 (m) 138.5 (s) 129.8 (d) 128.8 (d) 126.6 (d)
purified by HPLC (MeCN/H2O 30 : 70, flow rate: 3.6 ml/min, detection at
220 nm) to give 1 (2.4 mg). Fraction B (320 mg) was further separated using an RP-18 (LiChroprep, 40—63 mm, Merck) flash column (eluting with H2O,
90% MeCN/H2O, 70% MeCN/H2O, 50% MeCN/H2O, and 100% MeCN)
and afford five fractions. The subfraction B-2 was purified by preparative re-verse-phase HPLC (MeCN/H2O25 : 75, flow rate: 3.6 ml/min, detection at
225 nm) to obtain vaccaroside A (8.3 mg), dianoside A (4.8 mg), and di-anoside G (3.2 mg). The subfraction B-3 was purified by preparative reverse-phase HPLC (MeCN/H2O30 : 70, flow rate: 3.6 ml/min, detection at
225 nm) to give dianoside A (4.8 mg). The fraction D (125 mg) was purified by preparative HPLC (MeCN/H2O36 : 64, flow rate: 3.5 ml/min, detection
at 254 nm) to yield 3-(4-hydroxy-3-methoxy-phenyl)propionic acid methyl ester (2.3 mg). The fraction E (55 mg) was purified by silica gel column to yield p-hydroxybenzoic acid (4.2 mg), and p-hydroxybenzaldehyde (3.3 mg).
Hydrolysis and Derivatization of 111,12) Compound 1 (0.1 mg) was
hydrolysis following methods as described previously.5,10)
Electrospray Ionization Tandem Mass Spectrometry This experi-mental was processed using methods as described previously.5,10)
Cytotoxicity Assays This assay was performed using methods as de-scribed previously.5)
Longicalycinin A (1): Pale-yellow powder; [a]D2512° (c0.01, MeCN);
UV (MeCN) lmax (loge) 202 (4.01), 230 (3.75), 264 (sh, 3.52) nm; CD
(c1.6104M, MeOH) lmax (mdeg) 217 (sh, 3.45), 212 (3.80), 204
(2.39), 196 (4.72) nm; IR (KBr) nmax3407, 2925, 2857, 1685, 1604, 1518, 1449, 1280, 1090, 1017, 847, 772, 752 cm1; HR-FAB-MS m/z 612.2750 ([MH], Calcd for C34H37N5O6: 612.2744); 1H-NMR (400 MHz, C5D5N) and 13C-NMR (100 MHz, C
5D5N), see Table 1; ESI-MS (Full scan)
m/z 634 (100, [MNa]), 612 (25, [MH]); ESI-MS/MS m/z 612 (100, [MH]), 584 (33), 465 (20), 437 (9), 408 (12), 380 (9), 302 (15), 278 (14), 261 (48), 233 (62), 204 (6), 172 (6), 136 (6), 120 (12), 69 (5).
Acknowledgments This work was supported by a grant from the National Science Council of the Republic of China.
References
1) Oshima Y., Ohsawa T., Oikawa K., Konno C., Hikino H., Planta Med.,
50, 40—43 (1984).
2) Oshima Y., Ohsawa T., Hikino H., Planta Med., 50, 43—47 (1984). 3) Oshima Y., Ohsawa T., Hikino H., Planta Med., 50, 254—258 (1984). 4) Hikino H., Ohsawa T., Kiso Y., Oshima Y., Planta Med., 50, 353—355
(1984).
5) Hsieh P. W., Chang F. R., Wu. C. C., Wu K. Y., Li C. M., Chen S. L., Wu Y. C., J. Nat. Prod., 67, 1522—1527 (2004).
6) Koike K., Jia Z., Nikaido T., Phytochemistry, 47, 1343—1349 (1998). 7) Lam T. B. T., Iiyama K., Stone B. A., Phytochemistry, 31, 1179—1183
(1992).
8) Douglas E. D., Bovey F. A., J. Org. Chem., 38, 2379—2383 (1973). 9) Marfey P., Carlsberg Res. Commun., 49, 591—596 (1984).
10) Hsieh P. W., Chang F. R., Wu C. C., Wu K. Y., Li C. M., Wang W. Y., Gu L. C., Wu Y. C., Helv. Chim. Acta, 87, 55—66 (2004).
11) Johnson W. C., Tinoco I., J. Am. Chem. Soc., 94, 4389—4390 (1972). 12) Toniolo C., Polese A., J. Am. Chem. Soc., 118, 2744—2745 (1996). 13) Brahms S., Spach G., Brack A., Proc. Natl. Acad. Sci. U.S.A., 74,
3208—3212 (1977).
