全 文 ：短瓣花中的微量新环肽成分
程永现　周　俊＊　谭宁华
(中国科学院昆明植物研究所植物化学开放实验室 , 昆明 650204)
摘要:　从中国民间草药短瓣花(Brachystemma calycinum D.Don)干燥根的乙醇提取物中分离得到 4 个新的微量环八
肽 ,命名为短瓣花环肽 A、B 、C 和 D。经光谱分析鉴定 ,它们的结构分别为 cyclo(Pro1_Phe_Leu_Ala1_Thr_Pro2_Ala2_Gly)
(1)、cyclo(Pro1_Ala_Phe_Trp_Asp_Pro2_Leu_Gly)(2)、cyclo(Pro1_Ile_Gly_Pro2_Val_Ala1_Ala2_Tyr)(3)和 cyclo(Pro_OMet_Trp_
Ile_Gly_Ala_Leu_Asp)(4)。
关键词:　石竹科;短瓣花;环肽;短瓣花环肽 A、B、C、D
中图分类号:R914　　　文献标识码:A　　　文章编号:0577-7496(2001)07-0760-06
New Minor Cyclic Peptides from Brachystemma calycinum
CHENG Yong_Xian , ZHOU Jun＊ , TAN Ning_Hua
(Laboratory of Phytochemistry , Kunming Institute of Botany , The Chinese Academy of Sciences , Kunming 650204, China)
Abstract:　From the ethanol extract of the roots of Brachystemma calycinum D.Don , a Chinese folk herb ,
four new minor cyclic peptides namely brachystemin A , B , C and D(1-4)have been isolated.Their struc-
tures were established as cyclo (Pro1_Phe_Leu_Ala1_Thr_Pro2_Ala2_Gly)(1), cyclo (Pro1_Ala_Phe_Trp_Asp_
Pro
2_Leu_Gly)(2), cyclo (Pro1_Ile_Gly_Pro2_Val_Ala1_Ala2_Tyr)(3)and cyclo (Pro_OMet_Trp_Ile_Gly_Ala_
Leu_Asp)(4)respectively by means of extensive spectral methods.
Key words:　Caryophyllaceae;Brachystemma calycinum;cyclic peptide;brachystemin A , B , C , D
　　Brachystemma calycinum D.Don(Caryophyllaceae)
is the only species of the genus Brachystemma[ 1] .Among
the folks it has been used as a medicine for the treatment
of rheumatism , limb numbness , impotence and foot ede-
ma
[ 2] .Our previous studies on the family Caryophyllaceae
indicated the presence of cyclic peptides[ 3-5] .As a con-
tinuous research on Caryophyllaceae cyclic peptides , we
investigated the EtOAc extract of the title plant from
which four new cyclooctapeptides namely brachystemin A ,
B , C and D have been isolated and their structures were
elucidated by detailed spectral analyses.
1　Results and Discussion
Brachystemin A (1)was obtained as white amor-
phous powder and tested negative to ninhydrin reagent but
positive after hydrolysis with 6 mol/L HCl at 110 ℃ for 1
h[ 6] .The molecular formula C37H54N8O9was derived from
the high resolution positive FABMS at m/z 755.407 3 [M
+H] + , Calc.for C37H54N8O9 , 755.409 2 , which indi-
cated 15 unsaturations.IR absorptions at 3 413 , 3 309 ,
3 279 , 1 681 , 1 653 cm-1 were characteristic of amide
NH and amide carbonyl groups , respectively.The 1H and
13C_NMR spectra(Table 1)exhibited the presence of sev-
en Cαmethines , one Cαmethene , six amide NH signals
and eight amide carbonyl signals , respectively.The above
evidence indicated that compound 1was a cyclic octapep-
tide.The individual amino acid residues were established
to be one phenylalanine , one leucine , one threonine , one
glycine , two alanines and two prolines by analysis of 1H_
1
H COSY and TOCSY spectra.The linkage of these
amino acid residues was confirmed by HMBC correlations
(Fig.1)between each amide NH and amide CO of the
neighboring amino acid.Observations of 2J (C ,H)con-
nectives betweenδ7.73 and δ172.4(PheNH_Pro1CO), δ
7.10 andδ169.8(LeuNH_PheCO), δ10.61 andδ177.2
(Ala1NH_LeuCO), δ7.50 andδ174.2(ThrNH_Ala1CO), δ
7.96 andδ172.0(Ala2NH_Pro2CO)as well asδ8.96 andδ
173.7(GlyNH_Ala2CO)suggested the presence of two pep-
tide fragments _N_Pro1_Phe_Leu_Ala1_Thr_CO_ and _N_
Pro2_Ala2_Gly_CO_.Only one linkage is possible provided
that cyclic peptide was formed.Hence , compound 1 was
identified as cyclo (Pro1_Phe_Leu_Ala1_Thr_Pro2_Ala2_
Gly).
Brachystemin B (2)was obtained as a white solid
and showed negative reaction to ninhydrin reagent but
Received:2000-09-30　Accepted:2000-12-04
＊Author for correspondence.
植　物　学　报　2001 , 43(7):760-765 　 　 　 　 　 　 　
Acta Botanica Sinica
Table 1　1H and 13C_NMR spectral data of brachystemin A(1)in pyridine_d5
C O Cα Cβ Cγ Cδ HN Hα Hβ Hγ Hδ
Pro1 172.4 66.6 28.2 25.1 47.6 4.94 d 2.31 m 2.05 m 4.01 m
(4.4) 1.80 m 1.96 m 3.40 m
Phe 169.8 55.7 36.1 138.9 130.1 7.73 d 5.01 m 3.45 m 7.18-7.53 m
129.0 (5.6) 3.34 m
127.2
Leu 177.2 49.7 29.1 24.8 21.3 7.10 5.43 m 1.31 m 2.03 m 0.89 d
23.8 br s 1.25 m (6.8)
0.99 d
(6.0)
Ala1 174.2 53.6 16.8 10.61 d 4.30 m 1.55 d
(2.0) (5.6)
Thr 172.2 59.8 68.0 21.8 7.50 d 4.79 d 5.03 m 1.40 d
(7.6) (8.0) (6.4)
Pro2 172.0 64.0 30.0 26.4 47.2 4.61 m 2.02 m 1.55 m 3.16 m
1.32 m 3.38 m
Ala2 173.7 48.8 18.8 7.96 d 5.20 m 1.84 d
(9.6) (7.6)
Gly 169.6 44.1 8.96 t 4.54 dd
(6.4) (16.8 , 5.6)
3.87 dd
(16.8 , 5.6)
Fig.1.　Selective HMBC correlations(H※C)for brachystemin A
(1).
positive after hydrolysis by 6 mol/L HCl at 110 ℃ for 1
h
[ 6] .Its molecular formula C45H57N9O10 with 22 degrees
of unsaturation was achieved from the combination of posi-
tive FABMS at m/ z 906 [M +Na] + , 884 [M+H] + ,
13
C_NMR and DEPT spectra , and confirmed by high reso-
lution positive FABMS at m/z 884.477 5 [ M +H] + ,
Calc.for C45H57N9O10 , 884.480 6.The 1H and 13C_
NMR spectra (Table 2)exhibited the presence of seven
Cαmethines , one Cαmethene , six amide NH signals and
eight amide carbonyl signals , respectively.The above da-
ta suggested that compound 2 was also a cyclic octapep-
tide.Using 1H_1H COSY , TOCSY and HMBC spectra ,
eight amino acid residues were identified as two prolines ,
one alanine , one phenylalanine , one tryptophan , one as-
partic acid , one leucine and one glycine , respectively.
The sequence of these amino acid residues was deduced
by HMBC(J =10 Hz)and ROESY experiments.HMBC
spectrum furnished correlations(Fig.2)between δ9.79
and δ170.8 (PheNH_AlaCO), δ8.90 and δ175.5
(AlaNH_Pro1CO), δ8.47 andδ171.0 (AspNH_TrpCO), δ
9.18 and δ171.9(LeuNH_Pro2CO), suggesting the pres-
ence of three peptide fragments A:_N_Pro1_Ala_Phe_CO_,
B:_NH_Trp_Asp_CO_ and C:_N_Pro2_Leu_CO_.ROESY
spectrum indicated cross peaks(Fig.2)between AspNH_
TrpαH , AlaNH_Pro1αH , GlyNH_LeuαH , LeuNH_Pro2αH , Pro2δH_
AspβH , which suggested the peptide fragment D:_NH_
Trp_Asp_Pro2_Leu_Gly_CO_.One linkage between frag-
ments A and D is reasonable provided that cyclic peptide
was constructed.Hence , compound 2 was identified as
cyclo (Pro1_Ala_Phe_Trp_Asp_Pro2_Leu_Gly).The pro-
posed structure was further confirmed by the positive
FABMS fragments at m/ z 224 [Ala_Pro1_Gly] + and 406
[ Trp_Phe_Ala+2H] +.
Fig.2.　Selective HMBC(H※C)and ROESY correlations(H
H)for brachystemin B(2).
7 期 程永现等:短瓣花中的微量新环肽成分(英) 761　
Table 2　1H and 13C_NMR spectral data of brachystemin B(2)in pyridine_d5
C O Cα Cβ Cγ Cδ HN Hα Hβ Hγ Hδ
Pro1 175.5 61.7 29.3 25.0 47.5 4.61 m 2.24 m 1.78 m 3.54 m
1.68 m 3.50 m
Gly 173.2 44.4 9.50 4.53 m
br s 4.03 br d
(17.2)
Leu 174.3 55.2 42.1 25.4 23.2 9.18 d 5.20 m 2.20 m 3.70 m 0.86 d
21.6 (8.0) 1.80 m (6.4)
0.72 d
(6.0)
Pro2 171.9 61.7 29.7 25.3 47.7 4.90 m 2.00 m 1.80 m 3.82 m
1.79 m 1.50 m 3.59 m
Asp 174.5 50.5 41.5 175.5 8.47 d 5.28 m 3.25 br d
(6.8) (14.0)
3.12 d
(14.0)
Trp 171.0 55.9 25.8 111.3 126.3 9.11 5.05 m 3.70 m 12.01 s
119.4 br s (NH)
124.5 7.30 s
121.8 7.76 d
112.1 (7.6)
137.5 7.57-7.15 m
128.7
Phe 172.7 56.5 37.4 138.8 130.0 9.79 5.00 m 3.50 m 7.57-7.15 m
128.7 br s 3.40 m
126.8
Ala 170.8 50.2 18.0 8.90 4.82 m 1.68 d
br s (6.8)
　　Brachystemin C (3)was obtained as a white solid
and showed negative reaction to ninhydrin reagent but
positive after hydrolysis by 6 mol/L HCl at 110 ℃ for 1
h
[ 6] .Its molecular formula C38H56N8O9 possessing 15 de-
grees of unsaturation was derived from the combination of
positive FABMS at m/ z 769 [ M+H] + , 13C_NMR and
DEPT spectra.The 1H and 13C_NMR spectra (Table 3)
exhibited the presence of seven Cαmethines , one Cα
methene , six amide NH signals and eight amide carbonyl
signals , respectively.These data suggested that com-
pound 3 was a cyclic octapeptide.Amino acid residues
were identified as two prolines , one isoleucine , one
glycine , one valine , two alanines and one tyrosine by the
analysis of
1
H_1H COSY spectrum.The sequence of indi-
vidual amino acid residues was accomplished by detailed
analysis of HMBC spectrum and positive FABMS fragment
pattern.Two peptide f ragments _N_Pro2_Val_Ala1_Ala2_
Tyr_CO_ and _NH_Ile_Gly_CO_ were readily rationalized
by the correlations between δ8.40 andδ174.7 (ValNH_
Pro2CO), δ7.25 andδ170.0(Ala1NH_ValCO), δ9.96 and
δ175.6 (Ala2NH_Ala1CO), δ7.82 and δ173.6 (TyrNH_
Ala
2
CO), δ8.95 andδ172.9(GlyNH_IleCO)in its HMBC
spectrum (Fig.3).In positive FABMS spectrum , the ion
peak at m/ z 268 may be the fragment A:[ Pro2_Gly_Ile+
H] + or B:[ Pro2_Val_Ala1 +H] +.However , the ion
peaks at m/ z 365 [Val_Pro_Gly_Ile_H] + and m/ z 436
[Ala1_Val_Pro2_Gly_Ile_H] + tended to support that frag-
ment A was reasonable.Thus , the proposed structure of
compound 3 was identified as cyclo (Pro1_Ile_Gly_Pro2_
Val_Ala1_Ala2_Tyr).
Brachystemin D (4)was obtained as a white solid
and tested negative to ninhydrin reagent but positive after
hydrolysis with 6 mol/L HCl at 110 ℃ for 1 h[ 6] .The
molecular formula C42H61N9O11S with 17 unsaturations
was derived from the high resolution positive FABMS at
m/z 899.422 5 [ M ] + , Calc.for C42H61N9O11S ,
899.421 1.The 1H and 13C_NMR spectra(Table 4)ex-
hibited the presence of seven Cα methines , one Cα
methene , seven amide NH signals and eight amide car-
bonyl signals.These evidence indicated that compound 4
was a cyclic octapeptide.The individual amino acid
residues were established to be one proline , one S_oxome-
thionine , one tryptophan , one isoleucine , one glycine ,
one alanine , one leucine and one aspartic acid by ana-
lyses of 1H_1H COSY , HMQC and HMBC spectra.The
762　 植　物　学　报　　Acta Botanica Sinica 43 卷
Table 3　1H and 13C_NMR spectral data of brachystemin C(3)in pyridine_d5
C O Cα Cβ Cγ Cδ HN Hα Hβ Hγ Hδ
Pro1 172.1 64.5 29.7 25.5 47.3 4.81 m 2.39 m 1.70 m 3.40 m
1.80 m 2.20 m
Ile 172.9 57.9 37.1 25.1 12.3 8.11 d 5.42 dd 2.72 m 2.12 m 1.06 t
15.5 (10.4) (10.3 , 3.5) 1.27 d (7.4)
(7.5)
Gly 172.8 44.1 8.95 t 4.75 m
(6.1) 3.82 d
(11.6)
Pro2 174.7 64.3 28.4 26.3 47.3 4.68 m 2.32 m 1.60 m 4.27 m
3.26 m
Val 170.0 51.6 33.1 17.1 8.40 d 4.70 m 2.40 m 0.88 d
19.9 (4.5) (6.7)
1.18 d
(6.8)
Ala1 175.6 55.7 17.9 7.25 d 5.34 m 1.50 d
(9.8) (7.40)
Ala2 173.6 54.0 16.6 9.96 4.20 m 1.55 d
br s (6.6)
Tyr 169.4 55.1 36.5 129.9 131.1 7.82 d 5.22 m 3.29 d 7.54 d
157.1 (5.8) (6.28) (8.2)
116.1 7.18 d
(8.2)
Table 4　1H and 13C_NMR data of brachystemin D(4)in pyridine_d5
C O Cα Cβ Cγ Cδ HN Hα Hβ Hγ Hδ
Pro 175.1 62.9 30.3 24.8 48.2 4.78 t 2.33 m 1.98 m 3.90 m
(7.6) 2.02 m
OMet 174.6 56.5 24.5 51.0 38.3＊ 8.58 d 4.75 mb 2.80 mb 3.23 mb 2.54 s＊
(56.6)a (24.5)a (50.4)a (38.4)a (5.60) (2.57 s)a
(8.59 m)a
Trp 172.7 54.2 26.7 109.9 124.5 7.18 5.20 m 3.83 m 12.11 br s
119.1 Overlapped 3.17 (NH)
122.4 Overlapped 7.35 d
119.8 (2.0)
112.3 7.84 d
137.8 (8.0)
128.4 7.28 m
7.25 m
7.52 d
(8.0)
Ile 172.5 50.4 36.8 16.1 12.5 7.50 m 5.24 d 2.19 m 1.06 d 0.97 t
25.2 (6.8) (6.8) (3.6)
1.80 m
1.58 m
Gly 169.1 44.3 8.21 m 4.50 m
3.83 m
Ala 176.6 47.1 19.3 7.69 t 5.18 m 0.69 d
(1.2) (6.8)
Leu 172.9 55.4 40.3 25.2 22.6 9.93 4.28 m 1.70 m 1.80 m 0.76 d
22.1 br s (6.0)
0.72 d
(6.4)
Asp 171.7 57.7 36.4 174.5 7.78 d 5.22 m 3.71 m
(8.0) 3.15　
Overlapped
a , another group of signals exhibited in OMet.b , signals integrated to two protons.＊, presentedωorientation.
7 期 程永现等:短瓣花中的微量新环肽成分(英) 763　
Fig.3.　Selective HMBC correlations(H※C)for brachystemin C
(3).
Fig.4.　Selective HMBC correlations(H※C)for brachystemin D
(4).
linkage of these amino acid residues was disclosed by
HMBC correlations(Fig.4).Observations of 2J(C ,H)
connectives between δ7.78 and δ172.9 (AspNH_
LeuCO), δ9.93 and δ176.6 (LeuNH_AlaCO), δ7.69
and δ169.1 (AlaNH_GlyCO), δ8.21 and δ172.5
(GlyNH_IleCO), δ7.50 and δ172.7 (IleNH_TrpCO), δ
8.58 andδ175.1(OMetNH_ProCO)indicated two peptide
fragments_NH_Trp_Ile_Gly_Ala_Leu_Asp_CO_ and_N_Pro_
OMet_CO_.One linkage is rationalized provided that
cyclic peptide was formed.Hence , compound 4was iden-
tified as cyclo (Pro_OMet_Trp_Ile_Gly_Ala_Leu_Asp).
Interestingly , only the 1H_NMR and 13C_NMR data
of S_oxomethionine in compound 4 exhibited a pair of
peaks(Table 4), as observed with annosquamosin A[ 7] .
The reason remained unclear to date.
2　Experimental
2.1　General experimental procedures
Melting point was recorded on a XRC_1 apparatus ,
and uncorrected.Optical rotation was taken from a JAS-
CO_20C digital polarimeter.IR spectra were measured
with a Bio_Rad FTS_135 spectrometer.FABMS were run
on a VG Auto Spec_3000 spectrometer.1H and 13C_NMR
spectra and 2D_NMR experiments were carried out with a
Bruker AM_400 MHz and a DRX_500 MHz spectrometer ,
respectively.Silica gel (200-300 mesh and 10 -40
μm), RP_18 (40-63 μm)and Sephadex LH_20 were
used for column chromatography.
2.2　Plant material
The roots of Brachystemma calycinum D.Don were
collected in March 1999 in Xishuangbanna of Yunnan
Province , China , which were identified by Prof.
ZHUANG Xuan with a voucher specimen (No.1)pre-
served in the herbarium of Kunming Institute of Botany ,
the Chinese Academy of Sciences.
2.3　Extraction and isolation
The powdered roots of the plant(13.0 kg)were ex-
tracted with 95% ethanol under reflux for three times.
After concentration of the combined extracts in vacuum ,
the residue was suspended in water and partitioned suc-
cessively with petroleum ether(60-90 ℃), EtOAc and
n_BuOH (presaturated by water).The ethyl acetate por-
tion(50.0 g)was subjected to CC over silica gel(2 300
g , 200-300 mesh)eluted with CHCl3_MeOH (17∶1 to
8∶2)to afford subfraction , which was further purified by
alternating RP_18 (gradient MeOH_H2O 45%-70% as
eluent)and Sephadex LH_20 (MeOH_H2O 70% as elu-
ent)CC to furnish brachystemin A (1 , 8 mg), brachys-
temin B (2 , 4 mg), brachystemin C (3 , 5 mg), and
brachystemin D(4 , 5 mg).
2.4　Identification
Brachystemin A (1)　C37H54N8O9 , white amor-
phous powder (in methanol), mp >250 ℃, [ α] 21D
-33.8°(c 0.20 , methanol).IRνKBrmax cm-1:3 413 ,
3 309 , 3 279 , 1 681 , 1 653 and 1 621.Positive FABMS
m/z:755 [ M +H] + , HRFABMS [M +H] + at m/ z
755.407 3(calcd.755.409 2).1H and 13C_NMR data
see Table 1.
Brachystemin B (2)　C45H57N9O10 , white solid
(in methanol), mp 240-242 ℃, [ α] 25D -0.004°(c
0.40 , methanol).Positive FABMS m/ z:906 [ M +
Na] +(25), 884 [M +H] +(100), 776 , 678 , 577 ,
506 , 406 , 224 , HRFABMS [ M + H ] + at m/ z
884.477 5(calcd.884.480 6).1H and 13C_NMR data
see Table 2.
Brachystemin C (3)　C38H56N8O9 , white solid
(inmethanol), mp >250 ℃.[ α] 25D -21.0°(c 0.25 ,
methanol).Positive FABMS m/ z:769 [M+H] +(99),
662 , 606 , 481 , 436 , 382 , 365 , 308 , 268 , 86 , 70
(100).1H and 13C_NMR data see Table 3.
Brachystemin D(4)　C42H61N9O11S1 , white solid
(in methanol), mp 215-217.5 ℃, [ α] 28D -55.0°(c
0.15 , in methanol).Positive FABMS m/ z:899 [M] +
764　 植　物　学　报　　Acta Botanica Sinica 43 卷
(38), 769 , 616 , 486 , 415 , 358 , 301 , 242 , 184 , 159 ,
130 , 70 (100), HRFABMS [ M] + at m/ z 899.422 5
(calcd.899.421 1).1H and 13C_NMR data see Table 4.
Acknowledgements:　The authors heartily appreciate
the analytical group of Kunming Institute of Botany , the
Chinese Academy of Sciences , for the help in determining
the spectral data.
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7 期 程永现等:短瓣花中的微量新环肽成分(英) 765