Bioactive compounds from the aerial parts of brachystemma calycinum and structural revision of an octacyclopeptide

Article abstract of DOI:10.1021/np200048u

Four new cyclic peptides, brachystemins F - I (1 - 4), and 11 known compounds were isolated from the aerial parts of Brachystemma calycinum. The absolute configurations of compounds 1 - 4 were assigned using Marfey's method. The structure of compound 5 was revised from cyclo(Pro¹ - Phe ² - Leu³ - Ala⁴ - Thr⁵ - Pro ⁶ - Ala⁷ - Gly⁸) to cyclo(Pro¹ - Pro² - Ala³ - Gly⁴ - Leu⁵ - Ala ⁶ - Thr⁷ - Phe⁸) with QTOF/MS and X-ray diffraction analysis. The N-containing compounds were assessed for their inhibitory effects on the secretion of monocyte chemokine ligand 2 (CCL-2), interleukin 6 (IL-6), and collagen IV against high-glucose-stimulated mesangial cells. Compound 5 was evaluated for its effects on collagen I, reactive oxygen species (ROS), superoxide anion (O₂^?-) production, and cell viability in mesan ial cells, and on nitric oxide (NO) production in macrophage cells.

Full text of DOI:10.1021/np200048u

ARTICLE
pubs.acs.org/jnp
Bioactive Compounds from the Aerial Parts of Brachystemma
calycinum and Structural Revision of an Octacyclopeptide
†
,||
‡,||
†,§
^
,‡
,†
Jun Zhao, Li-Li Zhou, Xi Li, Hong-Bin Xiao, Fan-Fan Hou,* and Yong-Xian Cheng*
†
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, People’s Republic of China
‡
Division of Nephrology, Nanfang Hospital, Southern Medical University, Key Laboratory for Organ Failure Research,
Education Ministry, Guangzhou, 510515, People’s Republic of China
§
College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, People’s Republic of China
^Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People’s Republic of China
S Supporting Information
b
ABSTRACT: Four new cyclic peptides, brachystemins FꢀI
(
1ꢀ4), and 11 known compounds were isolated from the aerial
parts of Brachystemma calycinum. The absolute configurations
of compounds 1ꢀ4 were assigned using Marfey’s method. The
structure of compound
5
was revised from cyclo-
5 6 7 8
1
2
3
4
(
(
Pro ꢀPhe ꢀLeu ꢀAla ꢀThr ꢀPro ꢀAla ꢀGly ) to cyclo-
1
2
3
4
5
6
7
8
Pro ꢀPro ꢀAla ꢀGly ꢀLeu ꢀAla ꢀThr ꢀPhe ) with
QTOF/MS and X-ray diffraction analysis. The N-containing
compounds were assessed for their inhibitory effects on the
secretion of monocyte chemokine ligand 2 (CCL-2), inter-
leukin 6 (IL-6), and collagen IV against high-glucose-stimulated
mesangial cells. Compound 5 was evaluated for its effects on
collagen I, reactive oxygen species (ROS), superoxide anion
•
ꢀ
(
O2 ) production, and cell viability in mesangial cells, and on
nitric oxide (NO) production in macrophage cells.
iabetic nephropathy (DN) has become a public health
and the development of cartilagenous lesions in experimental dog
10
D
concern in recent years because it can develop into renal
failure, with a high risk of morbidity and mortality. Diabetes mellitus
is a primary driver of DN. With changes in lifestyle and dietary
patterns, it is estimated that 7% of the United States population
suffers from diabetes, and the incidence of diabetes mellitus
continues to grow worldwide, which means that the number of
osteoarthritis by inhibiting protease-activated receptor 2. Inflam-
matory and immune processes are implicated in rheumatism, and
it may be possible to isolate anti-inflammatory or immunoregula-
tory compounds from B. calycinum that may be useful in the
treatment of DN. Immunosuppressive alkaloids were character-
9
ized from the roots of this plant. As a continuation of our search
1
11,12
patients with DN will increase in the coming years. Although some
for bioactive agents that will benefit patients with DN,
17
preventive measures, such as glycemic and blood pressure control,
are currently used to reduce the kidney damage attributable to
kidney disease, the cause of DN is still not known, which makes the
treatment of DN challenging. Recent experimental and clinical
studies demonstrated that inflammation and oxidative stress are
compounds were isolated from the aerial parts of this plant. The
structure of a previously reported cyclic peptide was revised. The
selected compounds were evaluated for their inhibitory effects on
CCL-2, IL-6, and collagen IV secretion. In addition, the most
active compound, 5, was further tested for the effects on collagen I,
ROS, O2 inhibition, and cell viability in mesangial cells and on
NO production inhibition in macrophage cells.
•
ꢀ
1ꢀ5
involved in the development and progression of DN.
This
pathogenic perspective will open new therapeutic approaches to
DN, targeting inflammatory processes and oxidative stress.
Brachystemma calycinum D. Don (Caryophyllaceae) is the
single species of the genus Brachystemma. It is a Chinese folk
medicine used to treat rheumatoid arthritis, limb numbness,
’
RESULTS AND DISCUSSION
Compound 1 was isolated as a white powder. Its molecular
formula was determined to be C H N O by the HRESIMS at
40 60 8 10
6
impotence, and gonorrhea. Previous studies showed that it
7
ꢀ9
contains alkaloids and cyclic peptides. An extract of the whole
plant was recently found to be able to reduce disease symptoms
Received: January 20, 2011
Published: June 02, 2011
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
1392
dx.doi.org/10.1021/np200048u |J. Nat. Prod. 2011, 74, 1392–1400

Journal of Natural Products
ARTICLE
þ
13
m/z 835.4314 [M þ Na] (calcd 835.4330) and C NMR and
Compound 2 was isolated as an amorphous solid. It showed a
þ
DEPT spectra, indicating 15 degrees of unsaturation. The IR
absorptions at 3419, 1657, and 1526 cm were ascribable to
quasimolecular ion at m/z 880.3976 [M þ Na] in the HRE-
ꢀ
1
13
SIMS spectrum, in conjunction with the C NMR and DEPT
amino, amide carbonyl, and aromatic groups, respectively. The
spectra, suggesting the molecular formula C H N O Na
43 55 9 10
1
H NMR spectrum showed six amide NH resonances, and the
C NMR spectrum indicated eight amide carbonyls and eight R-
(calcd 880.3969) with 21 degrees of unsaturation. The IR
1
3
ꢀ1
spectrum showed absorption bands of amine (3417 cm ),
ꢀ
1
amino acid carbons (Table 1), suggesting that 1 is a peptide. A
negative response to the ninhydrin reagent but a positive
response after its hydrolysis with 6 N aqueous HCl implied that
amide carbonyl (1659 and 1628 cm ), and aromatic
1 1
ꢀ
(1513 cm ) groups. The H NMR spectrum showed seven
13
amide NH resonances, and the C NMR spectrum displayed
eight amide carbonyl carbons. These data were indicative of a
cyclic peptide. Using COSY, HMQC, and HMBC spectra, the
eight amino acid residues were identified as Phe, Trp, Gly, Ala,
Thr (ꢁ2), and Pro (ꢁ2). These residues accounted for 20
degrees of unsaturation, suggesting that 2 is a cyclic octapeptide.
The sequence of 2 was assembled with HMBC and ROESY
1
is a cyclic peptide. Analysis of 2D NMR data (COSY, HMQC,
HMBC, and NOESY-ROESY spectra) permitted the assignment
of the H and C NMR resonances and also allowed the
1
13
diagnosis of the eight amino acid residues as Pro (ꢁ2), Thr
(
ꢁ2), Leu, Gly, Val, and Phe.
2
experiments. The HMBC spectrum showed correlations of Ala -
1
3
2
4
3
5
NH/Pro -CO, Gly -NH/Ala -CO, Phe -NH/Gly -CO, Thr -
4
6
5
7
6
NH/Phe -CO, Thr -NH/Thr -CO, and Trp -NH/Thr -CO
1
(
Figure 2), corresponding to the peptide fragment Pro ꢀ
2
3
4
5
6
7
Ala ꢀGly ꢀPhe ꢀThr ꢀThr ꢀTrp . The observed ROESY
7
8
8
1
correlations of Trp -HR/Pro -Hδ, Pro -HR/Pro -Hδ, and
1
2
7
8
Pro -HR/Ala -NH suggested the fragment Trp ꢀPro ꢀ
1
2
Pro ꢀAla (Figure 2). These data implied that the sequence of
1
2
3
4
5
6
7
8
2
is cyclo(Pro ꢀPro ꢀAla ꢀGly ꢀPhe ꢀThr ꢀThr ꢀTrp ).
This conclusion was confirmed with QTOF/MS data, which
demonstrated a series of adjacent b (þ1) ions at m/z 672, 571,
n
4
70, 323, and 195, corresponding to the successive loss of Trp,
Thr, Thr, Phe, AlaꢀGly, and the terminal dipeptide ion Proꢀ
Pro. In addition, b (þ1) ions at m/z 266 and 195 were observed
n
(
Figure 1), in accordance with the peptide sequence Proꢀ
ProꢀAla (Figure 2). The geometry of the amide bonds of both
1
8
13
Pro and Pro was trans, on the basis of the difference in the
C
8
1
NMR chemical shifts of Pro (Δδ
= 2.9 ppm) and Pro
CβꢀCγ
13,14
(
Δδ
= 3.0 ppm).
CβꢀCγ
The molecular formula of compound 3 was established to be
1
3
C H N O by HRESIMS, C NMR, and DEPT spectra. The
4
5 58 10 9
ꢀ
1
IR absorptions at 3415, 3342, 1663, and 1524 cm indicated the
presence of amino, hydroxy, amide carbonyl, and aromatic
groups, respectively. The peptidic nature was suggested by the
presence of eight amide protons and nine amide carbonyl signals
in the H and C NMR spectra. The amino acid residues were
identified as Phe, Ile, Gly, Ala, Trp, Asn, and Pro (ꢁ2) by analysis
of the COSY, HMQC, and HMBC spectra. HMBC correlations
1
13
2
1
3
2
were observed between Phe -NH/Pro -CO, Ile -NH/Phe -CO,
4
3
5
4
4
3
Gly -NH/Ile -CO, Ala -NH/Gly -CO, and Gly -HR/Ile -CO,
1
2
3
4
5
The sequence of the amino acid residues was deduced with
indicating the peptide fragment Pro ꢀPhe ꢀIle ꢀGly ꢀAla .
8
1
6
HMBC, ROESY, and QTOF/MS experiments. A peptide frag-
ment, GlyꢀLeuꢀThr, was evident from the HMBC correlations
of Thr-NH/Leu-CO and Leu-NH/Gly-CO (Supporting Infor-
mation, S29). The ROESY spectrum demonstrated the correla-
tion Phe-NH/Pro-δH (Supporting Information, S36). The
Correlations in the ROESY spectra of Asn -HR/Pro -Hδ, Pro -
5 6 7
Hδ/Ala -HR, and Pro -HR/Trp -NH indicated the two peptide
5
6
7
8
1
units Ala ꢀPro ꢀTrp and Asn ꢀPro . The QTOF/MS data
demonstrated a series of adjacent b (þ1) ions at m/z 769, 583,
n
486, 415, 358, and 195, corresponding to the successive loss of
Asn, Trp, Pro, Ala, Gly, Ile, and the terminal dipeptide ion
ProꢀPhe (Figure 1). Thus, the structure 3 was determined to be
þ
protonated molecular ion [M þ H] of 1 (m/z 813) was
subjected to QTOF/MS analysis. The preferred ring-opening
1
2
3
4
5
6
7
8
occurred at PheꢀPro and gave a main series of adjacent b (þ1)
cyclo(Pro ꢀPhe ꢀIle ꢀGly ꢀAla ꢀPro ꢀTrp ꢀAsn ). The
n
ions at m/z 666, 565, 468, 367, 254, and 197 (Figure 1),
corresponding to the successive loss of Phe, Thr, Pro, Thr,
Leu, Gly, and the terminal dipeptide ion ProꢀVal. Taking these
data together, the sequence of 1 was determined to be cyclo-
trans configuration of the amide bonds in the two Pro residues
was deduced from their chemical shift differences in Δδ
values (4.4 ppm for Pro and 4.2 ppm for Pro ).
CβꢀCγ
1
6 13,14
Compound 4 had the molecular formula C H N O ,
43
56
9
10
1
2
3
4
5
6
7
8
þ
(
Pro ꢀVal ꢀGly ꢀLeu ꢀThr ꢀPro ꢀThr ꢀPhe ). Further-
deduced from the ion at m/z 858.4132 [M þ H] in its
1
3
1
13
more, the C NMR chemical shift differences of Pro
(
HRESIMS spectrum, C NMR, and DEPT spectra. The
6
1
13
Δδ
= 3.2 ppm) and Pro (Δδ
= 3.5 ppm) indicated
H and C NMR data were similar to those of compound 2.
The octapeptide skeleton was suggested by the presence of seven
CβꢀCγ
CβꢀCγ
13,14
that the amide bonds in the two Pro residues were trans.
1
393
dx.doi.org/10.1021/np200048u |J. Nat. Prod. 2011, 74, 1392–1400

Journal of Natural Products
ARTICLE
1
13
Table 1. H (400 MHz) and C (100 MHz) NMR Data for 1 and 2 in Pyridine-d5
1
2
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
1
a
1
Pro
R
5.03, overlap
2.30, m
64.3, CH
28.3, CH
Pro
R
4.57, t (8.4)
2.26, m
63.9, CH
29.3, CH
β
2
β
2
1
.80, m
1.80, m
γ
δ
2.27, m
4.09, m
25.1, CH
2
2
γ
δ
1.35, m
26.4, CH
2
2
b
47.4, CH
2.88, t (4.8)
1.92, m
47.0, CH
2
.27, overlap
c
CdO
R
172.8, qC
57.2, CH
29.9, CH
CO
R
172.0, qC
48.8, CH
2
2
Val
5.35, m
Ala
5.18, m
β
2.97, m
β
1.88, d (7.6)
8.20, d (9.6)
19.2, CH
3
Me γ
1.16, d (6.5)
19.2, CH
3
NH
CdO
R
1
.42, overlap
17.7, CH
3
173.2, qC
3
NH
CdO
R
7.85, d (10.4)
Gly
4.52, d (7.2)
43.5, CH
2
c
172.2, qC
3.76, dd (7.2, 6.4)
8.88, t (6.4)
3
d
Gly
4.64, overlap
43.9, CH
2
NH
CdO
R
3
.79, dd (17.2, 6.0)
169.8, qC
53.3, CH
4
NH
CdO
R
8.88, t (6.0)
Phe
5.89, m
169.8, qC
50.1, CH
β
3.69, d (13.6)
3.31, t (13.6)
41.4, CH
2
4
Leu
5.65, t (10.0)
3.44, m
d
β
43.8, CH
2
1
139.3, qC
129.7, CH
128.9, CH
127.0, CH
2
.28, overlap
2,6
3,5
4
7.17, d (7.2)
7.23, t (7.2)
7.12, d (7.2)
7.52, d (10.4)
γ
1.92, m
24.8, CH
23.7, CH
20.6, CH
Me δ
0.89, d (5.6)
3
3
0
.56, d (5.6)
NH
CdO
R
NH
CdO
R
7.18, d (8.8)
176.65, qC
64.6, CH
65.9, CH
5
6
7
177.8, qC
Thr
Thr
Trp
4.46, m
5
a
Thr
4.42, d (8.0)
4.73, m
64.5, CH
65.9, CH
21.9, CH
β
4.72, m
β
γ
1.39, d (6.4)
10.8, d (2.8)
21.7, CH
3
γ
1.39, d (6.4)
10.8, d (2.8)
3
NH
CdO
R
NH
CdO
R
172.5, qC
60.5, CH
66.6, CH
c
172.7, qC
4.91, d (6.8)
5.15, overlap
1.50, d (6.4)
7.64, d (7.6)
6
a
Pro
4.64, dd (9.5, 5.5)
2.38, m
64.4, CH
29.8, CH
26.3, CH
47.3, CH
β
γ
22.0, CH
3
β
γ
δ
2
NH
CdO
R
1
.64, m
1.56, m
.44, m
3.38, overlap
.18 d, (8.0)
172.8, qC
53.9, CH
2
2
5.39, m
1
β
3.68, d (13.6)
3.52, t-like (11.2)
11.99, s
27.2, CH
2
b
3
NH
2
c
CdO
172.5, qC
60.2, CH
66.5, CH
7.40, s
124.3, CH
111.9, qC
128.2, qC
120.3, CH
119.8, CH
122.4, CH
112.1, CH
137.7, qC
7
Thr
R
4.83, dd (7.2, 0.8)
5.03, overlap
1.44, overlap
7.48, d (7.2)
3
β
3a
4
γ
21.7, CH
3
7.89, d (7.6)
7.39, t (5.0)
7.36, t (5.0)
7.64, d (7.6)
NH
CdO
R
5
c
172.2, qC
55.9, CH
6
8
Phe
5.03, overlap
3.44, overlap
7
β
36.1, CH
2
7a
NH
CdO
R
1
139.3, qC
7.89, d (5.2)
4.99, m
2
3
4
,6
7.62, d (7.2)
7.32, t (7.2)
7.27, d (7.2)
7.81, d (5.6)
129.8, CH
128.8, CH
127.2, CH
170.4, qC
64.5, CH
8
,5
Pro
NH
β
2.24, m
1.67, m
28.0, CH
2
CdO
169.5, qC
1
394
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Journal of Natural Products
ARTICLE
Table 1. Continued
1
2
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
2.21, m
.85, m
4.06, m
.17, dd (6.4, 1.6)
δ
C
γ
25.0, CH
2
2
1
δ
47.5, CH
3
CO
172.2, qC
a,b,c,d
Resonances with the same superscripts are interchangeable.
Figure 2. HMBC and ROESY correlations in compounds 2 and 4.
corresponding to the successive loss of Trp, Pro, Thr, Thr, Phe,
Gly, and the terminal dipeptide ion ProꢀAla, and the other at m/z
656, 509, 452, 381, and 284, corresponding to the successive loss of
ThrꢀThr, Phe, Gly, Ala, Pro, and the terminal dipeptide ion
ProꢀTrp (Figure 1). These data confirmed the structure of peptide
1
2
3
4
5
6
7
8
4
as cyclo(Pro ꢀAla ꢀGly ꢀPhe ꢀThr ꢀThr ꢀPro ꢀTrp ).
13
1
The differences in the C NMR chemical shifts of Pro
7
(
Δδ
= 2.8 ppm) and Pro (Δδ
= 2.9 ppm) suggested
CβꢀCγ
7 13,14
CβꢀCγ
1
that the amide bonds in the Pro and Pro residues are trans.
The absolute configuration of the amino acid residues of
compounds 1ꢀ4 were determine by acid hydrolysis and analysis
15
of the hydrolysates with Marfey’s method. The Marfey’s deriva-
tives of authentic amino acids were prepared as standards and
compared with those of the hydrolysates by their co-injection onto
an HPLC apparatus. The results showed that all the amino acids
had L-configurations, except Gly. Because the Trp residues in the
structures of 2ꢀ4 were destroyed during hydrolysis, it was difficult
to clarify the configuration of Trp residues in these structures.
However, considering that naturally occurring amino acids from
higher plants are all L configured, the Trp residues in the com-
pounds 2ꢀ4 were tentatively proposed to be L.
Figure 1. QTOF/MS sequence ions (m/z) of the protonated molec-
ular ions of compounds 1ꢀ4.
amide protons (δ 7.49ꢀ10.52) and eight amide carbonyl reso-
1
13
nances in the H and C NMR spectra. Analysis of the COSY,
HMQC, and HMBC spectra demonstrated that the amino acid
residues were Phe, Trp, Gly, Ala, Thr (ꢁ2), and Pro (ꢁ2), which
are the same individual amino acids as those of 2. HMBC
The MS and NMR data of compound 5 were in accordance
with those of brachystemin A, an octacyclopeptide previously
isolated from the roots of the same plant. The structure of bra-
1
2
chystemin A was erroneously identified as cyclo(Pro ꢀPhe ꢀ
2
1
3
3
4
5
6
7
8 8
correlations were observed between Ala -NH/Pro -CO, Gly -
Leu ꢀAla ꢀThr ꢀPro ꢀAla ꢀGly ). The structure of 5
2
4
3
5
4
6
1
2
3
4
5
NH/Ala -CO, Phe -NH/Gly -CO, Thr -NH/Phe -CO, Thr -
should be revised to cyclo(Pro ꢀPro ꢀAla ꢀGly ꢀLeu ꢀ
5
8
7
6
7
8
NH/Thr -CO, and Trp -NH/Pro -CO, indicative of the partial
Ala ꢀThr ꢀPhe ) according to QTOF/MS analysis and X-ray
1
2
3
4
5
6
peptide sequences Pro ꢀAla ꢀGly ꢀPhe ꢀThr ꢀThr and
diffraction data. QTOF/MS data showed a series of b (þ1) ions
n
7
8
2
3
Trp ꢀPro . The ROESY correlations of Ala -NH/Gly -NH/
at m/z 608, 507, 436, 323, 266, and 195, indicating that the
preferred ring-opening occurred at the PheꢀPro amide bond,
and corresponded to the successive loss of Phe, Thr, Ala, Leu,
Gly, Ala, and the terminal dipeptide ProꢀPro. X-ray data further
confirmed the structure and assigned the relative configuration of
4
4
5
6
8
1
Phe -NH, Phe -H , /Thr -NH/Thr -NH, and Trp -H /Pro -
R β
R
Hδ supported the interpretation of the HMBC spectrum
Figure 2). Finally, two main series of b (þ1) ions were observed:
(
n
one at m/z 673, 575, 474, 373, 226, and 169 (Figure 1),
1
395
dx.doi.org/10.1021/np200048u |J. Nat. Prod. 2011, 74, 1392–1400

Journal of Natural Products
(Figure 3). The difference between the revised and previously
reported structure is that the sequence of ProꢀPhe and
PheꢀLeu should be revised to ThrꢀPhe and GlyꢀLeu. Such
confusion arises because of the fact that the carbonyl resonances
ARTICLE
17
18
5
loliolide (11), annuionone D (12), 9-O-D-glucopyranoside
19 20 21
(13), 7R-hydroxylambertianic acid (14), 2-minaline (15),
22
and 6,9-dihydroxy-4,7-megastigmadien-3-one (16) by compar-
ison with literature data. Compounds 12ꢀ17 were isolated from
this plant for the first time.
of Thr (δ 172.2), Pro (δ 172.4), Phe (δ 169.8), and Gly (δ
8
1
69.6) are often wrongly assigned.
On the basis of the traditional medicinal knowledge of this
herb, the cyclic peptides and alkaloids were evaluated in vitro for
their inhibitory effects on the secretion of CCL-2, IL-6, and
collagen IV by high-glucose-stimulated mesangial cells. The
results showed that compounds 1, 5, and 8 significantly inhibited
the secretion of IL-6, CCL-2, and collagen IV at concentrations
of 10 μM, compound 5 being the most active (Figure 4). There is
2
3
evidence showing that cytokines and ROS are interconnected,
and high-glucose-induced ROS overproduction has been directly
implicated in the pathogenic progress of DN. Thus, the anti-
oxidant effect of 5 was further investigated. The results showed
that high-glucose-induced ROS and O2• generation could be
significantly attenuated by 5 at 1 or 10 μM (Supporting
Information, S31, S32). Transforming growth factor-β (TGF-
β), one of the key mediators of extracellular matrix genes in
mesangial cells, has been implicated in the development of DN.
Elevated TGF-β levels have been shown to be related to the
ꢀ
The known compounds were identified as brachystemin C
7
1
6
7
7
7
(
6), brachystemidines C (7), A (8), B (9), and D (10),
24,25
accumulation of collagen I.
collagen I secretion in mesangial cells was tested and showed that
significantly decreased collagen I levels at 1 or 10 μM
Supporting Information: S33). To exclude the possibility that
Thus, the inhibitory effect of 5 on
5
(
the effects of 5 were related to its cell toxicity, the trypan blue
exclusion test was carried out. It showed that 5 exhibited no toxic
effect in mesangial cells at 10 μM (Supporting Information, S34).
The role of NO in the DN is complicated. There are studies
showing that the increased NO release contributes to the
hyperfiltration and microalbuminuria in the early stages of DN.
Several pathogenic factors of DN including hyperglycemia,
oxidative stress, and activation of protein kinase C may decrease
26
NO production in advanced nephropathy. In this sense,
compound 5 was further investigated for its effect on NO
production inhibition using LPS-stimulated RAW 264.7 macro-
phage cells. The results showed that compound 5 is inactive
Figure 3. ORTEP drawing of 5.
#
Figure 4. Inhibitory effects of compounds 1ꢀ11 on CCL-2, collagen IV, and IL-6 secretion. *P < 0.05 vs normal glucose; P < 0.05 vs high glucose.
1
396
dx.doi.org/10.1021/np200048u |J. Nat. Prod. 2011, 74, 1392–1400

Journal of Natural Products
ARTICLE
1
13
a
b
Table 2. H (500 MHz) and C (100 MHz for 3 and 125 MHz for 4) NMR Data for 3 and 4
3
4
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
1
1
Pro
R
4.14, t (9.5)
2.02, m
61.7, CH
29.3, CH
Pro
R
4.92, overlap
2.41, m
65.3, CH
28.9, CH
β
2
2
2
β
2
2
2
1
.63, m
1.94, m
.85, m
3.72, m
.37, m
2.35, m
γ
δ
24.9, CH
47.2, CH
γ
δ
2.35, m
26.1, CH
48.6, CH
1
2.18, m
4.05, t (9.0)
3.22, m
3
CdO
174.0, qC
55.5, CH
CdO
R
174.2, qC
49.9, CH
2
2
Phe
R
3.93, m
2.24, m
Ala
5.12, m
β
23.4, CH
2
β
1.93, d (7.0)
8.20, d (9.5)
19.9, CH
3
2
.11, m
NH
CdO
R
1
136.2, qC
123.3, CH
123.3, CH
127.0, CH
174.8, qC
3
2
3
4
, 6
7.17, d (7.2)
7.23, t (7.2)
7.12, d (7.2)
7.94, t (7.5)
Gly
4.40, dd (16.5, 6.5)
3.79, dd (16.5, 5.5)
8.80, t (6.5)
44.2, CH
2
, 5
NH
CdO
R
NH
CdO
R
171.2, qC
54.5, CH
4
171.7, qC
56.4, CH
35.5, CH
Phe
5.86, overlap
3
Ile
5.41, d (12.5)
2.06, m
β
4.03, overlap
41.7, CH
2
β
3.29, dd (13.5, 11.0)
γ
1.23, m
23.6, CH
2
1
137.3, qC
130.9, CH
130.0, CH
128.0, CH
1
.07, m
2,6
3,5
4
7.59, d (7.5)
7.42, m
Me γ
Me δ
NH
0.71, d (6.5)
0.76, t (7.5)
7.09, m
15.3, CH
12.1, CH
3
3
7.27, d (7.5)
7.49, d (10.0)
NH
CdO
R
CdO
R
171.1, qC
176.9, qC
65.7, CH
67.3, CH
4
5
Gly
3.82, dd (20.5, 7.5)
42.9, CH
2
Thr
4.47, dd (7.5, 2.5)
4.83, m
3
.28, overlap
β
NH
CdO
R
7.46, t (9.0)
γ
1.69, d (6.0)
10.52, d (2.5)
22.0, CH
3
167.7, qC
44.9, CH
NH
CdO
R
5
Ala
4.67, m
174.5, qC
61.6, CH
67.7, CH
6
β
0.72, d (7.6)
7.01, m
17.2, CH
3
Thr
4.90, overlap
5.16, m
NH
CdO
R
β
172.6, qC
62.3, CH
γ
1.62, d (6.0)
8.05, d (7.0)
22.4, CH
3
6
Pro
3.92, m
2.05, m
NH
CdO
R
β
28.5, CH
2
173.7, qC
64.8, CH
7
1
.62, m
Pro
4.64, t (9.5)
2.66, m
γ
1.79, m
3.51, m
24.3, CH
46.5, CH
2
2
β
30.2, CH
27.3, CH
48.2, CH
2
δ
1.81, m
CdO
171.8, qC
53.8, CH
γ
2.01, m
2
2
7
Trp
R
4.33, m
1.64, m
β
3.24, d (11.0)
25.5, CH
2
δ
2.93, m
3
.17, d (11.0)
CdO
174.0, qC
54.7, CH
8
NH
2
10.87, s
7.02, s
Trp
R
5.43, m
123.3, CH
111.6, qC
127.2, qC
121.3, CH
118.0, CH
118.6, CH
111.6, CH
137.7, qC
β
3.68, m
28.3, CH
2
3
3.59, t (13.0)
11.96, s
3
4
5
6
7
7
a
NH
2
7.04, m
7.87, s
113.4, CH
112.3, qC
128.8, qC
120.6, CH
120.9, CH
123.3, CH
7.45, d (9.5)
6.98, m
3
3a
4
7.28, d (9.0)
7.89, d (7.6)
8.61, d (7.5)
7.40, m
a
5
NH
7.18, d (5.2)
6
1
397
dx.doi.org/10.1021/np200048u |J. Nat. Prod. 2011, 74, 1392–1400

Journal of Natural Products
ARTICLE
Table 2. Continued
3
4
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
CdO
171.4, qC
48.6, CH
7
7.39, m
125.0, CH
138.6, qC
8
Asn
R
3.93, t (9.5)
2.61, m
7a
β
35.5, CH
2
NH
CdO
7.89, d (5.5)
2
.48, m
171.0, qC
γ
172.6, qC
NH
2
7.08, m
7.03, m
NH
CdO
170.2, qC
a
b
In DMSO-d . In Pyridine-d .
6
5
toward these cells. As mentioned above, the level of NO
decreased in advanced nephropathy, whereas the proper level
of NO is important for maintaining normal physical function.
Therefore, the negative effect of 5 on NO production may be
instead good for the later stages of DN patients.
silica gel, eluted with an increasing gradient of aqueous MeOH
(10ꢀ95%), to produce five fractions (B ꢀB ). Fraction B (600 mg)
was passed through a Sephadex LH-20 column (CHCl
1
5
3
ꢀMeOH, 6:4)
3
i
and then a silica gel column (petroleum etherꢀ PrOH, 15:1 and 10:1) to
yield compounds 11 (11.5 mg) and 12 (10 mg). Fraction E (3.3 g) was
subjected to reversed-phase CC over C18 silica gel (MeOHꢀH
2
O,
Cyclopeptides are an important group of compounds and have
27
5
:95ꢀ95:5) to produce two fractions, E and E . Fractions E (300 mg)
been isolated from plant, marine, and microbial sources.
1
2
1
and E
2
(180 mg) were purified by semipreparative HPLC (MeCNꢀ
and 8 (21 mg)
. Fraction F (6.0 g) was separated on reversed-
phase CC over C18 silica gel, eluted with aqueous MeOH (5ꢀ80%), to
yield fractions F and F . Fraction F (1.3 g) was separated on a
Although the biological effects of some cyclopeptides were
reported previously, few examples (such as cyclosporine A) have
been developed for clinical applications. The place of cyclopep-
tides in drug discovery requires further exploration. The present
study provides new perspectives on the biological significance of
cyclopeptides.
H
2
O, 65:35) to give (in this order) 10 (3.4 mg) from E
1
and 9 (8 mg) from E
2
1
2
1
Sephadex LH-20 column (MeOH) to produce 13 (62 mg), and fraction
F (720 mg) was purified on a silica gel column (CHCl ꢀMeOH, 10:1
2
3
and 5:1) to yield 7 (2.5 mg). Fraction G (8.5 g) was divided into six
’
EXPERIMENTAL SECTION
fractions (G
1
ꢀG
6
) by reversed-phase CC over C18 silica gel with a
(400 mg) was
gradient of aqueous MeOH (5ꢀ75%). Fraction G
2
General Experimental Procedures. Optical rotations were
separated on Sephadex LH-20 (MeOH), followed by vacuum liquid
measured with a Horiba SEPA-300 polarimeter. UV spectra were
determined on a Shimadzu double-beam 210A spectrometer. IR spectra
were measured on a Bio-Rad FTS-135 infrared spectrophotometer with
KBr disks. 1D and 2D NMR spectra were obtained on a Bruker AM-400
or a DRX-500 spectrometer, respectively, with TMS as the internal
standard. MS data were obtained on a VG Auto Spec-3000 mass
spectrometer. QTOF/MS was performed on an Agilent 1290 Infinity-
QTOF 6520 LC/MS system. Semipreparative HPLC was carried out on
an Agilent 1200 series pump equipped with a diode-array detector and a
Zorbax SB-C18 column (5.0 μm, φ 9.4 ꢁ 250 mm). Silica gel (80ꢀ100
and 300ꢀ400 mesh; Qingdao Marine Chemical Inc., People’s Republic
of China), C18 silica gel (40ꢀ60 μm; Daiso Co., Japan), and Sephadex
LH-20 (Amersham Biosciences, Sweden) were used for column chro-
chromatography (VLC) on silica gel (CHCl ꢀMe CO, 10:1), to
3
2
produce 1 (64 mg) and 2 (5.5 mg). Fraction G (4.0 g) was purified
3
i
by VLC on silica gel (CHCl ꢀ PrOH, 7:1) to yield 5 (2.1 g). Com-
3
pounds 3 (34 mg), 4 (39 mg), and 6 (30 mg) were purified from fraction
G (350 mg) by a combination of VLC on silica gel (CHCl ꢀMe CO,
4
3
2
5:1) and Sephadex LH-20 (MeOH). Fraction G (350 mg) was
6
separated into subfractions G6A and G6B by silica gel CC (CHCl ꢀ
3
MeOH, 5:1), and G6B (110 mg) was passed through a Sephadex LH-20
(MeOH) column to yield 14 (6 mg), 15 (11 mg), and 16 (30 mg).
2
5
D
Brachystemin F (1): white powder; [R] ꢀ69 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 250 (3.32), 203 (4.22) nm; IR (KBr) ν
3419, 2964, 2932, 2875, 1657, 1628, 1526, 1511, 1444 cm ; H and
max
ꢀ1 1
13
C
þ
NMR data, see Table 1; FABMS (positive) m/z 813 [M þ H] ;
þ
matography (CC). The plates were dipped into 5% H
heated to visualize the TLC spots.
2
SO
4
(EtOH) and
HRESIMS (positive) m/z 835.4314 [M þ Na] (calcd for C H N -
4
0 60 8
O Na, 835.4330).
1
0
2
5
Plant Material. The aerial parts of B. calycinum were collected in
Xishuangbanna, in Yunnan Province of People’s Republic of China, at
the end of March 2008, and identified by Prof. H. Peng at the Kunming
Institute of Botany. A voucher specimen (CHYX0572) was deposited at
the State Key Laboratory of Phytochemistry and Plant Resources in
West China, Kunming Institute of Botany, Chinese Academy of
Sciences, People’s Republic of China.
Brachystemin G (2): white powder; [R] ꢀ34 (c 0.1, MeOH);
D
UV (MeOH) λmax (log ε) 281 (3.61), 219 (4.36), 203 (4.43) nm; IR
ꢀ
1 1
13
(KBr) νmax 3417, 2933, 1659, 1628, 1512, 1449, 1304 cm ; H and
C
þ
NMR data, see Table 1; FABMS (positive) m/z 858 [M þ H] ;
þ
43 55
HRESIMS (positive) m/z 880.3976 [M þ Na] (calcd for C H -
N O Na, 880.3969).
9
10
2
5
Brachystemin H (3): white powder; [R] ꢀ42 (c 0.4, MeOH);
D
Extraction and Isolation. The sun-dried aerial parts of B.
calycinum (12 kg) were ground to a powder and extracted with 95%
aqueous EtOH (3 ꢁ 20 L, each 7 d) at room temperature. The crude
UV (MeOH) λmax (log ε) 290 (3.20), 280 (3.26), 203 (4.10) nm; IR
ꢀ
1 1
13
(KBr) νmax 3342, 2931, 1663, 1630, 1524, 1456, 1026 cm ; H and
C
þ
NMR data, see Table 2; FABMS (positive) m/z 883 [M þ H] ;
þ
extract was suspended in H O and then partitioned successively with
HRESIMS (positive) m/z 905.4386 [M þ Na] (calcd for C H -
2
45 58
EtOAc and n-BuOH. The EtOAc portion (42 g) was subjected to CC
over silica gel (200ꢀ300 mesh) using increasing amounts (2%) of
N O Na, 905.4385).
1
0 9
2
5
D
Brachystemin I (4): white powder; [R]
ꢀ13 (c 0.3, MeOH);
3
MeOH in CHCl and finally MeOH as the eluent to produce fractions
UV (MeOH) λmax (log ε) 281 (3.02), 219 (3.83), 203 (3.92) nm. IR
ꢀ
1 1
AꢀG. Fraction B (2.7 g) was subjected to reversed-phase CC on C18
(KBr) νmax 3431, 2923, 1656, 1639, 1629, 1512, 1451 cm ; H and
1
398
dx.doi.org/10.1021/np200048u |J. Nat. Prod. 2011, 74, 1392–1400

Journal of Natural Products
ARTICLE
1
3
þ
C NMR data, see Table 2; FABMS (positive) m/z 858 [M þ H] ;
Finland). The readings in each of the last 5 min were averaged and
expressed as counts per second. The data of each group were normalized
to the NG group and expressed as percent of control.
þ
HRESIMS (positive) m/z 858.4132 [M þ H] (calcd for C H -
4
3 55
N
9
O
10Na, 858.4150).
Crystallographic data for compound 5: C37
orthorhombic, space group P2 2 2 , a = 10.2490(8) Å,b = 16.5450(12)
H
54
N
8
O
9
, M
r
= 754,
Inhibition of NO Production in LPS-Stimulated RAW 264.7
Macrophage Cells. The inhibition of compound 5 on NO produc-
tion in LPS-stimulated RAW 264.7 macrophage cells was carried out as
1
1 1
3
ꢀ3
Å, c = 25.6543(19) Å; V = 4350.2(6) Å , Z = 4, D
= 1.263 g cm
,
calcd
3
35
crystal size 0.403 ꢁ 0.369 ꢁ 0.195 nm , F(000) = 1776. The final R
1
was previously reported. For details see the Supporting Information
value is 0.0542 (wR = 0.0627) for 8074 reflections [I > 2σ(I)].
2
(S36).
The crystallographic data for compound 5 have been deposited with
Trypan Blue Exclusion Test for Cell Viability. For details see
the Cambridge Crystallographic Data Centre (deposit number CCDC
the Supporting Information (S37).
807929). Copies of the data can be obtained, free of charge, on
application to the Director, CCDC, 12 Union Road, Cambridge CB2
’
ASSOCIATED CONTENT
1
EZ, UK (fax: þ44-(0)1223-336033 or e-mail: deposite@ccdc.cam.ac.uk).
Marfey’s Derivitization and HPLC Analysis of 1ꢀ4. Each
S
Supporting Information. HMBC and ROESY correla-
b
compound (0.5 mg) was dissolved in 6 N HCl (1 mL) in a sealed
container and heated at 110 ꢀC for 18 h. After cooling, the reaction
mixture was concentrated in vacuo to dryness. The hydrolysate was
tions for compounds 1 and 3. Retention times for amino acids
and X-ray crystallographic data in CIF format for compound 5.
1
D and 2D NMR spectra and MS spectra for the new com-
added to 20 μL of 1 M NaHCO
dinitrophenyl-5-L-alaninamide in acetone. The solution was reacted at
0 ꢀC for 1 h. The Marfey’s derivatives were analyzed by co-injection
into an HPLC apparatus (ODS, 5 μm, 250 ꢁ 9.4 mm i.d.; MeCNꢀH O
3
solution and 100 μL of 1% 1-fluoro-2,4-
pounds. Figures of analysis of intracellular ROS production,
NADPH-dependent superoxide anion production, and collagen I
secretion in mesangial cells. Cytotoxicity test in mesangial cells.
Procedure for determining ROS production and NO production.
These materials are available free of charge via the Internet at
http://pubs.acs.org.
4
2
(
3
0.05% TFA) = 10ꢀ60%; flow rate 1.5 mL/min; UV detection at
40 nm) and compared with the Marfey’s derivatives of authentic
amino acids.
Inhibition of IL-6, CCL-2, Collagen I, and Collagen IV
Secretion. Rat mesangial cells (American Type Culture Collection
no. CRL-2573) were grown in Dulbecco’s modified Eagle’s medium
’
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: 86-871-5223048. E-mail: yxcheng@mail.kib.ac.cn (Y.-X.C).
Tel: þ86-20-61641597. E-mail: ffhou@pub.guangzhou.gd.cn
(Invitrogen, Carlsbad, CA) containing 5.6 mM D-glucose (pH 7.4;
Sigma Chemical Co., St Louis, MO), supplemented with 20% fetal calf
serum (FCS; Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomy-
cin, and 10 mM HEPES. After the mesangial cells reached 80%
confluence, their growth was arrested in 0.5% FCS for 24 h. Exposure
of the mesangial cells to medium containing high-concentration glucose
induced the overproduction of CCL-2, IL-6, collagen I, and collagen IV,
(
F.-F.H).
Author Contributions
These authors contributed equally to this work.
28,29
as described in the previous reports.
To determine whether the
’
ACKNOWLEDGMENT
selected compounds inhibited the CCL-2, IL-6, and collagen over-
production triggered by high glucose, the mesangial cells were pre-
treated with 1 or 10 μM of each compound for 1 h and then stimulated
with high-concentration glucose for 24 h. The levels of supernatant
CCL-2, IL-6, and collagen were measured with a solid-phase quantitative
sandwich enzyme-linked immunosorbent assay (ELISA) kit for CCL-2
This work was financially supported by the following grants:
National Natural Science Foundation of China (No. 30700059),
Talent Scholarship of Yunnan Youth (No. 2007PY01-48), the
Open Research Fund of State Key Laboratory Breeding Base
of Systematic Research, Development and Utilization of
Chinese Medicine Resources, Key Project for Drug Innovation
(
1
BD Biosciences, San Diego, CA), specific for rat CCL-2 and sensitive to
0 pg/mL. The concentration in the culture supernatant was normalized
to the total amount of cell protein, quantified with the BCA method
(
2008ZX09401-004) from the Ministry of Science and Technol-
ogy of China, and Project of Natural Compound Library Construc-
tion from Chinese Academy of Sciences (KSCX2-EW-R-15).
30
(Pierce, Rockford, IL). Similar protocols were used for rat IL-6 (R&D
Systems, Abingdon, UK; sensitivity 0.25 ng/mL), rat collagen IV (R&D
Systems; sensitivity 0.13 ng/mL), and rat collagen I (EIAab; sensitivity
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