Rubipodanin A, the First Natural N-Desmonomethyl Rubiaceae-Type Cyclopeptide from Rubia podantha, Indicating an Important Role of the N9-Methyl Group in the Conformation and Bioactivity
Rubipodanin A, the First Natural N- Desmonomethyl Rubiaceae-Type Cyclopeptide from Rubia podantha,
Zhe Wang 0 1
Si-Meng Zhao 0 1
Li-Mei Zhao 0 1
Xiao-Qiang Chen 0 1
Guang-Zhi Zeng 0 1
Ning- Hua Tan 0 1
☯ These authors contributed equally to this work. 0 1
(NHT) 0 1
(GZZ) 0 1
0 1 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences , Kunming, 650201 , PR China , 2 Department of Natural Medicinal Chemistry & State Key Laboratory of Natural Medicines, China Pharmaceutical University , Nanjing, 210009 , PR China , 3 University of Chinese Academy of Sciences , Beijing, 100049 , PR China
1 Editor: Surajit Bhattacharjya, Nanyang Technological University , SINGAPORE
One new cyclic hexapeptide named rubipodanin A (1), which is the first identified natural Ndesmonomethyl Rubiaceae-type cyclopeptide, together with six known Rubiaceae-type cyclopeptides (2-7) were obtained using the TLC cyclopeptide protosite detection method with ninhydrin from the roots and rhizomes of Rubia podantha. The cyclopeptide structures were elucidated by extensive spectroscopic analysis, including 1D-NMR, 2D-NMR, IR, UV and MS. The solution conformation and biological activities of 1 and RA-V (4) were evaluated, and the results demonstrated that the N9-methyl group plays a vital role in the maintenance of the conformation and bioactivity.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Rubiaceae-type cyclopeptides (RAs) are homodicyclohexapeptides mainly composed of one
αD-alanine, one α-L-alanine, three N-methyl-α-L-tyrosines, and one other proteinogenic
α-Lamino acid. The most unusual feature is a 14-membered ring formed by a phenolic oxygen
linkage between two adjacent tyrosines with a cis peptide bond, and the 14-membered ring is
fused to a 18-membered cylic hexapeptide ring [
]. In 1977, bouvardin and deoxybouvardin
(RA-V), the first two RAs, were isolated from Bouvardin ternifolia (Rubiaceae) with potential
antitumor activities . Subsequently, an additional 34 RAs have been isolated from Rubia
codifolia, R. yunnanensis and R. akane, and more than 200 analogues have been synthesized
Competing Interests: The authors have declared
that no competing interests exist.
]. Studies on antitumor mechanisms of RAs have indicated that RA-VII suppresses
protein synthesis through interaction with eukaryotic 80S ribosomes . Furthermore, RA-V and
RA-XII inhibit the production of NO and inducible nitric oxide synthase (iNOS) [
Moreover, RA-VII has the ability to change the conformational structure of F-actin to induce G2
For the unique bicyclic structure and significant antitumor activities in vitro and in vivo
], RAs have recently attracted our interest. We have now isolated 28 RAs and
synthesized several RA analogues, including 11 novel natural RAs consisting of two new skeleton RAs
and one new O-seco-RA from R. yunnanensis and R. schumanniana, respectively [
have also evaluated their cytotoxic activities and performed 2D- and 3D-QSAR studies on 54
]. Our studies showed that RA-V exhibits anti-inflammatory activity by inhibiting NO
production and NF-κB activation induced by TNF-α [
]. Furthermore, RA-V significantly
suppresses angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and
HMEC1 endothelial cells [
]. In addition, RA-V kills human breast cancer cells by inducing
mitochondria-mediated apoptosis and inhibits cell adhesion and invasion via the PI3K/AKT and
NF-κB signaling pathways [
]. Importantly, the potential roles of RAs in cancer therapy
have been highlighted .
Some investigations have shown that N-methylated amino acids of cyclopeptides play an
important role in the maintenance of their conformation and bioactivity. Our previous NMR
studies have indicated that both natural and synthetic RAs have two or four conformers [
], which prompted us to obtain the natural N-desmethyl RAs and investigate the functional
role of amino acid N-methylation in conformation and bioactivity. In this study, we selected
another Rubia plant, Rubia podantha Diels, which has been used as a substitute for the
traditional Chinese medicine R. codifolia. The roots and rhizomes have been used to treat
tuberculosis, menoxenia, rheumatism, contusion, hematemesis, anemia and lipoma for a long time in
China. To the best of our knowledge, however, there is no literature on the chemical and
antitumor constitutes of this plant. To expand the distribution of plant resources containing RAs
and explore the functional role of N-methylated amino acids in natural RAs in the maintenance
of the conformation and bioactivity, we structurally and pharmacologically investigated
interesting RAs using the TLC cyclopeptide protosite detection method with ninhydrin  from
R. podantha. Fortunately, a new cyclic hexapeptide, rubipodanin A (1), which is the first
identified natural N-desmonomethyl RA, together with six known RAs (2–7) were obtained. Here,
the isolation and structural elucidation of 1–7 as well as cytotoxic and NF-κB signaling
pathway activities of 1 and 4 are described.
Materials and Methods
General experimental procedures
Optical rotations were obtained on a Jasco P-1020 polarimeter. IR spectra were measured by a
Tensor 27 spectrometer using KBr pellets. UV spectra were performed using a Shimadzu
UV-2401A spectrophotometer. 1D-NMR and 2D-NMR spectra were recorded on a Bruker
AVANCE III-600 and Ascend ™ 800 spectrometer at 298K. Chemical shifts (δ) were expressed
in parts per million (ppm) with reference to the solvent signals. Mass spectra were obtained on
a Waters XEVO-TQD spectrometer or an Agilent G6230 TOF Mass spectrometer. Analytical
or semi-preparative HPLC was performed on an Agilent 1100 with a Zorbax Eclipse-C18 (4.6
mm × 150 mm; 9.4 mm × 250 mm; 5 μm).
Column chromatography was performed with silica gel (100–200 mesh and 200–300 mesh,
Qingdao Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, China), Sephadex LH-20 (Pharmacia
Fine Chemical Co., Uppsala, Sweden) or Lichroprep RP-18 gel (40–63 μm, Merck, Darmstadt,
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Germany). Fractions were monitored by TLC (GF254, Qingdao Yu-Ming-Yuan Chemical Co.
Ltd., Qingdao, China), and orange spots were visualized on the plate by spraying with 2%
ninhydrin reagent after hydrolyzed in a drying incubator (110°C) for 30 min by HCl [
The roots and rhizomes of R. podantha were collected in November of 2013 from Mount Chang
Chong, Kunming, China. The locations/activities to collect R. podantha did not require specific
permission because this studied plant is common in the field in Yunnan, China and is not
cultivated in privately owned or protected areas. In addition, the filed studies did not involve
endangered and protected species. The material was identified by Professor Ning-Hua Tan of Kunming
Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KUN0359279) was
deposited in the Herbarium of Kunming Institute of Botany.
Extraction and isolation
The air-dried and powdered roots and rhizomes of R. podantha (13 kg) were extracted three
times with methanol (3 × 15 L) under reflux. After removal of the solvent under vacuum, the
methanol extract (1.63 kg) was subjected to silica gel column chromatography (CC) eluted
with petroleum ether-CHCl3 (1:0, 1:1, 0:1) and CHCl3-MeOH (95:5, 9:1, 8:2, 7:3, 0:1) to afford
eight fractions. Fractions containing cyclopeptides (150 g, CHCl3-MeOH, 9:1) were
rechromatographed on a silica gel CC eluted with a CHCl3-MeOH gradient system (70:1–8:2) to yield
six fractions (Fr.1-Fr.6).
Fr.2 (40 g) was applied to silica gel CC using petroleum ether-acetone (3:1–0:1) to yield five
subfractions (Fr.2-1 to Fr.2-5). Fr.2-3 (17 g) was separated using Sephadex LH-20 CC
(CHCl3MeOH, 1:1) and then purified over repeated silica gel CC eluting with petroleum ether-acetone
(3:2) to afford RA-V (4) (878 mg).
Fr.3 (37 g) was chromatographed on a silica gel CC using a CHCl3-MeOH system (100:1–
8:2) to yield six subfractions (Fr.3-1 to Fr.3-6). Fr.3-3 (21 g) was subjected to RP-18 CC, eluting
with MeOH-H2O (20–90%) to afford five subfractions (Fr.3-3-1 to Fr.3-3-5). Fr.3-3-2 (9 g)
were separated by Sephadex LH-20 CC (CHCl3-MeOH, 1:1) and purified over repeated silica
gel CC eluting with petroleum ether-acetone (2:1) to afford RA-VII (5) (658 mg). Fr.3-3-3 (3 g)
was also passed through Sephadex LH-20 CC (CHCl3-MeOH, 1:1) and then silica gel CC
(petroleum ether–acetone, 3:2) to yield four subfrations (Fr.3-3-3-1 to Fr.3-3-3-4). Fr.3-3-3-2
(32 mg) was further purified by semi-preparative HPLC (30% CH3CN) to yield rubipodanin A
(1) (9 mg). Fr.3-3-3-3 (43 mg) was also further purified by semi-preparative HPLC (35%
CH3CN) to yield RA-I (2) (7 mg) and RA-III (3) (8 mg).
Fr.4 (10 g) was subjected to an RP-18 CC, eluting with MeOH-H2O (20–90%) to afford four
subfractions. Fr.4-4-2 (4 g) were separated by Sephadex LH-20 CC (CHCl3-MeOH, 1:1) and
then further purified by semi-preparative HPLC (30% CH3CN) to yield Allo-RA-V (7) (4 mg).
Fr.5 (32 g) was submitted to silica gel CC eluting with gradient CHCl3-MeOH (50:1–8:2) to
yield five subfractions (Fr.5-1 to Fr.5-5). Fr.5-3 (13 g) was further purified by repeated silica gel
CC (CHCl3-MeOH, 20:1–8:2) followed by Sephdex LH-20 CC (CHCl3-MeOH, 1:1), and it was
further separated by repeated RP-18 CC (MeOH-H2O, 30–70%), which led to the isolation of
RA-XII (6) (927 mg).
Rubipodanin A (1): white amorphous powder; [α]18.5 D -215.5 (c 0.06, CHCl3); UV
(MeOH) λmax (log ε) 203 (4.84), 222 (4.58), 278 (3.73) nm; IR (KBr) νmax 3428, 2957, 2925,
2871, 1656, 1636, 1514, 1500, 1459, 1411, 1377, 1288, 1246, 1212, 1182, 1160, 1111, 1094, 1033
cm-1; 1H (800 MHz) and 13C (200 MHz) NMR data, see Table 1; positive ESIMS m/z 743.80
[M+H]+; positive HRESIMS m/z 765.3214 [M+Na]+, calcd for C39H46N6NaO9 765.3224.
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HeLa, A549, SGC7901 and HEK293T cell lines were purchased from the American Type
Culture Collection (ATCC, USA) and were cultured in Dulbecco’s modified Eagle’s medium
(Invitrogen, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Life
technologies, USA) and 1% (v/v) penicillin-streptomycin (Invitrogen, USA). The cells were
maintained at 37°C in a humidified incubator atmosphere of 5% CO2/95% air (v/v).
Cytotoxicity was tested by the Sulforhodamine B (SRB) method (Sigma, USA). The assays were
performed as described previously [
Cell transfection and luciferase assay
HEK293T cells were seeded in 24-well plates and transiently transfected with 5×κB-luciferase
and pTK-Renilla reporters using Lipofectamine 2000 (Invitrogen, USA) for 18 h. The cells
were then incubated with different concentrations of compounds for the indicated time, and
subsequently stimulated with 10 ng/ml TNF-α for 2 h. The luciferase activity of the cell lysate
was analyzed by the Dual Luciferase Reported Assay System (Promega, USA). Luciferase
reporter assays were performed as described previously [
Western blot analysis
Cells were lysed using RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1
mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, protease inhibitors and
phosphatase inhibitors. After being disrupted on ice for 30 min, the cell lysates were centrifuged at
12,000 rpm for 15 min and boiled with SDS sample buffer at 95°C for 5 min. The samples were
equally subjected to SDS-PAGE, electrophoresed, and transferred to PVDF membranes
(Millipore, USA). After blocking with 5% nonfat milk in TBST, the membranes were incubated with
the indicated antibody for 2 h or overnight at 4°C, and the membranes were then incubated
with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room
temperature. The protein bands were detected with a SuperSingal West Pico Chemiluminescence ECL
kit (Pierce, USA).
Student’s t-test was used for statistical analysis. A p value < 0.05 was considered to be
Results and Discussion
The methanol extract of the air-dried powdered roots and rhizomes of R. podantha was
subjected to fractionation using silica gel CC, which yielded the total cyclopeptide fraction guided
by the TLC cyclopeptide protosite detection method with ninhydrin [
]. This fraction was
repeatedly chromatographed over a series of silica gel, Sephadex LH-20, RP-18 CC, and ODS
HPLC to obtain seven RAs (Fig 1), including one new natural N-demonomethyl RA
(rubipodanin A, 1) and six known RAs, including RA-I (2), RA-III (3), RA-V (4), RA-VII (5), RA-XII
(6) and Allo-RA-V (7). The structure and stereochemistry of 1 was established by 1D-NMR,
2D-NMR, UV, IR and MS, and the structural identification of known compounds 2–7 was
performed by comparison with NMR data in the literature [
]. Furthermore, cytotoxic
activities of 1 and 4 against three tumor cell lines and inhibitory activities of 1 and 4 on the
NF-κB signaling pathway were investigated. The results of the solution conformation and
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Fig 1. Chemical structures of 1–7.
biological activities of 1 and 4 suggested that the N9-methyl group plays a vital role in the
maintenance of their conformation and bioactivity.
Characterization of Rubipodanin A (1)
Rubipodanin A (1) was obtained as white amorphous powder, and its molecular formula was
established as C39H46N6O9 by its positive HRESIMS (m/z 765.3214, [M+Na]+), indicating 20
degrees of unsaturation. The UV spectrum of rubipodanin A showed absorptions at 203, 222
and 278 nm, which demonstrated the existence of phenyl groups. The IR spectrum exhibited
absorption bands at 3428 and 1636 cm-1, which is indicative of OH, NH and CO groups. The
1H and 13C NMR spectra of 1 in C5D5N (Table 1) displayed characteristics of typical RAs
without any conformers, but these characteristics are common in natural RAs. Further analysis of
1D-NMR and 2D-NMR spectra data of 1 displayed the signals for the three methyls (δH/δC
1.55/21.8, 1.46/17.0, 1.40/19.5), two amide N-methyls (δH/δC 3.03/29.8, 3.00/30.4), one
Omethyl (δH/δC 3.71/55.3), three methylenes (δH/δC 3.85, 3.98/33.9; 2.60, 3.65/36.7; 3.34, 3.54/
36.5), six α-amino methines (δH/δC 5.77/54.6, 5.19/46.8, 5.18/48.3, 5.01/58.0, 4.91/49.0, 4.10/
57.8), two 1,4-disubstituted benzene rings (δC 158.9, 132.9 and δH/δC 7.33/131.1 × 2, 7.04/
114.4 × 2; δC 158.9, 136.2 and δH/δC 7.43/133.5, 7.24/131.0, 6.94/126.5, 6.89/124.6), one
1,2,4-trisubstituted benzene ring (δC 152.7, 145.5, 128.1, and δH/δC 7.22/117.9, 6.76/122.2,
4.60/115.1), six carbonyl signals (δC 173.0, 172.6, 172.0, 171.5, 169.9, 169.9) and four amide
protons (δH 10.59, 9.80, 8.90, 7.81). An extensive comparison of 1D-NMR and 2D-NMR
spectra data of 1 with those of RA-V (4) in C5D5N indicated that both compounds were similar,
except for the third amino acid residue (Tyr3). Further analysis of the HMBC and 1H-1H
COSY correlations confirmed the construction of the N-desmethyl Tyr3 residue (Fig 2). In the
1H-1H COSY spectrum, cross-peaks of NH-3/H-3α/H-3β suggested that the amide N9-methyl
was absent, which was also confirmed by the HMBC correlation from δH 10.59 (NH-3) to δc
173.0 (C-2-CO). In addition, the sequence of the amino acid residues in 1 was confirmed by
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Fig 2. Key HMBC, 1H-1H COSY, and ROESY correlations of 1.
the HMBC correlations from δH 9.80 (2-NH) to δc 172.6 (C-1-CO), from δH 10.59 (3-NH) to
δc 173.0 (C-2-CO), from δH 7.81 (4-NH) to δc 169.9 (C-3-CO), from δH 3.00 (H-5NCH3) to δc
172.0 (C-4-CO), from δH 3.03 (H-6NCH3) to δc 169.9 (C-5-CO) and from δH 8.90 (1-NH) to
δc 171.5 (C-6-CO). In the ROESY spectrum, NOE correlations were observed among 3-NH/
H-2α, H-3α, 5-NCH3/H-4α, and H-5α/H-6α, thereby indicating that the peptide bonds
between Ala2/Tyr3, Ala4/Tyr5 and Tyr5/Tyr6 were trans, trans, and cis, respectively. Because
previous have studies demonstrated that the absolute configurations of Ala1 and Ala4 in RAs
were generally D (R) and L (S) [
], the remaining configurations of Ala2, Tyr3, Tyr5 and Tyr6
were deduced as L (S), L (S), L (S) and L (S), respectively, by observing the following NOE
correlations: H-1α/2-NH; 3-NH/H-2α, H-3α; 5-NCH3/H-4α; and H-5α/H-6α. Collectively, the
structure of 1 was identified as shown in Fig 1, which is the first identified natural
The N-methylated amino acids of cyclopeptides play an important role for the
conformation in solution, and RAs generally possess two or four conformers, including natural RAs,
synthetic RAs and O-seco-RAs. In 1995, the DL Boger group synthesized several N-desmethyl
derivatives of RA-VII and investigated their conformation property by 1H-NMR spectra, and
they reported that both N9- and N15-methyl groups are essential for maintenance of their
], which prompted us to investigate the difference between 1 and 4 in NMR
spectra. Consistent with previous studies , the 1H NMR spectra of 4 in C5D5N displayed
the presence of two conformers in a ratio of 79:21, and 1 revealed a single solution
conformation (Fig 3). The similar phenomenon occurred in 13C NMR spectra (Figure N in S1 File),
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Fig 3. 1H NMR spectra comparison of rubipodanin A (1, top) and RA-V (4, bottom).
which demonstrated that the N9-methyl group plays a vital role in the conformational
property of RAs.
Some studies have shown that natural bioactive cyclopeptides contain at least one
N-methylated amino acid. For example, the N-methyl groups of cyclosporin A (CsA), a cyclic
undecapeptide containing seven N-methylated amino acids, play an important role for maintenance
of immunosuppressive activity [
]. Sansalvamide A, isolated from a marine fungus of the
genus Fusarium, is a cyclic tetrapeptide, and the N-methyl derivatives of sansalvamide A
exhibit better antitumor activity [
RAs are a type of potent antitumor agent and often contain three N-methylated tyrosines,
which promotes researchers to explore the functional role of N-methyl groups on their
biological activity. The DL Boger group evaluated the cytotoxicity of synthetic N-desmethyl RA
derivatives and showed that the N15-methyl group is essential for their biological activities [
]. In the
present study, we investigated the cytotoxicity against three tumor cell lines and inhibitory
activity against the tumor-associated NF-κB signaling pathway of rubipodanin A (1) compared to
RA-V (4) by using the SRB assay and the Dual Luciferse Reporter Assay System, respectively.
As shown in Fig 4, 1 had weak cytotoxicity against HeLa, A549 and SGC-7901 with IC50 values
of 7.22 ± 0.76, 7.14 ± 0.81, 3.80 ± 0.17 μM, respectively, which was 420–650 times less than 4
(Table 2). We also found that 4 exhibited a potent inhibitory activity against the NF-κB pathway
activation induced by TNF-α, which was approximately 700 times greater than that of 1. In
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Fig 4. The cytotoxicity and inhibitory activity of rubipodanin A (1) and RA-V (4) against the NF-κB
addition, we examined the effects of 1 and 4 on the expression of NF-κB-associated proteins
using western blot analysis. As expected, 1 also had an inhibitory effect on TNF-α-induced
IκBα phosphorylation, IκBα degradation and p65 phosphorylation only in the high
concentration up to 20 μM, while 4 exhibited similar inhibitory effect at a concentration 200 nM. Taken
together, these results suggested that the N9-methyl group is essential for maintenance of
biological activities of natural RAs and that down-regulation of the NF-κB pathway might account for
the mechanism of the antitumor activities of RAs. Of note, previous reports have shown that the
N9-methyl group of synthetic RAs is not critical for biological activities [
], which was not
consistent with our results. So more N-desmethyl derivatives of RAs from plants or by synthesis in
future are needed to confirm this difference based on the structure-activity relationship analysis.
The phytochemical studies of Rubia podantha were performed using the TLC cyclopeptide
protosite detection method with ninhydrin, which led to the identification of one new cyclic
7.22 ± 0.76
7.14 ± 0.81
3.80 ± 0.17
0.015 ± 0.0014
0.017 ± 0.0026
0.0058 ± 0.0016
hexapeptide (rubipodanin A, 1) and six known ones (2–7). Importantly, 1 is the first identified
natural N-desmonomethyl RA. Further studies revealed that 1 had only one solution
conformation in NMR but two in RA-V (4), and it also had lower cytotoxicity and inhibitory activity
against the NF-κB signaling pathway than 4, thus indicating that the N9-methyl group is
essential for the conformation and biological activities of RAs. These findings not only expanded the
distribution of plant resources containing RAs but also provided new insights into the role of
the N-methyl group in maintaining conformational and biological properties of RAs.
S1 File. Relevant spectra of Rubipodanin A (1) and RA-V (4). 1H NMR Spectrum of
Rubipodanin A (1) (Figure A). 13C NMR Spectrum of Rubipodanin A (1) (Figure B). HSQC
Spectrum of Rubipodanin A (1) (Figure C). COSY Spectrum of Rubipodanin A (1) (Figure D).
HMBC Spectrum of Rubipodanin A (1) (Figure E). ROESY Spectrum of Rubipodanin A (1)
(Figure F). ESI Mass Spectrum of Rubipodanin A (1) (Figure G). High Resolution Mass
Spectrum of Rubipodanin A (1) (Figure H). UV Spectrum of Rubipodanin A (1) (Figure I). IR
Spectrum of Rubipodanin A (1) (Figure J). [α]D Spectrum of Rubipodanin A (1) (Figure K).
1H NMR Spectrum of RA-V (4) (Figure L). 13C NMR Spectrum of RA-V (4) (Figure M). 13C
NMR spectra comparison of Rubipodanin A (1, top) and RA-V (4, bottom) (Figure N).
XQC. Wrote the paper: ZW NHT.
We would like to thank the members of the analytical group of the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany for all of the
Conceived and designed the experiments: ZW NHT. Performed the experiments: ZW GZZ.
Analyzed the data: ZW SMZ GZZ. Contributed reagents/materials/analysis tools: SMZ LMZ
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