Two new β-carboline alkaloids from the roots of Gypsophila oldhamiana

Article abstract of DOI:10.1080/14786419.2014.996755

Phytochemical investigation of the roots of Gypsophila oldhamiana afforded two new β-carboline alkaloids, oldhamiaines A and B (1 and 2), along with a known analogue (3). Their structures were elucidated by using spectroscopic and chemical methods. This is the first report of β-carboline alkaloids in the genus Gypsophila.

Full text of DOI:10.1080/14786419.2014.996755

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Two new β-carboline alkaloids from the
roots of Gypsophila oldhamiana
a
a
a
a
Yangmei Zhang , Gang Wang , Huawei Lv , Jianguang Luo &
a
Lingyi Kong
a
State Key Laboratory of Natural Medicines, Department of
Natural Medicinal Chemistry, China Pharmaceutical University, 24
Tong Jia Xiang, Nanjing 210009, People's Republic of China
Published online: 02 Jan 2015.
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To cite this article: Yangmei Zhang, Gang Wang, Huawei Lv, Jianguang Luo & Lingyi Kong (2015):
Two new β-carboline alkaloids from the roots of Gypsophila oldhamiana, Natural Product Research:
Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.996755
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Natural Product Research, 2014
http://dx.doi.org/10.1080/14786419.2014.996755
Two new b-carboline alkaloids from the roots of Gypsophila oldhamiana
Yangmei Zhang, Gang Wang, Huawei Lv, Jianguang Luo* and Lingyi Kong*
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
(
Received 3 September 2014; final version received 4 December 2014)
Phytochemical investigation of the roots of Gypsophila oldhamiana afforded two new
b-carboline alkaloids, oldhamiaines A and B (1 and 2), along with a known analogue
(3). Their structures were elucidated by using spectroscopic and chemical methods.
This is the first report of b-carboline alkaloids in the genus Gypsophila.
Keywords: Caryophyllaceae; Gypsophila oldhamiana; b-carboline alkaloids
1
. Introduction
The genus Gypsophila (Caryophyllaceae) comprises approximately 150 species that are widely
distributed in Asia and Europe. Of these, more than 18 species grow in mainland China, especially
in Xinjiang province (Lu 1994), and some of which have been used as pharmaceutical and
ornamental plants for a long time (Nie et al. 2010). Gypsophila oldhamiana Miq., a perennial
herbaceous plant, is mainly distributed in the north of China, and its roots have been used as a
traditional folk medicine Shan-Yin-Chai-Hu for the treatment of fever, consumptive disease and
infantile malnutrition syndrome (Jiansu College of New Medicine 1977). Previous phytochemical
investigations on the roots of G. oldhamiana have led to the isolation of a series of bioactive
natural products, including triterpenoid saponins (Luo & Kong 2006), sterols (Liu et al. 1995) and
cyclic peptides (Wang et al. 2013). Therefore, as a continuation of our studies, two new b-
carboline alkaloids (1–2), along with one known analogue (3), were obtained from the roots of this
plant. Herein, we report the isolation and elucidation of the new b-carboline alkaloids.
2
. Results and discussion
Compound 1 was obtained as a yellow powder, presumably being endowed with an N function
on the basis of TLC examinations by using the Dragendorff’s reagent. The molecular formula of
*
Corresponding authors. Email: luojg99@163.com; cpu_lykong@126.com
q 2014 Taylor & Francis

2
Y. Zhang et al.
1
showed an [M 2 H] ion peak at m/z 368.0894 (calcd for 368.0888). The IR spectrum showed
was determined as C H N O (13 unsaturation degrees) based on its HR-ESI-MS which
2
1
8 15 3 6
2
1
absorption bands at 3420, 1679 and 1669 cm assignable to amino, carbonyl and amide
functionalities. Absorption maxima in the UV spectrum of 1 were observed at 372, 285 and
2
1
19 nm, suggesting the presence of b-carboline chromophore (Kuo et al. 2003). In the H NMR
(
DMSO-d ) spectrum of 1, resonances for four mutually coupled, aromatic protons at [d 8.45
6 H
(
1H, d, J ¼ 8.0 Hz), 7.35 (1H, t, J ¼ 7.5 Hz), 7.63 (1H, t, J ¼ 7.5 Hz) and 7.84 (1H, d,
J ¼ 8.0 Hz)] were indicative of the occurrence of a disubstituted aromatic ring. In addition, a
broad singlet NH signal at d 12.19 (1H, br. s, D O exchangeable) and an aromatic singlet at d
H
H
2
9
.09 (1H, s) were also displayed. These signals were indicative of a b-carboline skeleton (G o¨ zler
Shamma 1990; Sun et al. 2004).
&
1
The C NMR (DMSO-d ) spectrum of 1, along with the information obtained from the
3
6
HSQC experiment, showed 18 C-atom signals, of which 11 carbons were assigned to the
b-carboline alkaloid skeleton. Furthermore, the following four carbon resonances (d 49.1 and
C
3
9.9; 200.6 and 25.5), as well as the presence of proton signals [d 4.47 (1H, br. m), 2.71 (1H,
H
dd, J ¼ 8.0, 16.0 Hz) and 2.80 (1H, dd, J ¼ 2.0, 16.0 Hz); 2.88 (3H, s)] suggested the
occurrence of a ZCHCH Z moiety and an acetyl group. In the HMBC spectrum, the
2
correlations from Me-15 (dH 2.88) to C-1 (d 133.9) and C-14 (d 200.6) were observed,
C
C
supporting the location of the acetyl group at C-1. The correlations of H-19 (d 2.68–2.82)
H
with C-18 (d 49.1), C-21 (d 172.7) and C-20 (d 173.4), as well as H-17 (d 9.12) with C-18
C
C
C
H
(
CH COOH. This side chain was located at C-3 as deduced from the J correlations of H-4 (d
dC 49.1) and C-16 (dC 163.1) led to the construction of the fragment CONHCH(COOH)
3
2
H
9
.09) and C-16 (d 163.1) (Figure S7). Acid hydrolysis of 1 liberated L-aspartic acid, which
C
was identified by comparing the retention time and optical rotation with the standard sample.
Thus, the absolute configuration at C-18 was determined as S (Matsumura et al. 1984) and
compound 1 was formulated as 1-acetyl-16-aspartyl-b-carboline-3-carboxylate named as
oldhamiaine A (Figure 1).
Compound 2, purified as a yellow amorphous solid, was also characterised as a b-carboline
1
derivative, and the molecular formula was established as C H N O from HR-ESI-MS. The H
2
0 19 3 6
1
3
and C NMR spectra displayed signals very similar to those of 1, with the major differences
being on the presence of two additional methoxyl signals at d 3.69, 3.64 (3H, s, each). The
H
3
HMBC J-correlations of H-22 (d 3.69) and C-20 (d 171.2), as well as H-23 (d 3.64) and C-
H
C
H
2
1 (d 171.1) indicated these two methoxyls were esterified with the C-20, C-21, respectively.
C
Similar to 1, compound 2 displayed a positive optical rotation, indicating that the absolute
configuration of C-18 was also S. On the basis of the above-mentioned results, compound 2 was
identified as 1-acetyl-20,21-dimethoxy-16-aspartyl-b-carboline-3-carboxylate, named as old-
hamiaine B. To verify that 2 is an authentic natural product, compound 1 was treated with MeOH
by simulating isolation situation, and compound 2 was not detected in the supernatant using
HPLC analysis (Supporting Information Figures S13–S15). This result indicated that 2 is indeed
a naturally occurring metabolite.
Compound 3 was identified as 1-acetyl-3-methoxycarbonyl-b-carboline by comparing its
spectroscopic data with the literature (Faini et al. 1978).
This is the first report of b-carboline-type alkaloids isolated from the genus Gypsophila,
which adds to the number of reports regarding the absence of alkaloids in some species of the
Caryophyllaceae family (Chen et al. 2010; Tian et al. 2012).
The cytotoxicity of compounds 1–3 against three human cancer cell lines MCF-7
human breast cancer), HepG-2 (human hepatoma cancer) and U2OS (human osteosarcoma
(
cancer) were tested using the methyl thiazol tetrazolium method (Lu et al. 2009).
However, the result showed that these compounds exhibited no cytotoxic activities
(
IC50 . 100 mM).

Natural Product Research
3
Figure 1. The structures of compounds 1–3.
3
3
. Experimental
.1. General experimental procedures
Optical rotations were measured with a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). UV
spectra were recorded using a UV-2450 UV–visible spectrophotometer (Shimadzu, Tokyo,
Japan). IR spectra (KBr disks) were recorded on a Bruker Tensor 27 spectrometer (Bruker,
1
Karlsruhe, Germany). NMR spectra were measured on a Bruker AV-500 NMR instrument ( H:
5
1
3
00 MHz, C: 125 MHz, Bruker), with tetramethylsilane as internal standard. HR-ESI-MS were
measured with an Agilent 6520B Q-TOF mass instrument (Agilent Technologies, Santa Clara,
CA, USA). Extracts were chromatographed on silica gel (Qingdao Marine Chemical Co., Ltd,
Qingdao, China), octadecyl silane (40–63 mM, Fuji, Tokyo, Japan), Sephadex LH-20
(Pharmacia, Uppsala, Sweden) and purified on an Agilent 1100 Series preparative HPLC
system (Agilent Technologies, Santa Clara, CA, USA).
3
.2. Plant material
The roots of G. oldhamiana were collected in Lianyungang City, Jiangsu Province, China, and
identified by Prof. Minjian Qin, the Department of Natural Medicinal Chemistry, China
Pharmaceutical University, Nanjing, China. A voucher specimen (No. 20110616) has been
deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
3
.3. Extraction and isolation
The air-dried roots of G. oldhamiana (2.0 kg) were refluxed with 80% EtOH three times
3 £ 10 L). The solution was concentrated, and the obtained crude extract (202.0 g) was
suspended in H O and successively partitioned with petroleum ether, dichloromethane
(
2
(
CH Cl ), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc portion (total
2 2
1
5
0.0 g) was fractioned on a silica gel column with a gradient of CHCl –MeOH (10:1, 5:1,
3
:3, 1:1, v/v) to afford four fractions (A–D). Subsequently, fraction B (2.6 g) was further
separated by Sephadex LH-20 column (MeOH) to give three subfractions (B1–B3).
Subfraction B2 (620.0 mg) was subjected to an RP-C18 CC with MeOH–H O (60:40 to
2
8
purified by preparative HPLC (MeOH–H O–trifluoroacetic acid (TFA), 60:40:0.1, 10 mL/
0:20, v/v) to yield three major subfractions B2a–B2c. Finally, fraction B2c was further
2
min), to yield 1 (5.0 mg). Subfraction B3 (580.0 mg) was performed on RP-C18 column
eluted with a step gradient of MeOH–H O (50:50 to 90:10), and then purified by
2
preparative HPLC (MeOH–H O–TFA, 60:40:0.1, 10 mL/min) to afford 2 (3.0 mg) and 3
2
(
2.0 mg).

4
Y. Zhang et al.
3
.4. Oldhamiaine A (1)
2
D
5
Yellow powder; ½aꢀ þ 14.0 (c ¼ 0.02, MeOH); UV (MeOH) l
(log 1): 376 (3.45), 286
4.29), 220 (4.20) nm; IR (KBr) n : 3420, 2919, 2851, 1679, 1669, 1593, 1385, 1205, 1140,
max
max
(
21 1
049, 841, 803, 724 cm ; H NMR (500 MHz, DMSO- d ): d 9.09 (1H, s, H-4), 8.45 (1H, d,
6
1
J ¼ 8.0 Hz, H-5), 7.35 (1H, t, J ¼ 7.5 Hz, H-6), 7.63 (1H, t, J ¼ 7.5 Hz, H-7), 7.84 (1H, d,
J ¼ 8.0 Hz, H-8), 12.19 (1H, br. s, H-9), 2.88 (3H, s, H-15), 9.12 (1H, d, J ¼ 7.0 Hz, H-17), 4.47
(
1H, br. m, H-18), 2.71 (1H, dd, J ¼ 8.0, 16.0 Hz, Ha-19), 2.80 (1H, dd, J ¼ 2.0, 16.0 Hz, Hb-
1
9). C NMR (125 MHz, DMSO- d ): d 133.9 (C-1), 138.3 (C-3), 117.7 (C-4), 122.2 (C-5),
3
1
1
2
6
20.8 (C-6), 129.3 (C-7), 113.3 (C-8), 134.9 (C-10), 132.0 (C-11), 120.3 (C-12), 142.4 (C-13),
00.6 (C-14), 25.5 (C-15), 163.1 (C-16), 49.1 (C-18), 39.9 (C-19), 172.7 (C-20), 173.4 (C-21);
2
2
2
ESI-MS: m/z 368 [M 2 H] , 251 [M 2 H 2 C H O ] , 165 [M 2 H 2 C H O N] ; HR-ESI-
4
5
4
7 9 6
2
MS: m/z 368.0894 [M 2 H] (calcd for C H N O , 368.0888).
1
8 14 3 6
3
.4.1 Acid hydrolysis of 1
A mixture of compound 1 (2.0 mg), 6 M HCl (1 mL) and dimethyl sulfoxide (DMSO, 1 mL) was
heated at 808C for 4 h, and then poured into ice water. The reaction mixture was extracted with
CHCl and the acid aqueous layer was neutralised with AgNO to obtain an AgCl precipitate.
3
3
After filtering, the aqueous mixture was concentrated under reduced pressure to dryness. The
residue was purified by preparative HPLC (MeOH–H O, 40:60) to yield a white powder, which
2
was identified to be L-aspartic acid by comparing the retention time on HPLC (MeOH–H O,
2
2
D
5
4
authentic sample.
0:60, t ¼ 6.9 min) and optical rotation dispersion (½aꢀ þ 13.1 (c ¼ 1.0, MeOH)) with an
R
3
.5. Oldhamiaine A (2)
2
D
5
Yellow amorphous solid; ½aꢀ þ 8.2 (c ¼ 0.02, MeOH); UV (MeOH) l
(log 1): 375 (3.25),
86 (4.10), 205 (4.38) nm; IR (KBr) n : 3483, 2952, 1735, 1702, 1681, 1533, 1450, 1201, 964,
max
2
7
7
max
2
1 1
94 cm ; H NMR (500 MHz, DMSO- d ): d 9.11 (1H, s, H-4), 8.46 (1H, d, J ¼ 8.0 Hz, H-5),
6
.35 (1H, t, J ¼ 7.8 Hz, H-6), 7.64 (1H, t, J ¼ 7.8 Hz, H-7), 7.85 (1H, d, J ¼ 8.0 Hz, H-8), 12.20
(
1H, br. s, H-9), 2.91 (3H, s, H-15), 9.17 (1H, d, J ¼ 8.0 Hz, H-17), 5.07 (1H, dt, J ¼ 6.0, 8.0 Hz,
H-18), 2.99 (1H, dd, J ¼ 6.0, 16.5 Hz, Ha-19), 3.09 (1H, dd, J ¼ 6.0, 16.5 Hz, Hb-19), 3.69 (3H,
1
s, H-22), 3.64 (3H, s, H-23). C NMR (125 MHz, DMSO- d ): d 133.9 (C-1), 137.6 (C-3), 118.2
3
6
(
C-4), 122.3 (C-5), 120.9 (C-6), 129.4 (C-7), 113.3 (C-8), 135.0 (C-10), 131.9 (C-11), 120.3 (C-
1
2
2), 142.3 (C-13), 200.7 (C-14), 25.7 (C-15), 163.9 (C-16), 48.6 (C-18), 35.8 (C-19), 171.2 (C-
2
0), 171.1 (C-21), 52.4 (C-22), 51.7 (C-23); ESI-MS: m/z 396 [M 2 H] , 353
2
þ
[
M 2 H 2 C H O] ; HR-ESI-MS: m/z 420.1169 [M þ Na] (calcd for C H N O Na,
2
3
20 19 3 6
4
20.1196).
3
.5.1 Simulating reaction between 1 and methanol in the isolation situation
A portion of compound 1 (0.5 mg) in MeOH (2 mL) was mixed with 1 g of silica gel in water
bath at 508C for 12 h. Then, the supernatant was collected and subjected to HPLC analysis, and
compound 2 was not detected.
4
. Conclusions
In this article, two new b-carboline alkaloids, named oldhamiaines A and B (1 and 2), together
with a known analogue, were isolated from the roots of G. oldhamiana. The structure of
oldhamiaines A and B were identified as 1-acetyl-16-aspartyl-b-carboline-3-carboxylate, 1-

Natural Product Research
5
acetyl-20,21-dimethoxy-16-aspartyl-b-carboline-3-carboxylate, respectively, by 1D, 2D NMR
and MS spectra. This is the first report on the isolation of b-carboline-type alkaloids from the
genus Gypsophila.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S15.
Funding
This research was supported by the National Natural Science Foundation of China [grant number
81073009], Huahai Graduate Innovation Fund [grant number CX13S-007HH] and the Program for
Changjiang Scholars and Innovative Research Team in University [grant number IRT1193].
References
Chen YF, Kuo PC, Chan HH, Kuo IJ, Lin FW, Su CR, Yang ML, Li DT, Wu TS. 2010. b-Carboline alkaloids from
Stellaria dichotoma L. var. lanceolata and their anti-inflammatory activity. J Nat Prod. 73:1993–1998.
Faini F, Castillo M, Torres R. 1978. A new b-carboline alkaloid from Vestia lycioides. Phytochemistry. 17:338.
G o¨ zler B, Shamma M. 1990. Four b-carboline alkaloids from Roemeria hybrid. J Nat Prod. 53:740–743.
Jiansu College of New Medicine. 1977. The Chinese medicine dictionary. Shanghai: People’s Publishing House.
Kuo PC, Shi LS, Damu AG, Su CR, Huang CH, Ke CH, Wu JB, Lin AJ, Bastow KF, Lee KH. 2003. Cytotoxic and
antimalarial b-carboline alkaloids from the roots of Eurycoma longifolia. J Nat Prod. 10:1324–1327.
Liu Z, Li D, Owen NL, Grant DM, Cates RG, Jia Z. 1995. Two triterpenoid saponins from Clinopodium chinensis. J Nat
Prod. 6:157–161.
Lu DQ. 1994. The classification and distribution of Gypsophila (Caryophyllaceae) in China. Bull Bot Res. 4:329–337.
Lu YY, Luo JG, Huang XF, Kong LY. 2009. Four new steroidal glycosides from Solanum torvum and their cytotoxic
activities. Steroids. 74:95–101.
Luo JG, Kong LY. 2006. A pair of new nortriterpene saponin epimers from the roots of Gypsophila oldhamiana. Helv
Chim Acta. 89:947–953.
Matsumura E, Kobayashi H, Nishikawa T, Ariga M, Tohda Y, Kawashima T. 1984. 3,5-Dinitro-1-(4-nitrophenyl)-4-
pyridone, a novel and convenient protecting reagent for primary amines. Bull Chem Soc Jpn. 57:1961–1965.
Nie W, Luo JG, Kong LY. 2010. New triterpenoid saponins from the roots of Gypsophila pacifica Kom. Carbohydr Res.
345:68–73.
Sun B, Morikawa T, Matsuda H, Tewtrakul S, Wu LJ, Harima S, Yoshikawa M. 2004. Structures of new b-carboline-type
alkaloids with antiallergic effects from Stellaria dichotoma. J Nat Prod. 67:1464–1469.
Tian JM, Shen YH, Li HL, Liu RH, Shan L, Gao JM, Zhang WD. 2012. Carboline alkaloids from Psammosilene
tunicoides and their cytotoxic activities. Planta Med. 78:625–629.
Wang G, Luo JG, Yang MH, Wang XB, Kong LY. 2013. Six new cyclic peptides from the roots of Gypsophila
oldhamiana. Chem Pharm Bull. 61:489–495.