Two new quinolone alkaloids from the nearly ripe fruits of Tetradium ruticarpum

Article abstract of DOI:10.1080/14786419.2019.1566819

Tetradium ruticarpum (Juss.) Benth. belong to the family of Rutaceae. The complete and nearly ripe fruits of T.ruticarpum is used as traditional Chinese medicine and phytochemical investigations have been conducted on extracts of the seeds of T.ruticarpum to provide scientific validation of its properties. In this study, we successfully isolated two new quinolone alkaloids (1-2) from the MeOH extractive of nearly ripe fruits of T.ruticarpum. The structure elucidation of these compounds was determined by one- and two-dimensional nuclear magnetic resonance, ultraviolet and electrospray ionisation time-of-flight mass spectrometry. This finding expands the understanding of the natural constituents of the Rutaceae, in particular, the Tetradium genera.

Full text of DOI:10.1080/14786419.2019.1566819

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Two new quinolone alkaloids from the nearly ripe
fruits of Tetradium ruticarpum
Yi-Heng Li, Xiao Liu, Min Yin, Fei Liu, Bi Wang, Xu Feng & Qi-Zhi Wang
To cite this article: Yi-Heng Li, Xiao Liu, Min Yin, Fei Liu, Bi Wang, Xu Feng & Qi-Zhi Wang
(2019): Two new quinolone alkaloids from the nearly ripe fruits of Tetradiumꢀruticarpum, Natural
Product Research, DOI: 10.1080/14786419.2019.1566819
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1566819
Two new quinolone alkaloids from the nearly ripe fruits
of Tetradium ruticarpum
Yi-Heng Lia,b, Xiao Liua,b, Min Yina,b, Fei Liua,b, Bi Wanga,b, Xu Fenga,b and Qi-
Zhi Wanga,b
aJiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany,
b
Jiangsu Province and Chinese Academy of Sciences, Nanjing, People’s Republic of China; The
Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Institute of
Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, People’s Republic of China
ABSTRACT
ARTICLE HISTORY
Received 4 September 2018
Accepted 26 December 2018
Tetradium ruticarpum (Juss.) Benth. belong to the family of
Rutaceae. The complete and nearly ripe fruits of T. ruticarpum is
used as traditional Chinese medicine and phytochemical investi-
gations have been conducted on extracts of the seeds of T. ruti-
carpum to provide scientific validation of its properties. In this
study, we successfully isolated two new quinolone alkaloids (1-2)
from the MeOH extractive of nearly ripe fruits of T. ruticarpum.
The structure elucidation of these compounds was determined by
one- and two-dimensional nuclear magnetic resonance, ultraviolet
and electrospray ionisation time-of-flight mass spectrometry. This
finding expands the understanding of the natural constituents of
the Rutaceae, in particular, the Tetradium genera.
KEYWORDS
Quinolone A; Quinolone B;
Rutaceae;
Tetradium ruticarpum
CONTACT Qi-Zhi Wang
wangqizhi@cnbg.net
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1566819
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-H. LI ET AL.
1. Introduction
Herbs have been widely used as important remedies all over the world (Anazarba
2005; Li 2006). Rutaceous plants, especially Tetradium ruticarpum (Juss.) Benth.
(Tetradium genus, old name is Evodia rutaecarpa (Juss.) Benth.; and Chinese name is
wuzhuyu.), have long been used for the treatment of postpartum hemo-rrhage, gastro-
intestinal disorders, amenorrhea, and headache in traditional oriental medicine (Chen
and Chen 1933; Liao et al. 1981). Various secondary metabolites have been isolated
from members of this family, and have been divided into fractions of alkaloids, essen-
tial oils, limonoids, carboxylic acids, flavonoids and polycose (Zhou et al. 2010; Cao
et al. 2018). Among them, quinolone alkaloids and indolequinazoline alkaloids are pre-
dominantly found in plants of the genus Evodia (Zhou et al. 2010). Indolequina-zoline
alkaloids, with their structural diversity, were reported to possess wide range of bio-
logical activities against different strains of bacteria, fungi, viruses, plasmodium
(Severino et al. 2015) and various cancer cell lines (Zhu et al. 2011; Zou et al. 2014).
T. ruticarpum is a member of the Rutaceae family, its dried and nearly ripe fruits
have been used as a traditional Chinese medicine to treat various diseases including
dysentery, headache, abdominal pain, and postpartum hemorrhage (China
PCotMohoPsRo 1964). In recent years, many new active ingredients have been isolated
from T. ruticarpum, which shows good anti-tumor, anti-inflammatory, hypoglycemic,
bacterial infections and lipid-lowering activity (Zhu 2011). A subsequent research by
Wang et al. revealed that T. ruticarpum exhibits significant inhibited activity against
tumor cell as well as inhibit proliferation of various tumor cells (Wang et al. 2014;
Huang et al. 2015; Zhong et al. 2015; Chen et al. 2016). Recently, Su et al. (Su et al.
2018) reported the isolation of three new quinazolines from the seeds of T. ruticar-
pum. In addition, Baburin Igor. et al. (Baburin et al. 2018) and Yang et al. (Yang et al.
2017) obtained and evaluated the cardiotoxicity of several compounds from the
T. ruticarpum.
Attracted by the bioactive alkaloids from the fruits of T. ruticarpum, we develop
the study of the fruits of T. ruticarpum were extracted exhaustively with Petroleum
ether, CH2Cl2, EtOAc and 90% aqueous MeOH sequentially. Then we found two new
quinolone alkaloids (1 and 2) from 90% aqueous MeOH sequentially, compound 1 is a
Quinolone ester, compound 2 is a quinolone alkaloid glucoside.
2. Results and discussion
Compound 1 was obtained as a white powder (MeOH), showing UV absorbance max-
ima at 213, 236, 322, and 334 nm characteristic for an N-methyl-4(1H)-quinolone skel-
eton (Wang et al. 2013). The HR-ESI-MS is m/z 246.1116 ([M þ H] þ, Calcd for
C14H16NO3, 246.1130) with 8 degrees of unsaturation. Examination of the NMR spectral
data (Table S1) of 1 revealed an ortho-disubstituted phenyl group [dH 8.18 (1 H, dd,
J ¼ 7.8, 1.7 Hz, H-5), 7.75 (1 H, m, H-6), 7.40 (1 H, dd, J ¼ 7.5, 7.3 Hz, H-7), 7.81 (1 H, d,
J ¼ 7.5 Hz, H-8); dC 125.7 (C-5), 132.5 (C-6), 123.4 (C-7), 117.2 (C-8)], a N-methyl group
(dH 3.78, s, 3H; dC 34.7), one carbonyl (dC 161.2) and one conjugated olefinic group [dH
(6.05 1 H, s, H-3); dC 110.0 (C-3), 154.2 (C-2)], which revalidate that 1 processes an N-
1
methyl-4(1H)-quinolone skeleton (Wang et al. 2013) (Blue in Figure 1). From the H-

NATURAL PRODUCT RESEARCH
3
Figure 1. Chemical structures of compounds 1 and 2.
NMR spectral data dH 2.77 (2 H, t, J ¼ 7.6), dH 3.10 (2 H, t, J ¼ 7.6) and dH 3.66 (3 H, s)
we can guess the existence of one methylene and one oxymethyl. In addition,
there is another carbonyl from the 13C-NMR spectral data of dC 172.4. The correla-
tions of this fragments were confirmed by the HMBC correlations from O-CH3 (dH 3.
66, 3 H, s; dC 52.0) to dC 172.4 (C-3’); H-1’ (2.77, t, 2 H J ¼ 7.6) to C-2 (dC 154.2), C-3’
(dC 172.4); H-2’ (3.11, t, 2 H J ¼ 7.6) to C-2 (dC 154.2), C-3 (dC 110.0), C-3’ (dC 172.4),
and the structure of 1 was elucidated as shown in Figure 1. Thus, 1 was elucidated
as methyl-3-[1-methyl-4(1H)-quinolone-yl] propanoate, for which named as
Quinolone A.
Compound 2 was obtained as a white powder (MeOH). The HR-ESI-MS displayed
a pseudomolecular ion peak at m/z 422.1799 [M þ H]þ, consistent with a molecular
formula of C21H28NO8 (Calcd for 422.1809), with 9 degrees of unsaturation. The UV
absorptions at 207, 351 nm. The 1H NMR spectrum of Compound 2 (Table S2)
showed resonances for an trisubstituted benzene moiety at dH 7.40 (1 H, d, J ¼ 8.
0 Hz, H-5), 7.34 (1 H, d, J ¼ 8.0 Hz, H-7) and 7.17 (1 H, dd, J ¼ 1.0, 8.0 Hz, H-6), a pri-
mary methoxy group at dH 3.90, one N-H at dH 10.87, one olefinic proton at dH 5.
22, two methylene protons at dH 3.28, and six methyl protons at dH 1.68 and 1.77.
The 13C NMR and distortionless enhancement by polarization transfer (DEPT) data
(Table S2) showed 21 carbon signals assigned to one methoxy at dC 62.0 and two
methyl at dC 25.9 and 18.2; two methylene at dC 61.2 (belonged to a sugar moiety)
and 23.4; nine homomethyl which including three aromatic protons resonated at
dC 116.4, 122.1 and 115.7, five belonged to a sugar moiety (dC 102.6, 77.8, 73.6, 70.
4, 75.9); seven quaternary carbons (five aromatic dC 161.2, 144.2,128.4, 123.0, 117.4,
one carbonyl dC 163.4). The structure of the 3,8-substituted 2-quinolone structure
was established by HSQC and HMBC spectra from dH 3.90 (O-CH3) to C-4 (dC 161.3);
from H-1’ (dH 3.28) to C-2 (dC 163.5), C-4 (dC 161.3); Acid hydrolysis of 2 with 1 M
trifluoroacetic acid (TFA) offered a sugar, which was identified as D-glucose by its
3
optical rotation and TLC comparison with an authentic sample. In addition, the J
(1, 2) coupling constant (7.7 Hz) indicated a b-D-glucoside by comparing the 13C
NMR data with those reported in the literature (Zhang et al. 2013; Xia et al. 2016).
The sugar unit was connected to C-1 through a C–O linkage as deduced by the
HMBC from H-1’ (dH 4.83, d, J ¼ 7.3 Hz) to C-8 (dC 144.2). Thus, the structure of 2
was established as 4-methoxy-3-(3-methylbut-2-en-1-yl)-2-quinolone-8-O-b-D-gluco-
pyranoside, for which named as Quinolone B.

4
Y.-H. LI ET AL.
3. Experimental
3.1. General experimental procedures
Melting points were measured using a (uncorrected) XT-4 Boetius micro-melting point
apparatus (Tektronix, USA). HR-ESI-MS spectra were measured on an Agilent 1260
UPLC-DAD-6530 ESI Q-TOF MS spectrometer (Agilent, USA) equipped with a ZORBAX
SB C18 column (1.8 lm, 100 ꢀ 4.6 mm) in m/z. NMR data were acquired on a Bruker
DRX 500 NMR spectrometer with 1H and 13C NMR observed at 500 and 125 MHz
(Bruker, Germany), with chemical shifts (d) given in ppm with reference to the solvent
and coupling constants (J) in Hz. HPLC analyses were performed with a YMC ODS-
5 mm (150 ꢀ 4.6 mm) column, an Agilent pump 1100 and an ELSD detector Alltech 500
(Alltech, USA). Preparative HPLC (LC-20AR, Shimadzu, Japan) was conducted on a
Shim-pack GIS C18 column (5 lm, 250 ꢀ 20 mm, Shimadzu). Medium pressure liquid
chromatography (MPLC, Buchi, Switzerland) was conducted on a C18 column (20 lm,
920 ꢀ 100 mm, Aglea Technologies, USA). (100–200, 200–300 mesh) Silica gel (Qingdao
Marine Chemical Factory, China) and Sephadex LH-20 were used for column chroma-
tography (Amersham Pharmacia Biotech, Sweden). TLC analyses were carried out on
silica gel 60 F254 (Merck, Germany) plates. The compounds were monitored by spraying
Dragendorff reagent and 5% vanillin-sulfuric acid reagent.
3.2. Plant material
Plant material was purchased from Xuancheng, Anhui Province, China, in September
2015, and identified as the dried and nearly ripe fruits of Tetradium ruticarpum (Juss.)
Benth. (old name is Evodia rutaecarpa (Juss.) Benth.; and Chinese name is wuzhuyu.)
by Prof. Chang-Qi Yuan (Institute of Botany, Jiangsu Province and Chinese Academy of
Sciences). A voucher specimen (NO. ER2015/9) was deposited in the Natural Product
Chemistry Research Center, IBJSPACAS repository.
3.3. Extraction and isolation
The dried fruits of Tetradium ruticarpum (100 kg) were reflux extracted with 80% etha-
nol (each for 300 L) at 80 ꢁC for three times (each for 3 h), yielding 25.7 kg of crude
extract. The extract (25.7 kg) was subjected to silica gel (100–200 mesh) column chro-
matography (CC), eluted successively with a petroleum ether, petroleum ether:CH2Cl2
(1:1, v/v), CH2Cl2, CH2Cl2: EtOAc (2:1, v/v), EtOAc, EtOAc: MeOH (1:1, v/v) and methanol
gradient to produce 7 fractions. The MeOH fraction (9.735 kg) was chromatographed
over silica gel (200–300 mesh) and eluted using CH2CI2: methanol mixtures (100:0 to
0:100, v/v) to obtain fr. A-H. Fr. E (90 g) was subjected to Sephadex LH-20 CC with
CH2Cl2: MeOH (1:1, v/v) to get six fr. E1-6. Further separation of fr. E3 (10.7 g) by RP
C18 CC with MeOH: H2O (Medium pressure liquid chromatography MPLC 30% to
100%, v/v 20 mL/min) to yield seven mixtures. The mixtures fr. E3-2 (2.8 g) preparative
HPLC (40% MeOH-H2O, 5 ml/min, tR = 25.1 min) to get compound 1 (8 mg). The mix-
tures fr. E3-3 (1.4 g) and preparative HPLC (55% MeOH- H2O, 5 ml/min, tR = 48.1 min)
to get compound 2 (7 mg).

NATURAL PRODUCT RESEARCH
5
Quinolone A: C14H15NO3, white powder, m.p.: 157.0 – 160.1. [k]25 = ꢂ5.8 (c 0.09,
CH3OH); HR-ESI-MS (m/z 246.1116 [M þ H]þ, Calcd for C14H16NO3, 246.1130); UV
1
(kCH3OH max nm): 213, 236, 322, and 334 nm; H (500 MHz) and 13C (125 MHz) NMR
data in DMSO-d6 (see in Table S1, recorded in supporting Information).
Quinolone B: C21H27NO8, white powder, m.p.: 181.8 - 186.2. HR-ESI-MS (m/z
422.1799 ([M þ H]þ, Calcd for C21H28NO8 422.1809)); UV (kCH3OH max nm): 207, 218,
1
235, 247, 279, 328, 341, 351 nm; H (500 MHz) and 13C (125 MHz) NMR data in DMSO-
d6 (see in Table S2, recorded in supporting Information).
3.4. Acid hydrolysis of compound 2
Compound 2 (1 mg) was dissolved in 1 M TFA (5 mL) and then heated in a H2O bath
at 80 ꢁC for 2 h. Then extraction with EtOAc (3 ꢀ 5 ml). the aq. layer was repeatedly
evaporated to dryness with CH3OH until neutral. Co-TLC analysis (EtOAc: CH3OH: H2O:
HAc = 13:2:1:3, 5% vanillin-sulfuric acid reagent, heating in 105ꢁC Rf = 0.35) in com-
parison to authentic glucose, indicated the presence of glucose.
4. Conclusion
In this study, two new naturally quinolone alkaloids, quinolone A and B were obtained
from the MeOH extract of the nearly ripe fruits of Tetradium ruticarpum. The two com-
pounds all beglong to quinolone alkaloids, which reportedly present in the Evodia
genus and other members of the Rutaceae family with various activities. But the bio-
activity of both isolated compounds remains under investigation. In addition, during
to the narrow distribution of quinolones with this kind of structure, it has the signifi-
cance of botany classification. So, our fndings expandour of the natural constituents of
the Rutaceae and, in particular, the Rutoideae genera.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the [National Natural Science Foundation of China #1] under Grant
[number 31470425 and 31570359]; [Science and Technology Foundation of Jiangsu Province #2]
under Grant [number BM2015019]; and [Social Development Projects from the Science and
Technology Foundation of Jiangsu Province #3] under Grant [number BE2016753].
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