Two new sesquiterpenoid glycosides from the stems of Zanthoxylum armatum DC

Article abstract of DOI:10.1080/14786419.2019.1607332

Two new sesquiterpenoid glycosides as dihydrophaseic acid 4′-O-[6″-O-(4″′-hydroxy-3″′, 5″′-dimethoxy) benzoyl)]-β-D-glucopyranoside (1) and dihydrophaseic acid 4′-O-[6″-O-(3″′-methoxy- 4″′-hydroxy) benzoyl)]-β-D-glucopyranoside (2), were isolated from the stems of Zanthoxylum armatum in the study. The compound 1 and 2 showed moderate scavenging activity in DPPH free radical assay with IC₅₀ values of 241 and 264 μM, respectively.

Full text of DOI:10.1080/14786419.2019.1607332

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Two new sesquiterpenoid glycosides from the
stems of Zanthoxylum armatum DC
Ya-Lin Liu, Liang-Liang Gao, Tong-Tong Song, Tao Guo & Jun Chang
To cite this article: Ya-Lin Liu, Liang-Liang Gao, Tong-Tong Song, Tao Guo & Jun Chang (2019):
Two new sesquiterpenoid glycosides from the stems of Zanthoxylumꢀarmatum DC, Natural Product
Research, DOI: 10.1080/14786419.2019.1607332
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1607332
Two new sesquiterpenoid glycosides from the
stems of Zanthoxylum armatum DC
a
b
Ya-Lin Liu , Liang-Liang Gao , Tong-Tong Songc,d, Tao Guoa , and
ꢀ
ꢀ
Jun Changc
b
aSchool of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, 450056, China; School of
c
life sciences and health, Anhui Science and Technology University, Fengyang, 233100, china; School
d
of Pharmacy, Fudan University, Shanghai, 201203, China; School of Life Science and Engineering,
Lanzhou University of Technology, Lanzhou, 730050, China
ABSTRACT
ARTICLE HISTORY
Received 11 November 2018
Accepted 25 March 2019
Two new sesquiterpenoid glycosides as dihydrophaseic acid
40-O-[600-O-(4000-hydroxy-3000, 5000-dimethoxy) benzoyl)]-b-D-glucopyr-
anoside (1) and dihydrophaseic acid 40-O-[600-O-(3000-methoxy-
4000-hydroxy) benzoyl)]-b-D-glucopyranoside (2), were isolated
from the stems of Zanthoxylum armatum in the study. The com-
pound 1 and 2 showed moderate scavenging activity in DPPH
free radical assay with IC50 values of 241 and 264 lM, respectively.
KEYWORDS
Zanthoxylum armatum;
sesquiterpenoid glycoside;
scavenging activity in DPPH
free radical
1. Introduction
Genus Zanthoxylum (Rutaceae) consists of about 250 species widely distributed in
the tropical and subtropical regions of Asia, Africa, Oceania and North America. There
are 39 species and 14 varieties in China, occurring nearly everywhere in the country
CONTACT Tao Guo
Zhengzhou, 450056, China; Jun Chang
gt010010@163.com
School of Pharmacy, Henan University of Chinese Medicine,
jchang@fudan.edu.cn School of Pharmacy, Fudan University,
Shanghai, 201203, China.
ꢀ
The both authors contributed equally to this work.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1607332.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-L. LIU ET AL.
Figure 1. Chemical structures of compound 1 and 2.
(Guo et al. 2012). Zanthoxylum armatum DC. is a common specie in the genus. Its root
and stem were used as Traditional Chinese medicine, and its fruit was also often used
as food condiment and aromatic preservatives (Guo et al. 2017). Zhuyejiao tablets as a
Chinese patented drug are mainly made up of raw herb powder and ethanol extract
from Z. armatum (Guo et al. 2011). Modern pharmacological studies have shown that
Z. armatum possess xanthine oxidase inhibitory (Ranjana et al. 2019), antiasthmatic
(Sharma et al. 2018), hypolipidemic and hypoglycemic (Alam et al. 2018), anti-inflam-
matory (Nooreen et al. 2017), anticancer (Alam et al. 2017), antidiabetic (Karki et al.
2014) and hepatoprotective (Verma and Khosa 2010) activities. Phytochemical investi-
gations (Bhatt et al. 2015; Guo et al. 2012, 2015, 2017; Samad et al. 2014) have shown
that Z. armatum contains a variety of chemical constituents, such as alkaloid, lignan,
amide, flavone and phenol glycoside. As part of our systematic investigations on
chemical constituents from Z. armatum, the present study investigated the water frac-
tion of the ethanol extract of Z. armatum, resulting in isolation of two new com-
pounds, 40-O-[600-O-(4000-hydroxy-3000,5000-dimethoxy) benzoyl)]-b-D-glucopyranoside (1)
and dihydrophaseic acid 40-O-[600-O-(3000-methoxy-4000-hydroxy) benzoyl)]-b-D-glucopyra-
noside (2) (Figure 1).
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. The HR-ESI-MS showed
quasimolecular ion at m/z 647.2295 [M þ Na]þ (calcd. for C30H40O14Na, 647.2310), indi-
1
cating a molecular weight of 624. According to the HR-ESI-MS, H and 13C-NMR spec-
tral data, the molecular formula of 1 was determined to be C30H40O14. In the low field
1
of H-NMR spectrum (Table S1), the signals at d 7.18 (2H, s) defined a 1,3,4,5-substi-
tuted aromatic ring, which was further confirmed in the 13C-NMR spectrum by four
quaternary aromatic carbons at d 119.9 (C-1000), 147.2 (C-3000, C-5000), 139.6 (C-4000) and
two methine carbons at d 107.1 (C-2000, C-6000). The signals at d 7.47 (1H, d, J ¼ 15.9 Hz)
and 6.18 (1H, d, J ¼ 15.9 Hz) indicated the trans-double bond, while 5.79 (1H, s)

NATURAL PRODUCT RESEARCH
3
1
indicated another double bond. The high field of H-NMR spectrum showed the pres-
ence of three methyl groups attached to the quaternary carbon at d ¼ 1.93 (3H, s), 1.
1
06 (3H, s), and 0.45 (3H, s). Further analysis of H-1H COSY, HMBC, and HMQC spectra
of 1 established the ionone-type skeleton with two double bonds and one conjugated
carboxyl group, substituted benzoic acid moiety, and a glucose moiety (Youn et al.
2011). The structure of ionone-type skeleton was established to be dihydrophaseic
1
acid. In the sugar part of the H-NMR spectrum, the anomeric proton d 4.46 (1H, d,
J ¼ 7.9 Hz) indicated the presence of glucose, which was confirmed by acid hydrolysis
and comparison with authentic sample. The stereochemistry of the anomeric carbon
of glucose was determined as b according to the coupling constant of the anomeric
proton and the chemical shift of C-100 (102.1). The glycosidic site was established
unambiguously by a HMBC experiment in which long-range correlation between H-100
(d 4.46) and C-40 (d 74.9) was observed. The down-field shift of hydroxymethylene sig-
nal C-600 (d 63.9) and long-range correlation between H-600a, H-600b (d 4.66, 4.49) and C-
7000 (d 167.4) indicated the esterification of hydroxymethyl. The relative configuration
was deduced from the results of the NOESY spectrum, in which NOE signals were
observed between H-40 (d 3.95) and H-50a (d 2.10), H-80b (d 3.20). As a steric assump-
tion for the successful ether formation in ionone-type skeleton (Youn et al. 2011), the
hydroxymethylene C-80 and oxygen at C-20 must have an axial orientation. The relative
configuration at C-40 was also determined to be b-stereochemistry by the coupling
constant (J4’-5’b ¼ 10.6 and J4’-5’a ¼ 6.8). Consequently, the structure of compound 1
was established as dihydrophaseic acid 40-O-[600-O-(4000-hydroxy-3000, 5000-dimethoxy) ben-
zoyl)]-b-D-glucopyranoside.
Compound 2 was obtained as a white amorphous powder. HR-ESI-MS showed qua-
simolecular ion at m/z 617.2197 [M þ Na]þ (calcd. for C29H38O13Na, 617.2205).
1
According to the HR-ESI-MS, H and 13C-NMR spectral data, the molecular formula of 2
was determined to be C29H38O13. The molecular mass of 2 was 30 Da lower than that
1
of 1. Further analysis of H-1H COSY, HMBC, and HMQC spectra of 2 also established
the ionone-type skeleton with two double bonds and one conjugated carboxyl group,
substituted benzoic acid moiety, and a glucose moiety. The difference between 1 and
2 is the substituted benzoic acid moiety. Consequently, the structure of compound 2
was established as dihydrophaseic acid 40-O-[600-O-(3000-methoxy-4000-hydroxy) benzoyl)]-
b-D-glucopyranoside.
The results of pharmacological experiments in vitro showed that compound 1 and
2 displayed moderate scavenging activity in DPPH free radical assay with IC50 values
of 241 and 264 mM, respectively.
3. Experimental
3.1. General experimental procedures
Optical rotation data were obtained on a Perkin-Elmer 241 automatic digital polarim-
eter. UV spectral data were obtained on a Shimadzu UV-260 instrument. IR spectral
data were acquired on a Perkin–Elmer 599B instrument with KBr disks. Melting points
(mp) were determined using an X-4 microscope melting point apparatus (Shanghai
Precision Science Instrument Co., Ltd.) and were uncorrected. 1H, 13C NMR, 1H-1H

4
Y.-L. LIU ET AL.
COSY, HMQC, HMBC and NOESY spectra were recorded on a Bruker DRX-400 spec-
trometer (1H 400 MHz and 13C 100 MHz). The carbon multiplicities were obtained by
DEPT experiment. HRESI-MS data were measured on a SCIEX QTOF-5600 with Triple
Quad technology. Reversed-phase chromatography utilized TSK gel Toyopearl HW-40F
(30–60 lm, Toso Co., Ltd.), MCI gel CHP 20P (75–150 lm, Mitsubishi Chemical
Industries Co., Ltd.) and Cosmosil 75 C18-OPN (42–105 lm, Nacalai Tesque Inc.) col-
umns. TLC was performed using precoated silica gel 60 F254 plates (0.2 mm, Merck).
3.2. Plant material
The stems of Z. armatum were collected from Nanning (located at 22.8ꢁ N and 108.3ꢁ
E, Altitude: 400 m - 600 m), Guangxi Province of China in 2014 and identified by Prof.
Ze-Hao Huang (Fujian University of Traditional Chinese Medicine). A voucher specimen
of the plant (#1409-21) was deposited in the Herbarium of School of Pharmacy, Fudan
University, Shanghai, China.
3.3. Extraction and isolation
Dried stems of Z. armatum (20 kg) were extracted with 95% ethanol (50 L ꢂ 3) at room
temperature. The combined EtOH solvent was filtered and concentrated under
reduced pressure to give 680 g of residue. The residue was then suspended in water
(2.0 L) and extracted 3 times with petroleum ether. The remaining aqueous solution
was concentrated to 1 L. And then, adjusted the pH of the aqueous solution to 2 ꢃ 3
with 2% hydrochloric acid and extracted with ethyl acetate (EtOAc) (1 L) for three
times to obtain ethyl acetate extract (9.4 g) and water part extract (83 g). The water-
soluble extract (83 g) was purified by MCI gel CHP 20P (8 ꢂ 60 cm) and eluted with a
mixture of MeOH and H2O (0:10, 1:9, 3:7, 5:5, 7:3, 10:0, 600 mL for each step) to yield 2
fractions (Fr.A and Fr.B) . Fraction A was further chromatographed on Toyopearl HW-
40F (6 ꢂ 60 cm, eluted with H2O!10% MeOH) to yield Fr.A 1 and Fr.A 2. Fr.A 1 were
purified using Cosmosil 75 C18-OPN (4 ꢂ 30 cm, eluted with H2O!20% MeOH) to yield
compound 1 (13.2 mg) and 2 (11.9 mg).
3.4. Characterization
3.4.1. Compound 1
White amorphous powder; [a]20D -5.714ꢁ (c 0.10, H2O); mp:135 ꢁC; UV (MeOH): kmax
256, 266 nm; IR mmax (KBr): 3300, 2920, 1700, 1620, 1450 cmꢄ1. HR-ESI-MS m/z 647.2295
1
[M þ Na]þ (calcd. for C30H40O14Na, 647.2310); H and 13C NMR: see Table S1.
3.4.2. Compound 2
White amorphous powder; [a]20D -8.333ꢁ (c 0.12 H2O); mp:133 ꢁC; UV (MeOH): kmax
256, 267 nm; IR mmax (KBr): 3310, 2918, 1710, 1625, 1455 cmꢄ1. HR-ESI-MS m/z 617.2197
1
[M þ Na]þ (calcd. for C29H38O13Na, 617.2205); H and 13C NMR: see Table S1.

NATURAL PRODUCT RESEARCH
5
3.5. Acid hydrolysis
A solution of compound 1–2 (1 mg each) in 5% HCl (0.5 ml) was heated (90 ꢁC) for 2 h.
After removing HCl by evaporation in vacuum, the mixture was diluted with H2O and
extracted with EtOAc. The aqueous layer was neutralized with 0.1 M NaOH and sugars
were detected by TLC analysis with authentic sugar: TLC conditions: CHCl3-MeOH-H2O
(14:6:1), Rf 0.13 (glucose); n-BuOH-pyridine-H2O (6:4:3), Rf 0.37 (glucose). (C¸alis and
Kırmızıbekmez 2004)
3.6. DPPH radical-scavenging assay
DPPH free radical-scavenging method were adopted as previously described (Wang
et al. 2014). The reaction mixtures containing 2.5mL of test samples solution and
DPPH (0.025 mg/mL) were incubated for 30 min. Following that, the absorbance at
517 nm was measured and the inhibition (%) of DPPH radical formation was calcu-
lated. The concentration required for 50% inhibition (IC50) of DPPH was determined
graphically. Ascorbic acid was used as a positive control.
4. Conclusion
The chemical investigation of the stems of Z. armatum has resulted in the isolation of
two new sesquiterpenoid glycosides, dihydrophaseic acid 40-O-[600-O-(4000-hydroxy-
3000,5000-dimethoxy) benzoyl)]- b-D-glucopyranoside (1) and dihydrophaseic acid 40-O-
[600-O-(3000-methoxy-4000-hydroxy) benzoyl)]-b-D- glucopyranoside (2).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
Financial support by the Shanghai Municipal Committee of Science and Technology (No:
14431900300, 15431900400), Development Project of Shanghai Peak Disciplines-Integrative
Medicine (No: 20150407) and National Natural Science Foundation of China (No: 81360476) are
acknowledged.
ORCID
Tao Guo
http://orcid.org/0000-0003-4502-5570
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