A new triterpenoid and a new flavonoid glycoside isolated from Bupleurum marginatum and their anti-inflammatory activity

Article abstract of DOI:10.1080/14786419.2019.1582037

This study to investigate chemical constituents from the aerial of Bupleurum marginatum led to the isolation of a new trierpenoid and a new flavonoid, namely 3β-hydroxy-cycloart-24-en-26-acetyloxy (1), and 3, 3′, 5′-trimethoxyl-myricetin 7-O-β-D–glucopyranoside (2) along with eight known compounds (3–10). Their structures were established by spectral data analyses (MS, 1D and 2D NMR), as well as by comparison of spectral data with those of the related known compounds. The 24-en-lanostane type triterpenoid with a cyclopropane ring (1 and 3) was firstly reported from this specie, which might be chemotaxonomic markers of this specie. In addition, compounds 1 and 2 were examined for their anti-inflammatory activity. Compounds 1 and 2 inhibited the NF κB induction by 60.61% and 24.30%.

Full text of DOI:10.1080/14786419.2019.1582037

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new triterpenoid and a new flavonoid glycoside
isolated from Bupleurum marginatum and their
anti-inflammatory activity
Zhiqiang Lei, Guoming Zou, Ying Gao, Yuqiao Yao, Caiying Peng, Jicheng Shu
& Ming Yang
To cite this article: Zhiqiang Lei, Guoming Zou, Ying Gao, Yuqiao Yao, Caiying Peng, Jicheng
Shu & Ming Yang (2019): A new triterpenoid and a new flavonoid glycoside isolated from
Bupleurumꢀmarginatum and their anti-inflammatory activity, Natural Product Research, DOI:
10.1080/14786419.2019.1582037
To link to this article: https://doi.org/10.1080/14786419.2019.1582037
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1582037
A new triterpenoid and a new flavonoid glycoside
isolated from Bupleurum marginatum and their
anti-inflammatory activity
a
a
a
b
a
ꢀ
ꢀ
Zhiqiang Lei , Guoming Zou , Ying Gao , Yuqiao Yao , Caiying Peng ,
Jicheng Shua and Ming Yanga
aKey Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine,
b
Ministry of Education, Nanchang, China; The First Affiliated Hospital of Jiangxi University of
Traditional Chinese Medicine, Nanchang, China
ABSTRACT
ARTICLE HISTORY
Received 7 November 2018
Accepted 7 February 2019
This study to investigate chemical constituents from the aerial of
Bupleurum marginatum led to the isolation of a new trierpenoid and a
new flavonoid, namely 3b-hydroxy-cycloart-24-en-26-acetyloxy (1), and
3, 30, 50-trimethoxyl-myricetin 7-O-b-D–glucopyranoside (2) along with
eight known compounds (3–10). Their structures were established by
spectral data analyses (MS, 1D and 2D NMR), as well as by comparison
of spectral data with those of the related known compounds. The 24-
en-lanostane type triterpenoid with a cyclopropane ring (1 and 3) was
firstly reported from this specie, which might be chemotaxonomic
markers of this specie. In addition, compounds 1 and 2 were examined
for their anti-inflammatory activity. Compounds 1 and 2 inhibited the
NF jB induction by 60.61% and 24.30%.
KEYWORDS
Bupleurum marginatum;
trierpenoid; flavonoids; anti-
inflammatory
1. Introduction
The Bupleurum genus is widely distributed in the northern hemisphere and commonly
used in Asian and North Africa for their medicinal properties, contains about 180 species
CONTACT Jicheng Shu
of Traditional Chinese Medicine, Ministry of Education, Nanchang 330004, China; Ming Yang
yangming16@126.com Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese
shujc210@163.com
Key Laboratory of Modern Preparation of TCM, Jiangxi University
Medicine, Ministry of Education, Nanchang 330004, China
ꢀ
Co-authors.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1582037.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Z. LEI ET AL.
(Luo and Li 2005). Among these species, three species, B. chinense, B. scorzonerifolium
and B. marginatum, are major medicinal source of “Caihu” in traditional Chinese medicine
used in treatment of various ailments (Pan 1996). However, B. chinense and B. scorzoneri-
folium have been included in Chinese Pharmacopoeia (Pharmacopoeia Commission of
PRC 2015). In the past decade, more and more researches were focused on the two spe-
cies. For example, they are chemically characterized by presence of flavonoids, trierpe-
noids, and polysaccharides (Pistelli et al. 2005; Zhang et al. 2007; Achouri et al. 2017).
They have been reported to exhibit various pharmacological activities, such as, anti-
inflammatory, antioxidant, antitumor, immunomoduulatory and kidney-protective bioac-
tivities (Cheng et al. 2003; Sun 2006; Zhao et al. 2012). However, until now, there is
relatively little information pertaining to isolation, purification, and hepatoprotective
activity determination of B. marginatum. Therefore, in order to relieve insufficient basic
researches, and fully develop the medical plant resources and extend the potential use
of B. marginatum, we have systematically investigated the constituents of it. In this paper,
we reported the isolation and structure identification of a new trierpenoid (1) and a new
flavonoid glycoside (2), as well as eight known compounds (4–10), and anti-inflamma-
tory activity of two new compounds in vitro.
2. Results and discussion
The ethyl acetate soluble part partitioned from the 75% ethanol extract of B. margina-
tum was isolated by a combination of silica gel column chromatography and
Sephadex LH-20 column chromatography to give two trierpenoids and eight flavo-
noids, including a new trierpenoid, namely 3b-hydroxy-cycloart-24-en- 26-acetyloxy (1),
and a new flavonoid glycoside, namely 3, 30, 50-trimethoxyl-myricetin 7-O-b-D-gluco-
pyranoside (2), and eight known compounds, cycloartenal (3) (Flach et al. 2004), quer-
cetin (4) (Deyama et al. 1987), myricetin (5) (Zhou et al. 2010), Quercitrin (6) (Li et al.
2016), 7-methoxyl-qurecetin (7) (Yan et al. 2012), catethin (8) (Cheng et al. 2014), 30-
metyoxyl-catehin (9) (Cheng et al. 2014) and Phloretin 40-O-b-D- glucopyranoside (10)
(Gao et al. 2016), respectively (Figure 1).
Compound 1, a colorless amorphous solid, showed a molecular formula of
C32H52O3 with 7 degrees of unsaturation as determined by HRESIMS at m/z 485.3997
[M þ H]þ (calcd for C32H53O3, 485.3995). The IR spectrum exhibited the characteristic
1
absorption of carbonyl group at 1739 cmꢁ1. In the H NMR spectrum (Table 1), five
singlet methyl signals (dH 0.81, 0.89 and 1.65, each 3 H, 0.96, 6H), one secondary
methyl signal (dH 0.89 (d, J ¼ 6.4 Hz)), one olefinic proton signal (dH 5.45 (t, J ¼ 6.8 Hz)),
one oxygenated methine proton singnal (dH 3.27), an oxygenated methylene group
signals (dH 4.45, s), and the methyl signal of acetyl group (dH 2.07), as well as the
methylene group signals of characteristic 9,19-cyclo-functional group (dH 0.33, 1 H, d,
J ¼ 4.1 Hz and dH 0.55, 1 H, d, J ¼ 4.1 Hz) were observed. The 13C NMR spectrum
(Table 1) showed 32 carbon signals, which were attributed to 7 methyl, 12 methylene,
six methane (including a sp2 one), and 7 quaternary carbons (including a characteristic
carbonyl) by HSQC spectra. Aforementioned data suggested that compound 1 is a 24-
en-lanostane type triterpenoid with a cyclopropane ring (Nes et al. 1998). Comparison
of the NMR data of 1 with those of Cycloartenal (3) (Flach et al. 2004), which was

NATURAL PRODUCT RESEARCH
3
isolated at the same time, revealed that they had a similar structure. The differences
between them are that C-26 of compound 1 is substituted by an acetoxy group other
than aldehyde group. This deduction could be confirmed by the HMBC experiment. In
HMBC spectrum (Figure 2), the correlations from H-26 and H-20 to C-10 (dC 171.1) sug-
gested the acetyoxy group was placed at C-26. In NOESY spectrum (Figure 2), the
NOESY correlations of H-19 with H-2b, H-18, and H-29, of H-28 with H-5 and H-6a, of
H3-30 with H-15a, H-16a, and H-17, and of H3-18 with H-20 suggested the normal C-
5/C-17/C-20 configuration of lanostane skeleton for compound 1(Wang et al. 2015; Su
et al. 2018). In addition, the NOESY correlations from H-3 to H-29, indicated that the
3-OH was an b-orientation. Consequently, compound 1 was identified as 3b-hydroxy-
cycloart-24-en-26- acetyloxy.
Compound 2, obtained as a yellowish gum, showed a molecular formula of
C24H26O13 with 12 degrees of unsaturation as determined by HRESIMS at m/z
523.1459 [M þ H]þ (calcd for C24H27O13, 523.1452). The IR spectrum exhibited the char-
acteristic absorption of hydroxyl (3468 cmꢁ1) and carbonyl (1650 cmꢁ1) groups. The UV
spectrum of 2 showed the characteristic absorptions of a flavonoid skeleton
(Markham, 1982). The 13C NMR spectrum (Table 1) showed 21 signals for 24 carbons,
corresponding to three methoxyls, one methylene, nine methane groups and 11 qua-
1
ternary carbons. The H NMR spectrum (Table 1) showed an exchangeable proton at
dH 12.65 (5-OH), two coupled one-proton doublets (J ¼ 2.1 Hz) at dH 6.45 (H-6) and
6.90 (H-8), a singlet at dH 7.42 (H-20 and H-60), integrating for two protons.
Aforementioned data suggested 2 was a derivative of myricetin (Fossen et al. 1999).
Furthermore, the signals of a particular the doublet (J ¼ 7.5 Hz) at dH 5.05 of the
anomeric hydrogen and the characteristic chemical shifts (dC 77.4, 76.5, 73.2, 69.7 and
1
60.7) of glucose in H-NMR and 13C NMR suggested 2 possessed one b-configuration
glucose (Mohamed 2001). It further was confirmed GC-MS analysis after acid hydrolysis
(Xin et al. 2009). The connectivity of glucose part was identified by HMBC correlations
1
between the H-100 (dH 5.05) and the C-7 (dC 163.0). In addition, the H NMR also exhib-
ited the signals for three methoxyl groups at dH 3.85 (2 ꢂ 3 H, s) and dH 3.83 (3 H, s).
The methoxyl group at dH 3.85 were placed at C-30 and C-50 based on HMBC correla-
tions of these protons with C-30 and C-50 at dC 147.9. It also further confirmed the
deduction of the symmetric tetra-substitution pattern of ring B of myricetin. The other
methoxyl group at dH 3.83 was placed at C-3 based on HMBC correlations of these
protons with C-3 at dC 138.2. On the basis of the above evidence, the structure of 2
was elucidated to be 3, 30, 50-trimethoxyl-myricetin 7-O-b-D- glucopyranoside.
Compounds 1 and 3 belong to 24-en-lanostane type triterpenoids with a cyclopro-
pane ring. This was the first report on the occurrence of a 24-en-lanostane type triter-
penoid from Bupleurum marginatum. To our best of knowledge, the presence of 24-
en-lanostane type triterpenoid with a cyclopropane ring is unusual in Apiaceae family,
except found in a few species Thapsia laciniata (Drew et al. 2013), Centella asiatica
(Kumar et al. 2013), Bupleurum falcatum (Kim et al. 2011), etc al, which implies that
they might be chemotaxonomic markers for Bupleurum marginatum.
Compound 1 (51.9 lM) and 2 (48.9 lM) inhibit the TNK-induction in 60.61 2.95%
and 24.30 3.18% (n ¼ 3), respectively. This suggested that the compounds 1 and 2
have modest anti-inflammatory effects through suppression of NF jB production.

4
Z. LEI ET AL.
3. Experimental
3.1. General experiment procedures
1H (600 MHz), 13 C (150 MHz) and 2 D NMR spectra were obtained on Bruker AV-600 with
TMS as internal reference, and using methanol-d4 as solvents. Electrospray ionisation
(ESI) mass spectra were acquired in the positive ion mode on a LCQ DECAXP instrument
(Thermo Finnigan, San Jose, CA, USA) equipped with an ion trap mass analyser. HR-ESI-
MS were obtained in the positive ion mode on Waters UPLC Premir Q-TOF (Waters Inc.,
Milford, MA, USA). The IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer
(Thermo Fisher Scientific, Sparks, NV, USA). Optical rotations were acquired on
Shenguang SGW-1 digital polarimeter (Jingke, Shanghai, China). GC-MS were obtained
ꢀ
ꢀ
on Thermo Finnigan Trace DSQ (TR-5MS column: 60 m 0.25 mm 2.5 lm). TLC plates
were HSGF254 SiO2 obtained from Yantai Jiangyou Silica Gel Development Co., Ltd.,
China. Column chromatography (CC) silica gel (SiO2; 200–300 mesh; Qingdao Haiyang
Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (GE-Healthcare Bio-Sciences AB,
Uppsala, Sweden).
3.2. Plant material
The aerial of Bupleurum marginatum were collected from Zigong City, Sichuan Province,
China in April, 2016, and identified by Professor Xiaomei Fu (College of Pharmacy, Jiangxi
University of TCM). A voucher specimen (No. 20160510) was deposited at the Key
Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese
Medicine, China.
3.3. Extraction and isolation
The aerial of Bupleurum marginatum (15 kg) were extracted with 75% (v/v) aqueous
ethanol. The 75% EtOH extract (920 g) was stirred with diatomite and silica gel, then
extracted separately with petroleum ether, EtOAc and n-BuOH and MeOH. The ethyl
acetate extract was concentrated to yield a residue (487 g), which was subjected to sil-
ica gel column chromatography with gradient mixtures CHCl2/MeOH (100:1, 50:1, 25:1,
10:1, 5:1 and 1:1). Six fractions (Frs.Y1-Y6) were obtained. Fr.Y2 (40.8 g) was further
subjected to silica gel column chromatography with gradient mixtures CHCl3/MeOH
(50:1, 25:1, 10:1). 28 fractions (Frs. Y2-1 ꢃ 28) were obtained. Frs. Y2-(8-11) (259 mg)
was further subjected to silica gel column chromatography with mixtures solvent
CHCl3/MeOH (20:1). 24 fractions (Frs. Y2-8-1 ꢃ 24) were obtained. Frs. Y2-8-9 (51 mg)
was further purified by Sephadex LH-20 column chromatography using CHCl3/MeOH
(1:1) solvent, to yield 1 (5.3 mg). Frs. Y2-8-16 (42 mg) was further purified by Sephadex
LH-20 column chromatography using CHCl3/MeOH (1:1) solvent, to yield 3 (6.8 mg).
Frs. Y2-(19-20) (106 mg) was further purified by Sephadex LH-20 column chromatog-
raphy using CHCl3/MeOH (1:1) solvent, to yield 4 (15.4 mg) and 7 (8.3 mg). Fr.Y3
(50.0 g) was further subjected to silica gel column chromatography with gradient mix-
tures CHCl3/MeOH (30:1, 15:1, 10:1, 5:1). 43 fractions (Frs. Y3-1 ꢃ 43) were obtained.
Frs. Y3-(10-13) (169 mg) was further purified by Sephadex LH-20 column

NATURAL PRODUCT RESEARCH
5
chromatography using CHCl3/MeOH (1:1) solvent, to yield 5 (8.0 mg). Frs. Y3-(19-20)
(34 mg) was further purified by Sephadex LH-20 column chromatography using CHCl3/
MeOH (1:1) solvent, to yield 9 (6.8 mg). Fr.Y4 (62.0 g) was further subjected to silica gel
column chromatography with gradient mixtures CHCl3/MeOH (20:1, 15:1, 10:1, 5:1). 52
fractions (Frs. Y4-1 ꢃ 52) were obtained. Frs. Y4-(12-16) (216 mg) was further purified
by Sephadex LH-20 column chromatography using CHCl3/MeOH (1:1) solvent, to yield
2 (3.7 mg) and 6 (9.8 mg). Frs. Y4-(26-29) (83 mg) was further purified by Sephadex LH-
20 column chromatography using MeOH solvent, to yield 8 (11.4 mg) and 10 (7.5 mg).
3.4. 3b-hydroxy-cycloart-24-en- 26-acetyloxy (1)
Colorless amorphous solid; a2D0 þ25.6ꢄ (c 0.01, CHCl3); UV (CHCl3) kmax 208, 236 nm; IR
1
ꢀmax 3669, 2941, 1739, 1445, 1374, 1238, 1023, 741 cmꢁ1; H NMR and 13C NMR data,
see Table 1; HRESIMS m/z 485.3997 [M þ H]þ (calcd for C32H53O3, 485.3995).
3.5. 3,30,50-trimethoxyl-myricetin 7-O-b-D–glucopyranoside (2)
Yellowish gum; UV (MeOH) kmax 254, 356 nm; IR ꢀmax 3468, 2925, 1727, 1650, 1383,
1
1287, 1124, 1075, 668 cmꢁ1; H NMR and 13C NMR data, see Table 1; HRESIMS at m/z
523.1459 [M þ H]þ (calcd for C24H27O13, 523.1452).
3.6. Acid hydrolysis of 2
The absolute configuration of glucose was determined as described by Xin (Xin et al.
2009). Compounds 2 (2 mg) were heated with 1 mol/L HCl (2 mL) at 105 ꢄC for 4 h. The
mixture was cooled, neutralised (1 mol/L NaOH) and partitioned between AcOEt (2 mL)
and H2O (2 mL). The aqueous layer was evaporated and contained in a vial. The follow-
ing solvents were added: (a) (2S)-1- aminopropan-2-ol/MeOH 1:8 (20 ll); (b) AcOH/
MeOH 1:4 (17 ll); (c) 3% Na[BH3CN] in MeOH (13 ll). The vial was capped, and the mix-
ture was allowed to react for 2 h at 65 ꢄC. After cooling, 3 mol/L aq. CF3COOH was
added drop-wise until the pH dropped to pH 1 ꢁ 2. The mixture was evaporated and
co-evaporated with H2O (3 ꢂ 0.5 mL) and MeOH (5 ꢂ 0.5 mL). The residue was dried
overnight in a vacuum desiccator and treated with pyridine/Ac2O 1:1 for 1 h at 100 ꢄC.
After cooling, the mixture was extracted with CHCl3 and the extract washed with H2O
(3 ꢂ 1 mL) and saturated NaHCO3 solvent (3 ꢂ 1 mL). The org. phase was dried
(Na2SO4) and subjected to GC/MS.
3.7. Anti-inflammatory activity in vitro (luciferase assay)
Assay was performed as described by Clavin (Clavin et al. 2007). The J-170 cells were
obtained from Chinese Academy of Sciences, Shanghai, China. J-170 cells were pre-
incubated for 20 min with the compounds 1 and 2 at sublethal doses (doses causing
<20% of cell death as determined in the cytotoxic assay), followed by stimulation
with TNFa (2 ng/mL) for 6 h at 37 ꢄC. Briefly, the cells were lysed in 25 mM Tris-phos-
phate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 7% glycerol. Luciferase

6
Z. LEI ET AL.
activity was measured using a Berthold LB 960 (YPH, Beijing, China), following the
instructions of the ‘luciferase assay kit’ (Biovision, San Francisco, CA, USA). The back-
ground obtained with the lysis buffer was subtracted in each experimental value and
the specific transactivation was expressed as the percentage of inhibition with respect
to the value obtained with control TNFa only treated cells. All the experiments were
repeated three times. The data of anti-inflammatory activity was recorded as the mean
of inhibition percentage standard deviation (SD; n ¼ 3).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the National Natural Science Foundation of China (NSFC)
[grant numbers 81760703 and 81760721] and Natural Science Foundation of Jiangxi Province
[grant numbers 20161BAB205219 and 20181BAB205077].
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