Natural Product Research

Formerly Natural Product Letters

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Chemical constituents of Sophora flavescens Ait.

and cytotoxic activities of two new compounds

Guo-Qing Long, Dong-Dong Wang, Jing Wang, Jing-Ming Jia & An-Hua Wang

To cite this article: Guo-Qing Long, Dong-Dong Wang, Jing Wang, Jing-Ming Jia & An-Hua
Wang (2020): Chemical constituents of Sophora�flavescens Ait. and cytotoxic activities of two new
compounds, Natural Product Research, DOI: 10.1080/14786419.2020.1765340

To link to this article: https://doi.org/10.1080/14786419.2020.1765340

View supplementary material

Published online: 14 May 2020.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20
NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1765340

Chemical constituents of Sophora flavescens Ait. and

cytotoxic activities of two new compounds
Guo-Qing Long, Dong-Dong Wang, Jing Wang, Jing-Ming Jia and
An-Hua Wang
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang,
People’s Republic of China

ABSTRACT ARTICLE HISTORY

A chemical investigation of Sophora flavescens Ait. identified 6 Received 21 January 2020
compounds. On the basis of spectroscopic data, they were deter- Accepted 25 April 2020
mined to be flavonoids and their analogues, among which were
two previously undescribed compounds, sophoflavanone G (1) KEYWORDS
and sophoflavanone H (2). The inhibitory effects of new com- Sophora flavescens;
flavonoid; isopentene
pounds against five human tumour cell lines were evaluated group; cytotoxicity
in vitro by MTT assays, which revealed potential inhibitory effects
with IC50 values < 20 mM, in particular, compound 1 has shown
significant cytotoxicity for several tumour cells with IC50 values
around 20 mM, which was similar to cisplatin and potential to be
used as tumour drugs.

1. Introduction
Sophora flavescens Ait., a perennial herbaceous plant in the Leguminosae family, is
mainly distributed throughout East Asia, mainly in China, Korea and Japan. It has a
long history in traditional Chinese Medicine to utilise for the treatment of diarrhoea,
gastrointestinal haemorrhage, and eczema, etc, and is often used as a diuretic in folk
(Chinese Pharmacopoeia Commission. 2015). Modern pharmacological studies have
shown that the plant also has obvious inhibitory activities for several tumour cells

CONTACT Jing-Ming Jia [email protected] School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang, 110016, People’s Republic of China; An-Hua Wang [email protected]
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People’s
Republic of China.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1765340
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 G.-Q. LONG ET AL.

Figure 1. Structures of compounds 1–6 from the root bark of Sophora flavescens Ait.

(Zhou et al. 2009.). Previous chemical studies of this plant have revealed the presence
of alkaloids (Zhang et al. 2018; Wang et al. 2018; Wang et al. 2014) and flavonoids (Ma
et al. 2019; Huang et al. 2017).
In the present investigation of the chemical composition of S. flavescens, two previ-
ously undescribed compounds (1 and 2) were identified together with 4 known com-
pounds (3
6) (Figure 1). The structures of the isolated compounds were determined
from various spectroscopic data. Finally, the inhibitory effects of the new compounds
against five tumour cells were evaluated in vitro using MTT assays.

2. Result and discussion

Compound 1, yellow amorphous powder, was found to have a molecular formula of
C19H26O5 as showed by its HRESIMS at m/z 333.1684 [M-H]‾ (calcd. for C19H25O5,
333.1702) and m/z 369.1444 [M þ Cl]‾ (calcd. for C19H26O5Cl, 369.1469). Analyses of the
spectroscopic data of compound 1 suggested the structure to be a prenylflavonoids
analogue just like the structure of kushenol F (Wang et al. 2010). The 1H and 13C NMR
spectra showed a phloroglucinol unit (dC 91.1, 94.7, 106.5, 159.6, 161.8 and 162.1) with
a methyl formyl (dC 171.5 and 52.0; dH 3.80, s, 3H) and of a methoxy group (dC 55.5;
dH 3.70, s, 3H), as well as two connected isopentene groups (dC 26.9, 46.3, 30.8, 123.4,
130.5, 25.6, 17.7, 147.8, 110.9 and 18.5). The free phenolic hydroxyl was positioned on
C-2 and C-4 based on the formation of an intramolecular hydrogen bond at dH 11.91
and 10.25. The isopentene group and methoxyl were ascribed to C-3 and C-6, respect-
ively, as determined by the HMBC correlations between the methoxyl (dH 3.70) with
C-6(dC 159.6), and between H-10 (dH 2.52) with C-3 (dC 106.5). Comparison of experi-
mental and calculated ECD data (Supplementary Material, Figure S6) confirmed that
the absolute configuration of 1 was 2’R (The experimental value is the same as 1a in
the Figure S6, namely 2’R). Therefore, the structure of compound 1 was determined to
be as shown and was determined to be sophoflavanone G.
Compound 2 was obtained as a light-yellow amorphous powder, and its molecular
formula was established as C12H16O3 from the HRESIMS at m/z 207.1026 [M-H]- (calcu-
lated for C12H15O3, 207.1021). It showed characteristic signals for a 5,7-disubstituted
chromone analogue at dH 5.77 (1H, d, J ¼ 2.4 Hz, H-6) and 5.92 (1H, d, J ¼ 2.4 Hz, H-8)
 NATURAL PRODUCT RESEARCH 3

in the 1H NMR spectrum. Whereas, a pair of coupled methylene signals at dH 1.67 (2H,
t, J ¼ 6.6 Hz) and 2.43 (2H, t, J ¼ 6.6 Hz) in upfield, as well as a quaternary carbon signal
at dC 73.6 (C-2) in the 13C NMR spectrum, revealed the structure of chromone was
missing a carbonyl group of C-4. There was a strong long-range couple between dH
1.22 (6 H, s) and dC 73.6 (C-2) in the HMBC spectrum, which indicated the two methyl
were connected with C-2, meanwhile, based on the long-range correlation of dH 3.60
(OCH3)/dC 158.6 (C-5) observed in the HMBC data, the methoxy group was connected
on the position of C-5. Thus, the structure of compound 2 was determined to be as
shown and was determined to be sophoflavanone H.
According to the previous reports, the other compounds were identified to be
maackiain (3) (Abdel-Kader 2001), (þ)-4-Hydroxy-3-methoxy-8,9-methylenedioxyptero-
carpan (4) (Swapan et al. 2004), (2S)-liquiritigenin 7-methyl ether (5) (Achenbach et al.
1988; Fu et al. 2016) and hexadecyl ferulate (6) (Kim et al. 2001) on the basis of spec-
troscopic data and past literature.
The inhibitory effect of new compounds against A549, H460, H1299, Hela and MCF-
7 tumour cell lines were investigated using an in vitro bioassay. The results indicated
that the new compound 1 exerted inhibitory effects against five tumour cell lines. In
particular, compound 1 showed cytotoxicity on A549, H460, H1299 and Hela tumour
cells with IC50 around 20 lM, which were equivalent to the positive drug cisplatin.
(Supplementary Material, Table 1).

3. Experimental
3.1. General experimental procedures
Melting points were measured using a micro-melting point apparatus (Keyidianguang
Factory, Beijing, China). A PerkinElmer 341 MC instrument was used to record optical
rotations. UV spectra were measured by an Agilent 1260 infinity II UV–vis spectropho-
tometer in methanol. HRESIMS data were obtained using an Agilent 1290 series 6540
UHD accurate mass Q-TOF mass spectrometer using direct injection. NMR spectra
were run in DMSO-d6 on a Varian Mercury NMR spectrometer. Analytical HPLC data
were collected on Agilent 1260 infinity II instrument (Thermo Scientific dionex).
Preparative HPLC was given by a Saipuruisi MH-LC 52 instrument with an Elite UV2300
detector and a YMC C18 column (250  20 mm, 5 lm). Column chromatographic sepa-
rations were carried out on silica gel H-60 (Qingdao Marine Chemical Group
Corporation, Qingdao, China), ECD spectra were recorded on a Bio-Logic Science MOS-
450 spectrometer. Acetonitrile and Methanol were used of chromatographic grade
purchasing from Fisher in American. All other solvents were used in chemical grade
(Da-Mao Chemical Co. Ltd., Tianjin, China).

3.2. Plant material

The dried root bark of S. flavescens (air-dried at the shade in room temperature) was
collected from Ling-yuan city in Liao-Ning province and identified by Prof. Jia Jing-
Ming, Shenyang Pharmaceutical University. The specimen was deposited in the
4 G.-Q. LONG ET AL.

specimen room of traditional Chinese Medicine in Shenyang Pharmaceutical University

(SPU-2018-1014-06).

3.3. Extraction and isolation

The powdered root bark of S. flavescens (4.5 kg) was extracted with 95% ethanol (aq,
45 L/time) three times and obtained extracts (1.2 kg) by vacuum distillation. The
extracts were suspended again with water (24 L) and partitioned three times with
equal volume ethyl acetate. The ethyl acetate extracts (750 g) were chromatographed
on silica gel with CH2Cl2-MeOH (100:0-0:100) by gradient elution and yielded six frac-
tions (Fraction A to Fraction F).
Afterwards, fifty milligrams from fraction A (11.8 g) was dealt with a thin-layer chro-
matography by elution with PE: EtOAc (20:1) to give compound 6 (30 mg). The fraction
B (20 g) was dealt with a silica gel column by gradient elution with PE: EtOAc
(20:1–0:100) in sequence to give fractions 1–7. Sub-fraction 7 (500 mg) was purified
directly by a preparative HPLC with CH3OH–H2O (50%, 0.03% TFA) to obtained com-
pound 5 (4.7 mg, tR ¼ 37.4 min). The fraction C (152 g) was chromatographed on poly-
amide with EtOH/H2O in sequence to give fractions 1–5 (Fr.0, Fr.30, Fr.50, Fr.70, Fr.95).
Fr.50 (5.0 g) was separated by medium pressure ODS CC (0.03% TFA, 40%–90% CH3OH
aqueous) to obtained 17(Fr.50-1–Fr.50-17). Fr.50-15 (292 mg) was purified with
CH3CN–H2O (40%, 0.03% TFA) to get compound 4 (23.0 mg, tR ¼ 24.4 min). Fr.70
(128 g) and Fr.95 (44 g) were merged and dealt with silica gel (PE-EtOAc, 100:1-0:100)
to give 21(Fr.70-1–Fr.70-21). Fr.70-4(2.0 g) was separated by medium pressure ODS CC
(0.03% TFA, 60%–100% CH3OH aqueous), and purified with CH3OH–H2O (60%, 0.03%
TFA) to get 2 (2.1 mg, tR ¼ 15.2 min), 3 (280 mg, tR ¼ 28.2 min). Fr.70-5(2.0 g) was sepa-
rated by medium pressure ODS CC (0.03% TFA, 50%–100% CH3OH aqueous), and puri-
fied with CH3CN–H2O (62%, 0.03% TFA) to get compound 1 (27.3 mg, tR ¼ 43.6 min).

3.3.1. Sophoflavonoid G (1)

A yellow amorphous powder; UV (CH3OH) kmax 198, 224, 274 and 306 nm. HRESIMS
m/z 333.1684 [M-H]‾ (calcd for C19H25O5, 333.1702). 1H NMR (600 MHz, DMSO-d6), dH
6.04(1H, s, H-5), 2.52 (2H, m, H-10 ), 2.43 (1H, m, H-20 ), 1.99 (2H, t, J ¼ 7.2 Hz, H-30 ), 4.97
(1H, t, J ¼ 6.6 Hz, H-40 ), 1.50 (3H, s, H-60 ), 1.59 (3H, s, H-70 ), 1.64 (3H, s, H-100 ), 3.80 (3H,
s, a-OCH3), 3.70 (3H, s, b-OCH3), 11.91 (1H, s, 2-OH) and 10.25 (1H, s, 4-OH). 13C NMR
(150 MHz, DMSO-d6), dC 94.7 (C-1), 161.8 (C-2), 106.5 (C-3), 162.1 (C-4), 91.1 (C-5), 159.6
(C-6), 26.9 (C-10 ), 46.3 (C-20 ), 30.8(C-30 ), 123.4 (C-40 ), 130.5 (C-50 ), 25.6 (C-60 ), 17.7 (C-70 ),
147.8 (C-80 ), 110.9 (C-90 ), 18.5 (C-100 ), 55.5 (a-OCH3), 52.0 (b-OCH3), 171.5 (C ¼ O). ECD
(0.5 mg/mL, CH3OH): 7.8 (207 nm), 12.7 (220 nm), 6.3 (229 nm) and 2.7 (238 nm).

3.3.2. Sophoflavonoid H (2)

A light-yellow amorphous powder; UV (CH3OH) kmax 206 and 270 nm. HRESIMS m/z
207.1026 [M-H]‾ (calcd for C12H15O3, 207.1021). 1H NMR (600 MHz, DMSO-d6), dH 5.92
(1H, d, J ¼ 2.4 Hz, H-8), 5.77 (1H, d, J ¼ 2.4 Hz, H-6), 2.43 (2H, t, J ¼ 6.6 Hz, H-4), 1.67 (2H,
t, J ¼ 6.6 Hz, H-3), 1.22 (6H, s, 2  CH3), 3.60 (3H, s, OCH3). 13C NMR (150 MHz, DMSO-
 NATURAL PRODUCT RESEARCH 5

d6), dC 73.6 (C-2), 31.9 (C-3), 16.4 (C-4), 158.6 (C-5), 92.7 (C-6), 156.1 (C-7), 93.4 (C-8),
154.9 (C-9), 100.8 (C-10), 54.7 (OCH3), 26.4 (2  CH3).

3.4. Cytotoxicity assay

New compounds 12 were tested for cytotoxicity against A549, H460, H1299, Hela
and MCF-7 cell lines by means of the MTT method as described (Peng et al. 2018).

4. Conclusions
The present paper has obtained six compounds from the herb of S. flavescens, includ-
ing two new compounds. On the basis of the structure analysis, the cytotoxicity activ-
ities of new compounds have been evaluated in vitro using MTT assay. Isopentenyl
flavonoids and their analogues were another group of compounds in S. flavescens
besides alkaloids, which have very important biological activities. Compared with the
positive control cisplatin, compound 1 displayed moderate cytotoxic activities, while
the other compounds were weak in this study, which further indicated the positive
effect of isopentenyl on tumour cells.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Key R&D Program of China (2017YFC1701200) and the
National Natural Science Foundation of China (No. 81374061, 81903789); the Program for
Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative
Research Team in University.

ORCID
Jing-Ming Jia http://orcid.org/0000-0002-0779-4013

References
Achenbach H, Sto €cker M, Constenla M-A. 1988. Flavonoid and other constituents of Bauhinia
manca. Phytochemistry. 27(6):1835–1841.
Chinese Pharmacopoeia Commission. 2015. National commission of Chinese pharmacopoeia.
Beijing: Chinese Medicine and Technology Publishing House.
Fu KL, Li X, Ye J, Lu L, Xu XK, Li HL, Zhang WD, Shen YH. 2016. Chemical constituents of
Narcissus tazetta var. chinensis and their antioxidant activities. Fitoterapia. 113:110–116.
Huang R, Liu Y, Zhao LL, Chen XX, Wang F, Cai W, Chen L. 2017. A new flavonoid from Sophora
flavescens Ait. Nat Prod Res. 31(19):2228–2232.
Kim JS, Han SJ, Byun JH, Xu YN, Yoo SW, Kang SS, Son KH, Chang HW, Kim HP. 2001. Minor con-
stituents from the roots of Sophora flavescens. Nat Prod Res. 7(1):5–8.
6 G.-Q. LONG ET AL.

Abdel-Kader MS. 2001. Phenolic constituents of Ononis vaginalis roots. Planta Med. 67(4):
388–390.
Ma JY, Zhao DR, Yang T, Liu D, Li RT, Li HM. 2019. Prenylflavanones isolated from Sophora flaves-
cens. Phytochem Lett. 29:138–141.
Peng HL, Huang WC, Cheng SC, Liou CJ. 2018. Fisetin inhibits the generation of inflammatory
mediators in interleukin-1b–induced human lung epithelial cells by suppressing the NF-jb
and ERK1/2 pathways. Int Immunopharmacolo. 60:202–210.
Swapan KC, Li H, Fekadu F, Daniel MB, Mansukh CW, Monroe E-W, John CT, Christopher WW, A,
Douglas K. 2004. Isolation and structure identification of an active DNA strand-scission agent,
(þ)-3,4-di-hydroxy-8,9-methylenedioxypterocarpan. J Nat Prod. 58(12):1966–1969.
Wang X, Zheng R, Huang X, Mao Z, Wang N, Li H, Wen C, Shou D. 2018. Effects of alkaloids
from Sophora flavescens on osteoblasts infected with Staphylococcus aureus and osteoclasts.
Phytother Res. 32(7):1354–1363.
Wang L, Wu XD, He J, Li GT, Peng LY, Li Y, Song LD, Zhao QS. 2014. A new quinolizidine alkaloid
from Sophora flavescens. Chem Nat Compd. 50(5):876–879.
Wang XL, Wang NL, Gao H, Zhang G, Qin L, Wong MS, Yao XS. 2010. Phenylpropanoid and fla-
vonoids from osteoprotective fraction of Drynaria fortunei. Nat Prod Res. 24(13):1206–1213.
Zhou HP, Lutterodt H, Cheng ZH, Yu LL. 2009. Anti-inflammatory and antiproliferative activities
of trifolirhizin, a flavonoid from Sophora flavescens roots. J Agric Food Chem. 57(11):
4580–4585.
Zhang SY, Li W, Nie H, Liao M, Qiu B, Yang YL, Chen YF. 2018. Five new alkaloids from the roots
of Sophora flavescens (Article). Chem Biodiversity. 15(3):e1700577.