Flavonoids with hepatoprotective activity from the leaves of Cleome viscosa L.

Article abstract of DOI:10.1080/14786419.2017.1283497

One new flavonol glycoside named visconoside C (1), together with seven known flavonol glycosides, quercetin 3-O-β-d-glucopyranoside 7-O-α-l-rhamnopyranoside (2), quercetin 7-O-α-l-rhamnopyranoside (3), astragalin (4), kaempferol 3-O-(4-O-acetyl)-α-l-rham

Full text of DOI:10.1080/14786419.2017.1283497

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Flavonoids with hepatoprotective activity from the
leaves of Cleome viscosa L.
Tan Phat Nguyen, Cong Luan Tran, Chi Hung Vuong, Thi Hong Tuoi Do, Tien
Dung Le, Dinh Tri Mai & Nhat Minh Phan
To cite this article: Tan Phat Nguyen, Cong Luan Tran, Chi Hung Vuong, Thi Hong
Tuoi Do, Tien Dung Le, Dinh Tri Mai & Nhat Minh Phan (2017): Flavonoids with
hepatoprotective activity from the leaves of Cleome viscosa L., Natural Product Research, DOI:
10.1080/14786419.2017.1283497
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Date: 01 February 2017, At: 06:44

Natural Product research, 2017
http://dx.doi.org/10.1080/14786419.2017.1283497
Flavonoids with hepatoprotective activity from the leaves of
Cleome viscosa L.
Tan Phat Nguyena, Cong Luan Tranb, Chi Hung Vuongc, Thi Hong Tuoi Dod,
Tien Dung Lee, Dinh Tri Maia and Nhat Minh Phana
aInstitute of chemical technology, Vietnam academy of science and technology, ho chi Minh city, Viet Nam;
btay do university, can tho city, Viet Nam; cdalat research center for cultivation and Processing of Medicinal
Plants, Ministry of health, Viet Nam; dFaculty of Pharmacy, department of Pharmacology, university of
Medicine and Pharmacy, ho chi Minh city,Viet Nam; eInstitute of applied Materials science, Vietnam academy
of science and technology, ho chi Minh city, Viet Nam
ABSTRACT
ARTICLE HISTORY
received 8 November 2016
accepted 26 december 2016
One new flavonol glycoside named visconoside C (1), together with
seven known flavonol glycosides, quercetin 3-O-β-
d
-glucopyranoside
-rhamnopyranoside
-rhamnopyranoside
7-O-α-
l
-rhamnopyranoside (2), quercetin 7-O-α-
l
KEYWORDS
(3), astragalin (4), kaempferol 3-O-(4-O-acetyl)-α-
l
Cleome viscosa; capparaceae;
visconoside c; cytotoxicity
activity; hepatoprotective
activity
(5), kaempferol 7-O-α-l-rhamnopyranoside (6), kaempferitrin (7) and
kaempferol 3-O-β-d-glucopyranoside 7-O-α-l-rhamnopyranoside (8)
were isolated by various chromatography methods from the leaves of
Cleome viscosa L. Their structures were elucidated by IR, UV, HR-ESI-MS
and NMR (1D & 2D) experiments.The cytotoxicity and hepatoprotective
activities using HepG2 human hepatoma cell line of 1 were measured
by MTT assay. At the concentration of 25 μM and 50 μM, 1 showed
cytotoxic activity against HepG2 cells (cell viability was decreased to
22.2 and 23.0%, respectively, compared with doxorubicin control),
while at the concentration of 100 μM, 1 showed hepatoprotective
activity against CCl4-induced hepatotoxicity on HepG2 cells (34.3%,
compared with quercetin control).
CONTACT Nhat Minh Phan
pnhatminh@gmail.com
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2017.1283497.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
T. P. NGUYEN ET AL.
Figure 1. structure of compounds 1–8.
1. Introduction
Cleome viscosa L. is used as drugs in the traditional medicine for the treatment of diarrhea,
fever, inflammation, liver diseases, bronchitis, skin diseases and malarial fever (Mali 2010).
This plant has been scientifically screened for various pharmacological activities such as
antimicrobial, anti-inflammatory, hepatoprotective (Gupta & Dixit 2009; Mali 2010), antifi-
brotic (Kumar et al. 2009), anticonvulsant (Mishra et al. 2010), antioxidant (Gupta et al. 2011),
antinociceptive, antibacterial (Bose et al. 2011), antitumor (Gopal et al. 2012) and antibacterial
activities (Donkor et al. 2014). Phytochemical studies on C. viscosa led to the isolation of
flavanone (Srivastava et al. 1979), flavonol (Chauhan et al. 1979; Senthamilselvi et al. 2012),
sterol (Srivastava 1980), coumarinolignoid (Ray et al. 1985; Kumar et al. 1988), diterpene
(Jente et al. 1990), lactam derivative (Jana & Biswas 2011), dipyridodiazepinone derivative
(Chatterjee et al. 2013) and triterpenoid (Farimani et al. 2016). In our previous study, two
new flavonol glycosides from the leaves of this species were reported (Phan et al. 2016). This
paper described the isolation and structure elucidation of flavonoids (1–8) and the evaluation
of cytotoxicity and hepatoprotective activity of new compound from C. viscosa L. collected
in Ben Cat, Binh Duong province, Viet Nam.
2. Results and discussion
The MeOH extract from the dried leaves of C. viscosa L. was subjected to column chroma-
tography over silica gel normal-phase and reversed-phase RP-18 to give two new flavonol
glycosides named visconoside C (quercetin 3-O-[β-
d
-glucopyranosyl-(1 → 3)]-α- -rhamnopy
l

NATURAL PRODUCT RESEARCH
3
ranoside 7-O-α-l-rhamnopyranoside) (1) and seven known flavonol glycosides (Figure 1),
quercetin 3-O-β-d-glucopyranoside 7-O-α-l-rhamnopyranoside (2) (Kerhoas et al. 2006),
quercetin 7-O-α-l-rhamnopyranoside (3) (Ailian et al. 2006), astragalin (4) (Wei et al. 2011),
kaempferol 3-O-(4-O-acetyl)-α-l-rhamnopyranoside (5) (Masuda et al. 1991), kaempferol
7-O-α-l-rhamnopyranoside (6) (Mobiya et al. 2010), kaempferitrin (7) (Olszewska & Wolbis
2002) and kaempferol 3-O-β-d-glucopyranoside 7-O-α-l-rhamnopyranoside (8) (Song et al.
2007).
1 was obtained as yellow amorphous powder. The molecular formula was established as
C33H40O20 by HR-ESI-MS data ([M + Na]+ m/z 779.1986). The aglycone of 1 was identified as
quercetin, according to observation of 15 carbons in 13C NMR and DEPT spectrum, including
1 carbonyl carbon at δC 177.9 (C-4), 7 oxygenated aromatic carbons, 2 quaternary aromatic
carbons and 5 non-oxygenated aromatic carbons together with 2 AX-type aromatic protons
at δH 6.44 (d, 2.0, H-6) and 6.76 (d, 2.0, H-8); 3 ABX-type aromatic protons at δH 6.92 (d, 8.5,
H-5′), 7.31 (dd, 2.0 and 8.0, H-6′) and 7.38 (br s, H-2′) in 1H NMR data. Moreover, three anomeric
carbons at δH 5.32 (br s, H-1′′), 4.36 (d, 7.5, H-1′′′), 5.56 (br s, 1.0, H-1′′′′) corresponded to three
anomeric carbons at δC 101.6 (C-1′′), 104.8 (C-1′′′), 98.4 (C-1′′′′) were assigned to α-l-rhamnose
(Rha I), β-d-glucose (Glc) and α-l-rhamnose (Rha II) units, respectively. The COSY and HSQC
spectrum allowed analysis of their spin systems and assignment of their proton resonances
to determine every sugar unit clearly. Beside, the sugar moiety was identified as l-rhamnose
and d-glucose by the acidic hydrolysis and using TLC to compare the hydrolysate with the
authentic sugars (see Experimental). The HMBC spectrum (Figure 1), showed correlations
between anomeric proton at δH 5.32 (H-1′′) of Rha I and carbon at δC 134.2 (C-3) of aglycone;
between anomeric proton at δH 4.36 (H-1′′′) of Glc and carbon at δC 81.2 (C-3′′) of Rha I; between
anomeric proton at δH 5.56 (br s, 1.0, H-1′′′′) of Rha II and carbon at δC 161.7 (C-7). Based on
data of HR-ESI-MS, 1D, 2D NMR and compared with previous published data (Phan et al. 2015
& 2016), the structure of 1 was determined as quercetin 3-O-[β-d-glucopyranosyl-(1→3)]-α-l-
rhamnopyranoside 7-O-α-l-rhamnopyranoside, and named visconoside C (Figure 1).
The cytotoxicity and hepatoprotective activities were evaluated for 1 using HepG2 cell
(Table S1). At the concentration of 25 μM and 50 μM, 1 showed cytotoxic activity against
HepG2 cells (cell viability was decreased to 22.2 and 23.0%, respectively, compared with
doxorubicin control), while at the concentration of 100 μM, 1 showed hepatoprotective
activity against CCl4-induced hepatotoxicity on HepG2 cells (34.3%, compared with quercetin
control).
One new flavonol glycoside (1) together with seven known flavonol glycosides (2–8) were
isolated from the methanol extract of the dried leaves of C. viscosa L. For the first time, com-
pounds 4 and 5 were reported from the genus Cleome. Their structures were elucidated by
intensive spectroscopic and spectrometric analyses.
3. Experimental
3.1. General experimental procedures
The high-resolution electrospray ionisation mass spectroscopy (HR-ESI-MS) was recorded
on a Bruker MicrOTOF-QII spectrometer. The 1H NMR (500 MHz), 13C NMR (125 MHz), DEPT,
COSY, HSQC and HMBC spectra were recorded on a Bruker AM500 FT-NMR spectrometer
using tetramethylsilane (TMS) as internal standard. Column chromatography was carried

4
T. P. NGUYEN ET AL.
out using Merck Silica gel normal-phase (230–240 mesh) and reversed-phase C18 (Merck).
Analytical TLC was carried out in silica gel plates (Merck DC-Alufolien 60 F254). Compounds
were visualised by spraying aqueous 10% H2SO4 and heating for 3–5 min.
3.2. Chemicals
Eagle’s Minimum Essential Medium (EMEM), fetal calf serum (FCS), trypsin-EDTA were
purchased from Gibco, USA; L-glutamine, penicillin-streptomycin, phosphate buffer,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin, quercetin,
carbon tetrachloride (CCl4) from Sigma–Aldrich, USA; dimethylsulfoxide (DMSO), isopropanol
from Merck, Germany. All chemicals met cell culture standards.
3.3. Plant material
The leaves of C. viscosa were collected from Ben Cat, Binh Duong province, Viet Nam, in May
2015; and identified by Prof. Vo Van Chi. A voucher specimen (No. VH/MINH-0515) was depos-
ited in the Institute of Chemical Technology, Vietnam Academy of Science and Technology.
3.4. Extraction and isolation
Powdered leaves of C. viscosa (8 kg) were extracted with 95% EtOH for three times (3 × 30 L,
total amount 90 L) at room temperature, residue was filtered , solvents were removed under
low pressure and the crude extract (980 g) was obtained. Then, crude extract was applied
to solid-phase extraction procedures and successively partitioned into n-hexane (70 g), CHCl3
(150 g), EtOAc (260 g) and MeOH (450 g). The MeOH extract was subjected to silica gel col-
umn chromatography and eluted with gradient solvent system of chloroform–methanol
(95:5–5:95) to afford seven fractions: M1 (25 g), M2 (30 g), M3 (86 g), M4 (75 g), M5 (60 g),
and M6 (72 g).
Fraction M2 (5 g) was chromatographed on silica gel and eluted with CHCl3–MeOH (10:1)
to obtain compounds 3 (72 mg) and 7 (68 mg). Fraction M3 (5 g) was separated by silica gel
chromatographic column using CHCl3-MeOH (5:1), and further separated by RP-18 using
gradient mixtures of MeOH–H2O (5:1, v/v) to affrord compound 8 (268 mg). The same manner
was applied to fraction M4 (75 g), eluted CHCl3-MeOH (6:1 → 3:1) to give four subfractions
(M4.1–M4.4). Subfraction M4.3 (25 g) was further purifed by RP-18 with MeOH-H2O (4:1, v/v)
to give compounds 5 (16 mg) and 6 (72 mg). Subfraction M4.2 (20 g) was done in the same
manner, further separated by RP-18 with MeOH-H2O (3:1, v/v) to affrord 1 (72 mg) and 3
(7 g). Fraction M5 (5 g) was applied on a silica gel chromatographic column and eluted with
CHCl3-MeOH (2:1) in silica gel column chromatography to yield compound 2 (40 mg).
Visconoside C (1): pale yellow needles (MeOH); [ꢀ]2D5 − 0.63 (c 0.01, MeOH); HR-ESI-MS
m/z 779.1986 [M + Na]+ (Calcd for C33H40O20Na, 779.2011). 1H NMR (DMSO-d6, 500 MHz, J in
Hz): 6.44 (1H, d, J = 2.0 Hz, H-6), 6.76 (1H, d, J = 2.0 Hz, H-8), 7.38 (1H, br s, H-2′), 6.92 (1H, d,
J = 8.5 Hz, H-5′), 7.31 (1H, dd, J = 2.0 and 8.0 Hz, H-6′), 5.32 (1H, br s, H-1′′), 4.20 (1H, br s, H-2′′),
3.63–3.68 (1H, m, H-3′′), 3.28–3.51 (1H, m, H-4′′), 3.06–3.09 (1H, m, H-5′′), 0.86 (3H, d, J = 5.0 Hz,
H-6′′), 4.36 (1H, d, J = 7.5 Hz, H-1′′′), 3.06–3.09 (1H, m, H-2′′′), 3.20–3.22 (1H, m, H-3′′′), 3.28–3.51
(1H, m, H-4′′′), 3.20–3.22 (1H, m, H-5′′′), 3.63–3.68 (1H, m, H-6′′′a), 3.50–3.53 (1H, m, H-6′′′b),
5.56 (1H, br s, H-1′′′′), 3.85 (1H, br s, H-2′′′′), 3.63–3.68 (1H, m, H-3′′′′), 3.28–3.51 (1H, m, H-4′′′′),

NATURAL PRODUCT RESEARCH
5
3.50–3.53 (1H, m, H-5′′′′), 1.12 (3H, d, J = 6.0 Hz, H-6′′′′). 13C NMR (DMSO-d6, 125 MHz): 157.8
(C-2), 134.2 (C-3), 177.9 (C-4), 160.9 (C-5), 99.5 (C-6), 161.7 (C-7), 94.5 (C-8), 156.1 (C-9), 105.7
(C-10), 120,4 (C-1′), 115.8 (C-2′), 145.3 (C-3′), 148.8 (C-4′), 115.7 (C-5′), 121.3 (C-6′), 101.6 (C-1′′),
69.2 (C-2′′), 81.2 (C-3′′), 71.6 (C-4′′), 70.1 (C-5′′), 17.5 (C-6′′), 104.8 (C-1′′′), 73.9 (C-2′′′), 76.5
(C-3′′′), 69.9 (C-4′′′), 76.3 (C-5′′′), 60.8 (C-6′′′), 98.4 (C-1′′′′), 69.8 (C-2′′′′), 70.4 (C-3′′′′), 71.8 (C-4′′′′),
70.3 (C-5′′′′), 17.9 (C-6′′′′).
3.5. Acid hydrolysis
Each new compounds (2 mg) was refluxed with 2N aq. CH3COOH (5 ml) for 2 h at 100°C. After
extraction with CHCl3 (3 × 5 ml), the aqueous layer was repeatedly evaporated to dryness
with MeOH until neutral, and then analysed byTLC over silica gel (MeCOEt–isoPrOH–Me2CO–
H2O 20:10:7:6) by comparison with authentic samples (L-rhamnose Rf 0.65; -glucose Rf 0.40)
d
(Nguyen et al. 2015; 2016).
3.6. The cytotoxicity and hepatoprotective assay
HepG2 cells were seeded and cultured in EMEM containing 10% FCS, 2 mM L-glutamine,
100 IU/ml penicillin, 100 μg/ml streptomycin at 5% CO2 at 37°C to attain confluency, then
harvested and seeded in 96-well plates at 2.5 × 104 cells/cm2. Then, cells were treated with
EMEM containing 2 mM CCl4 and compound 1 alone or combined at different concentrations.
Cell viability was evaluated as mitochodrial succinate dehydrogenase activity, a marker of
viable cells using MTT test [Yao et al. 2011; Pareek et al. 2013]. Doxorubicin was used as a
positive control for cytotoxicity and quercetin for hepatoprotective effect.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research work has been financially supported by the Vietnam Academy of Science andTechnology,
Project No. VAST.DLT.09/16-17.
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