Bioactive compounds from Physalis angulata and their anti-inflammatory and cytotoxic activities

Article abstract of DOI:10.1080/10286020.2020.1825390

A new compound, physalucoside A (1), together with seven withanolides (2–8) and three flavonoids (9–11), were isolated from Physalis angulata L. (Solanaceae), a medicinal plant native to Vietnam. The chemical structures of these compounds were elucidated by one- and two-dimensional NMR spectra, high-resolution electrospray ionization mass spectrometry analyses, and chemical reactivity. The anti-inflammatory and cytotoxic activities of isolated compounds were also evaluated. These data suggest that the anti-inflammatory activity of P. angulata is due primarily to its withanolide content. This study demonstrates the potential of withanolides as promising candidates for the development of new anti-inflammatory drugs.

Full text of DOI:10.1080/10286020.2020.1825390

Journal of Asian Natural Products Research
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Bioactive compounds from Physalis angulata and
their anti-inflammatory and cytotoxic activities
Hoang Le Tuan Anh , Vinh Le Ba , Thi Thao Do , Van Kiem Phan , Hai Yen
Pham Thi , Long Giang Bach , Manh Hung Tran , Phuong Anh Tran Thi &
Young Ho Kim
To cite this article: Hoang Le Tuan Anh , Vinh Le Ba , Thi Thao Do , Van Kiem Phan , Hai Yen
Pham Thi , Long Giang Bach , Manh Hung Tran , Phuong Anh Tran Thi & Young Ho Kim (2020):
Bioactive compounds from Physalisꢀangulata and their anti-inflammatory and cytotoxic activities,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2020.1825390
To link to this article: https://doi.org/10.1080/10286020.2020.1825390
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1825390
Bioactive compounds from Physalis angulata and their
anti-inflammatory and cytotoxic activities
c,d
Hoang Le Tuan Anha,b , Vinh Le Ba , Thi Thao Dob,e, Van Kiem Phanc,
Hai Yen Pham Thib, Long Giang Bachf, Manh Hung Trang,
Phuong Anh Tran Thib and Young Ho Kimd
ꢀ
ꢀ
aMientrung Institute for Scientific Research, Vietnam Academy of Science and Technology, 321
b
Huynh Thuc Khang, Hue City, Thua Thien Hue 531600, Vietnam; Graduated University of Science
c
and Technology, Vietnam Academy of Science and Technology, Ha Noi 10307, Vietnam; Institute of
Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Ha Noi
d
10307, Vietnam; College of Pharmacy, Chungnam National University, Daejeon 34134, Korea;
eInstitute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Ha
f
Noi 10307, Vietnam; Nguyen Tat Thanh University, 300A Nguyen Tat Thanh, Ho Chi Minh City
g
748000, Vietnam; Medicinal Chemistry Division, Faculty of Chemistry, University of Science, Vietnam
National University Hochiminh city, 227 Nguyen Van Cu, Ho Chi Minh City 748000, Vietnam
ABSTRACT
ARTICLE HISTORY
Received 26 June 2019
Accepted 16 September 2020
A new compound, physalucoside A (1), together with seven with-
anolides (2–8) and three flavonoids (9–11), were isolated from
Physalis angulata L. (Solanaceae), a medicinal plant native to
Vietnam. The chemical structures of these compounds were eluci-
dated by one- and two-dimensional NMR spectra, high-resolution
electrospray ionization mass spectrometry analyses, and chemical
reactivity. The anti-inflammatory and cytotoxic activities of iso-
lated compounds were also evaluated. These data suggest that
the anti-inflammatory activity of P. angulata is due primarily to its
withanolide content. This study demonstrates the potential of
withanolides as promising candidates for the development of
new anti-inflammatory drugs.
KEYWORDS
Physalis angulate;
solanaceae; physalucoside
A; withanolides;
anti-inflammatory;
cytotoxic activity
CONTACT Young Ho Kim
Korea; Hoang Le Tuan Anh
yhk@cnu.ac.kr
College of Pharmacy, Chungnam National University, Daejeon 34134,
Mientrung Institute for Scientific Research,
hoangletuananh@hotmail.com
Vietnam Academy of Science and Technology, 321 Huynh Thuc Khang, Hue City, Thua Thien Hue 531600, Vietnam
ꢀ
These authors contributed equally to this work.
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1825390.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
H. L. TUAN ANH ET AL.
1. Introduction
Inflammatory diseases and disorders include autoimmune diseases, cancers, metabolic
syndromes, and cardiovascular diseases. Diverse pro-inflammatory cytokines and
mediators, such as interleukin 1b, tumor necrosis factor a, interleukin 6, prostaglan-
dins, and nitric oxide (NO), are released mainly by activated macrophages [1] in
response to exogenous stress or endogenous signals including external pathogens and
damage-associated molecular patterns [2]. The inhibition of cyclooxygenase-2 (COX-
2) and inducible nitric oxide synthase (iNOS) plays an important role in many
inflammatory disease therapies. Hence, selective iNOS and COX-2 inhibitors are
potential candidates for new therapeutic agents against inflammation.
The plant Physalis angulata L. belongs to the genus Physalis (Solanaceae), which
includes approximately 120 species distributed primarily within South and North
America, with a few species found in Europe and southeastern and central Asia [3].
In Vietnam, four species of Physalis are identified with the local name Tam Bop [4].
It is used widely in Vietnamese folk medicines for treating fever, cough, vomiting,
hiccups, and diabetes. Physalis fruit is eaten in China, Korea, Japan, and Vietnam for
its high levels of vitamin C, vitamin B1, and pro-vitamin A [5]. The fruits are also
used to prevent urinary tract infections, kidney inflammation, and gout. Interestingly,
the branches, fruits, or leaves of P. angulata are used as one of the ingredients in a
traditional anti-cancer (cervical, throat, lung, and colon) medicine that also includes
Atractylodes macrocephala, Platycodon grandiflorus, Ophiopogon japonicas,
Scruphularia buergeriana, Scutellaria baicalensis, and Glycyrrhiza uralensis [4]. As
with other Physalis sp., P. angulata produces withanolide triterpenoids containing a
steroidal lactone possessing 28 carbon atoms [6,7]. These compounds have exhibited
antimicrobial, anticancer, anti-inflammatory, hepatoprotective, immunomodulatory,
and insect-repelling properties [8–11].
In our anti-inflammatory screening program evaluating Vietnamese medicinal and
natural plants, an ethanol extract of P. angulata exhibited significant inhibition of NO
production. This report describes the isolation and structural elucidation of a new
alkaloid, physalucoside A (1), from the branches of P. angulata, along with 10 known
compounds comprising withanolides (2–8) and flavonoids (9–11). The anti-inflamma-
tory and cytotoxic activities of these compounds were also investigated. Moreover, their
effects on protein expression were determined by Western blot analysis.
2. Results and discussion
Bioassay-guided separation of the MeOH extract of P. angulata resulted in the
isolation and structural elucidation of eleven compounds (1–11) (Scheme S1, and
Extraction and isolation), including new compound 1. The known compounds were
identified as physagulin B (2), (20S,22R)-15a-acetoxy-5a-chloro-6b,14b-dihydroxy-
1-oxowitha-2,24-dienonide (3), physalin F (4), physalin B (5), physalin G (6), and
physagulide Q (7), physagulin D (8) [6,12–15], and three flavonoid glucosides as
quercetin 3-O-b-rutinoside (9), kaempferol 3-O-b-rutinoside (10), and kaempferol
3-O-b-rutinoside-7-O-b-D-glucopyranoside (11) [16] by comparison of their NMR
spectroscopic data to those previously reported values.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1H and 13C NMR spectral data of new compound 1.
a,b
a,c
No.
dC
dH (mult., J in Hz)
1
2
3
4
5
6
7
8
128.0
111.7
149.3
149.9
116.5
123.3
142.4
118.6
169.3
132.3
129.2
115.9
158.2
115.9
129.2
81.8
–
7.03 (d, J ¼ 1.5 Hz)
–
–
6.69 (d, J ¼ 8.5 Hz)
6.93 (dd, J ¼ 1.5, 8.5 Hz)
7.34 (d, J ¼ 15.5 Hz)
6.32 (d, J ¼ 15.5 Hz)
–
9
10
20
30
40
50
60
70
80
–
7.15 (d, J ¼ 8.5 Hz)
6.65 (d, J ¼ 8.5 Hz)
–
6.65 (d, J ¼ 8.5 Hz)
7.15 (d, J ¼ 8.5 Hz)
ꢀ
4.75-4.77 (m)
47.7
3.46 (dd, J ¼ 7.0, 13.5 Hz)
3.63 (ddd, J ¼ 5.5, 13.5 Hz)
30-0OMe
1
56.4
104.5
75.5
78.1
71.5
77.9
62.6
3.79 (s)
4.40 (d, J ¼ 7.5 Hz)
3.20-3.22 (m)
3.25 (d, J ¼ 9.0 Hz)
3.18 (dd, J ¼ 5.0, 9.0 Hz)
3.09 (dd, J ¼ 2.5, 5.5 Hz)
3.46-3.48 (m)/3.60-3.62 (m)
200
300
400
500
600
aMeasured in CD3OD.
b125 MHz.
c500 MHz.
ꢀ
This assignment may be interchanged.
Compound 1 was obtained as a white amorphous powder and showed a positive
reaction with Dragendorff’s reagent. The molecular formula of 1 was C24H29NO10,
based on positive-mode high-resolution electrospray ionization mass spectrometry,
which yielded a protonated molecular ion at m/z 492.1838 [M þ H]þ. Infrared
absorption spectrum indicated the presence of hydroxyl (3380 cmꢁ1), carboxylic acid
1
(1635 cmꢁ1), and aromatic ring (1,60,51,520 cmꢁ1) moieties. The H-NMR spectrum
of 1 indicated the presence of an ABX proton system with peaks at d 7.03 (1H, d,
J ¼ 1.5 Hz, H-2), 6.69 (1H, d, J ¼ 8.5 Hz, H-5), and 6.93 (1H, dd, J ¼ 1.5, 8.5 Hz, H-6).
An AABB system was also evident, with peaks at d 7.15 (2H, d, J ¼ 8.5 Hz, H-20, H-
60) and 6.65 (1H, d, J ¼ 8.5 Hz, H-30, H-50) indicating the presence of a 1,3,4-trisubsti-
tuted aromatic system and a p-substituted aromatic ring, respectively. Two doublets
proton signals at d 7.34 (1H, d, J ¼ 15.5 Hz, H-7) and 6.32 (1H, d, J ¼ 15.5 Hz, H-8)
indicated the presence of a trans-substituted double bond. Additional signals indi-
cated the presence of an oxygenated methine proton at d 4.75-4.77 (1H, m, H-70),
methylene protons at d 3.46 (1H, dd, J ¼ 7.0, 13.5 Hz, H-80a) and 3.63 (1H, dd,
J ¼ 5.5, 13.5 Hz, H-80b), a methoxyl at d 3.79 (3H, s), in addition to a glucosyl anome-
ric proton at 4.40 (1H, d, J ¼ 7.5 Hz, H-100). 13C-NMR and DEPT spectra of 1
revealed 24 carbon signals corresponding to 12 aromatic carbon atoms (for two aro-
matic rings), one oxymethine group at d 81.8 (C-70), one methylene group at 47.7
(C-80), one double bond at d 142.4 (C-7) and 118.6 (C-8), one carbonyl at d 169.3
(C-9), one methoxyl group at d 56.4 (3-OCH3), and six carbon atoms belonging to a
glucosyl moiety at d 62.6 to d 78.1 (Table 1). These spectroscopic data suggest that 1

4
H. L. TUAN ANH ET AL.
Figure 1. Selected HMBC and COSY correlations of 1.
contains a feruloyl group and a 2-amino ethanoyl side chain, indicating that the main
skeleton is cinnamic acid amide with a glycoside moiety. Acid hydrolysis of 1 in 1 N
methanolic hydrochloric acid (HCl), together with the observed coupling constant of
the anomeric proton (J ¼ 7.6 Hz), confirmed the presence of D-(þ)-glucose. Further
confirmation was provided by direct comparisons with a reference sample in co-spot-
ted (co)-TLC and GC analyses (Supplementary data). COSY, HSQC, and HMBC
spectra confirmed the planar structure of 1 (Figure 1). The COSY spectrum revealed
couplings between the oxymethine proton H-70 and the two nitrogenated methylene
protons H-80, and between the two olefinic protons H-7 and H-8. The HMBC spec-
trum of 1 showed long-range correlations between H-7 and the carbonyl carbon C-9
and methine carbons C-2 and C-6, indicating the presence of a feruloyl group.
Interestingly, long-range correlations between H-80 protons and the carbonyl carbon
C-9 and oxygenated methine carbon C-70 confirmed the presence of a 2-hydroxy-2-
(4-hydroxyphenyl) ethyl amine group (octopamine) linked to C-9 of the ferulic unit.
A single methoxyl group at C-3 was evidenced by an HMBC correlation between dH
3.79 and dC 150.9 (C-3). HMBC correlations between dH 4.40 (H-100) and dC 81.8
indicated that the glucopyranosyl unit was linked to the oxygen atom at C-70. In add-
ition, the optical rotation value of aglycon of 1 was determined as negative (aD ꢁ3.0),
indicating the absolute configuration of S at the chiral center C-70 [17]. Based on all
of the above evidence, the structure of 1 was characterized as physalucoside A.
NO is a key mediator of inflammatory responses and pathogenesis. The inhibition
of NO by each of the isolated compounds (1-11) was evaluated in RAW246.7 cells by
measuring NO production after LPS stimulation (1 lg/ml). The data in Table 2 show
that physagulin B (2), (20S,22R)-15a-acetoxy-5a-chloro-6b,14b-dihydroxy-1-oxowi-
tha-2,24-dienonide (3), physalin B (5), and physagunin R (7) strongly inhibited NO
production, with IC50 values less than 1.0 lM, followed by physalin F (4, IC50
1.06 0.68 lM) and physalin G (6, IC50 3.74 0.29 lM). The new compound, physa-
lucoside A, also exhibited significant inhibitory activity, with an IC50 value of
2.69 0.17 lM, and the isolated flavonoid glucosides showed weak inhibition, with
IC50 values greater than 30 lM. The positive control, cardamonin, showed anti-NO
production activity, with an IC50 value of 1.17 0.10 lM. These results indicate that
withanolides play an active role in the anti-inflammatory activity of P. angulata.
Interestingly, all of the withanolide compounds (1–7) were good inhibitors of NO
production. iNOS and COX-2 are two main targets typically evaluated in anti-inflam-
mation studies [18]. Physalin F (4) and physalin B (5) were selected for use in our
subsequent experiments. RAW 264.7 cells were pretreated with the indicated concen-
trations of physalin F (4) and physalin G (5) for 2 h and then incubated with LPS

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 2. Inhibition of NO production in macrophage RAW264.7 cells by 1–11.
Compounds
IC50 (mM)a
1
2
3
4
5
6
7
8
2.69 0.17
0.24 0.09
0.68 0.02
1.06 0.68
0.28 0.10
3.74 0.29
0.57 0.18
12.5 1.70
> 30
9
10
> 30
> 30
1.17 0.10
11
Cardamoninb
aThe inhibitory effects are represented as giving 50% inhibition (IC50) relative to the vehicle control. The values are
expressed as the means S.D. of three individual experiments.
bPositive control for NO production.
Figure 2. Effect of physalin F (A) and physalin B (B) on LPS-induced iNOS and COX-2 expressions
in RAW 264.7 macrophages. Expressions of iNOS and COX-2 proteins were determined by Western
blot analysis and tubulin was chosen as positive control.
(1 lg/ml) for 24 h. Equal amounts of total protein were subjected to SDS-PAGE, and
the levels of iNOS and COX-2 proteins were determined by Western blot analyses.
The data in Figure 2 show that iNOS and COX-2 protein and mRNA expression
were almost undetectable in unstimulated (no LPS activation) RAW 264.7 cells.
However, after LPS treatment, the protein levels of iNOS and COX-2 increased mark-
edly. Pretreating the cells with physalin F (4, 0.1–3.0 lM) and physalin G (5,
0.01–0.3 lM) attenuated LPS-induced iNOS and COX-2 protein production in a con-
centration-dependent manner. These data suggest that both of these compounds,
along with the other withanolides, may downregulate LPS-induced iNOS and COX-2
expression at the transcription level. These results are consistent with those of previ-
ous studies [19,20] and provide evidence for the anti-inflammatory properties of P.
angulata. Furthermore, experiments to confirm the anti-inflammation mechanism of
these compounds are now in process.
The potentially cytotoxic activities of compounds 1–11 were investigated using
three human cancer cell lines: A-549 (human lung carcinoma cells), HeLa (human
cervical cancer cells), and Panc-1 (human pancreatic cancer cells). Compounds 3, 4,
5, and 6 exhibited particularly strong cytotoxic activities against A-549 cancer cell
lines, with IC50 values of 1.03 0.09, 0.68 0.05, 0.95 0.04, and 6.88 2.41 mM,
respectively. These results are comparable with that for the positive control, etoposide
(IC50 2.68 0.89 mM) (Table 3). Therefore, the withanolide constituents (3–6) of P.
angulata are potentially candidate anticancer therapeutic agents.

6
H. L. TUAN ANH ET AL.
Table 3. Cytotoxic effect of selected compounds.
Compounds
A-549
IC50 (mM)
Hela
Panc-1
3
4
5
6
1.03 0.09
0.68 0.05
0.95 0.04
6.88 2.41
2.68 0.89
29.89 1.15
0.23 0.03
13.84 1.27
>30
11.53 0.36
>30
12.77 1.07
>30
Etoposidea
3.29 0.05
0.08 0.11
aPositive control. The values are presented as the means S.D (n ¼ 3).
Natural products are a consistent source of active pharmacological compounds.
Previously, withanolides isolated from P. angulata exhibited remarkable anti-inflam-
matory, antinociceptive, and anti-cancer activities [21,22]. In this research, 11 com-
pounds (1–11), including the new compound physalucoside A, were isolated from a
methanol extract of P. angulata via combined column chromatography. Compounds
3, 4, 5, and 6 exhibited significant anti-inflammatory and cytotoxic effects. Among
these, compounds 5 and 6 exhibited iNOS and COX-2 expression in Western blot
analyses. Our results indicate that the withanolides in P. angulata may potentially be
used to treat inflammation, cancer, and other related diseases.
3. Experimental
3.1. General experimental procedures
The optical rotation values were recorded with a JASCO P-1020 polarimeter (JASCO,
Oklahoma, OK, US). FT-IR spectra were performed on a Bruker TENSOR 37 FT-IR
spectrometer (Bruker, Billerica, MS, USA). 1 D and 2 D NMR spectra were carried
out on a Bruker Avance III 500 spectrometers (MA, USA) with TMS as the internal
standard. HR-ESI-MS results were afforded by Agilent 6530 Accurate-Mass Q-TOF
LC/MS system (Agilent, Santa Clara, CA, USA). Silica gel (Kieselgel 60, 70-230, and
ꢀ
230-400 mesh, Merck, Darmstadt, Germany), YMC GEL (ODS-A, 12 nm S-150 mm,
YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (Sigma-Aldrich, MO, USA), and
Diaion HP-20 (Supelco, PA, USA) were chosen for the column chromatography by
similarly described [23]. All solvents were supplied by SK chemical Company, Korea.
Sigma-Aldrich provided the chemical reagents, and tubulin antibodies while iNOS,
and COX-2 were afforded from Cell Signaling Technology (Beverly, MA, USA).
3.2. Plant material
The plant material of Physalis angulata was obtained from Thai Binh province,
Vietnam, in June 2018. Dr. Tran Thi Phuong Anh and Prof. Young Ho Kim taxo-
nomically identified the plant species. A voucher specimen (QTB 16 2018) was stored
at the herbarium of the Natural Product Laboratory, Chungnam National University.
3.3. Extraction and isolation
The dried and powdered form of whole plants of P. angulata (2.0 kg) were extracted
by methanol at 50 ꢂC using ultrasonic for three times (4.0 L each). After removal of

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
the solvent, the methanol extract (90 g) was suspended in distilled water (1.5 L) and
successively partitioned with dichloromethane and ethyl acetate (three times, 1.5 L
each) to give corresponding soluble extracts, dichloromethane (PAD, 24.5 g), ethyl
acetate (PAE, 4.6 g), and water-soluble layers (PAW). The PAD fraction was then
subjected through a silica gel column and eluted with dichloromethane/methanol
(0%, 25%, 50%, 75%, and 100% volume of methanol, each 1.5 L, stepwise) to give five
fractions PAD1 - PAD5. Fraction PAD3 (1.8 g) was chromatographed on a silica gel
column, eluting with dichloromethane/methanol (15:1) to give five sub-fractions
PAD3.1-PDA3.5. Fraction PAD3.2 was purified on an RP-18 reverse phase column
and eluted with methanol/water (1:1) to yield compound 6 (5.3 mg). Fraction PAD3.3
was further separated on a silica gel column, eluting with dichloromethane/acetone
(15:1 and 15:2) to obtain compounds 1 (5.6 mg) and 2 (6.5 mg). Sub-fraction PAD3.5
was further chromatographed on a silica gel column, eluting with dichloromethane/
methanol (10:1, and 10:2), and then further purified on a silica gel column, eluting
with dichloromethane/acetone (8:1) to give compounds 4 (12.0 mg) and 5 (10.7 mg).
Fraction PAD5 (4.5 g) was separated on an RP18 reversed phase column using metha-
nol/water (2:1) to give three smaller fractions (PAD5.1 - PAD5.3). Fraction PAD5.2
was purified on a silica gel column using dichloromethane/acetone (6:1 and 3:1) to
yield 3 (7.0 mg) and 7 (4.5 mg). The water-soluble layers were passed through the
Diaion column HP-20, washed with distilled water, then eluted with increasing vol-
ume of methanol in water (25%, 50%, 75%, and 100% of methanol) to obtain four
fractions PAW1 - PAW4. The fraction PAW2 (3.8 g) was chromatographed on a silica
gel column, eluting with dichloromethane/methanol/water (3:1:0.01) to yield two sub-
fractions PAW 2.1 and PAW2.2. The sub-fractions PAW2.2 (0.8 g) was continuously
separated on an RP18 reversed phase column using methanol/water (1:2); then further
purified on a Sephadex LH-20 column, eluting with methanol/water (3:1) to yield com-
pound 8 (10mg). Fraction PAW4 (3.1 g) was subjected to a silica gel column and
eluted with dichloromethane/methanol/water (2:1:0.01) to give two smaller fractions
PAW4.1 and PAW4.2. Fraction PAW4.1 (1.1 g) was passed through an RP18 reversed
phase column using acetone/water (1:2); and then purified on a silica gel column, elut-
ing with ethyl acetate/methanol/water (3:1:0.05) to give compounds 9 (6.2 mg) and 10
(7.5 mg). Fraction PAW4.2 (0.6 g) was purified on a silica gel column using dichloro-
methane/methanol/water (2:1:0.01) as eluent to afford compound 11 (10.1mg).
3.3.1. Physalucoside A (1)
20
White amorphous powder; ½aꢃD ꢁ0.080 (c 0.25, CHCl3); IR (KBr) ꢀmax 3380, 1635,
1
1605, and 1520 cmꢁ1; H (500 MHz in CD3OD) and 13C-NMR (125 MHz in CD3OD)
þ
spectral data, see Table 1; HR-ESI-MS: m/z 492.1838 [M þ H]
(calcd for
C24H30NO10, 492.1870).
3.4. Anti-inflammatory effects
The anti-inflammatory assay was carried out by previously reported protocol with
small modification [24]. RAW264.7 cells were obtained from American Type Culture
Collection (ATCC, TIB-71, Rockville, MD, USA) and cultured in DMEM

8
H. L. TUAN ANH ET AL.
supplemented with 10% fetal bovine serum (FBS, 16000-044, Gibco, Grand Island,
NY, USA) and 100 U/ml penicillin-streptomycin (Gibco) at 37 ꢂC in incubator con-
taining 5% CO2. Cells were plated at 5 ꢄ 104 cells/well in 96-well culture plates and
then were incubated with compounds for 1 h before LPS (0.1 mg/ml) stimulation for
18 h. Cell supernatant (100 ml) was mixed with an equal amount of Griess reagent
consisting of 1% sulfanilamide (Sigma, St. Louis, MO, USA) and 0.1% N-(1-naphthy-
l)ethylenediamine (Sigma) in 5% phosphoric acid. Its absorbance was measured at
550 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific Inc.,
Waltham, MA, USA).
3.5. Cytotoxic activity
Cytotoxic activity was performed using MTT assay by previously reported method
[25]. Cell viability was evaluated by the MTT assay. Briefly, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) was added to the cells for 2 h.
The supernatant was removed and then the MTT formazan crystals were dissolved in
dimethyl sulfoxide (DMSO, Sigma). Absorbance was recorded at 570 nm using a
microplate reader (Thermo Fisher Scientific Inc.).
3.6. Western blotting analysis
According to our previous protocol with minor modification, Western blot analysis
was carried out to identify iNOS, and COX-2 protein expression in the RAW264.7
cells [18,23]. Briefly, RAW264.7 cells (2 ꢄ 106 cells/well) were treated with LPS
(0.1 mg/ml) for 16 h after pre-incubated with compounds 4 and 5 for 1 h. The cells
lysate was extracted using Cell Lysis Buffer (Cell Signaling Technology) according to
the manufacturer’s protocol.
3.7. Statistical analysis
The results were calculated by using GraphPad Prism 5 software (GraphPad Software,
San Diego, CA, USA). The significant difference between compounds treated group
and LPS alone group was evaluated by one-way analysis of variance (ANOVA) fol-
ꢀ
lowed by Dunnett’s test ( p < 0.05).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was financially supported by Vietnam National Foundation for Science and
Technology Development (NAFOSTED) under grant number 104.01-2016.26.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
ORCID
Young Ho Kim
http://orcid.org/0000-0002-7441-9459
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