Biological Activities of Oleanolic Acid Derivatives from Calendula officinalis Seeds

Article abstract of DOI:10.1002/ptr.5589

Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds identified as 28-O-β-D-glucopyranosyl-oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS1) and oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2). Biological evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid production activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2 has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the production of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells and increased cell viability against H₂O₂. These activities (melanin biosynthesis stimulatory and protective effect against H₂O₂ of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported for the first time.

Full text of DOI:10.1002/ptr.5589

PHYTOTHERAPY RESEARCH
Phytother. Res. 30: 835–841 (2016)
Published online 16 February 2016 in Wiley Online Library
(
wileyonlinelibrary.com) DOI: 10.1002/ptr.5589
Biological Activities of Oleanolic Acid Derivatives
from Calendula officinalis Seeds
2
,3
1,2
1,2
1
1
Ahmed Zaki, Ahmed Ashour, Amira Mira, Asuka Kishikawa, Toshinori Nakagawa,
1
1
Qinchang Zhu and Kuniyoshi Shimizu *
Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
National Center for Natural Product Research, University of Mississippi, University, MS 38677, USA
1
2
3
Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds
identified as 28-O-β-D-glucopyranosyl-oleanolic acid 3-O-β-D–glucopyranosyl (1→3)-β-D-glucopyranosiduronic
acid (CS1) and oleanolic acid 3-O-β-D–glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2). Biological
evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid produc-
tion activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective
effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2
has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the pro-
duction of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not
show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells
and increased cell viability against H O . These activities (melanin biosynthesis stimulatory and protective effect
2
2
against H O of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported
2
2
for the first time. Copyright © 2016 John Wiley & Sons, Ltd.
Keywords: Calendula officinalis; triterpene; melanin; hyaluronic acid production; anti obesity.
flower extract of this plant, as well as pure compounds iso-
INTRODUCTION
lated from it, has been demonstrated to possess multiple
pharmacological activities such as anti-HIV, cytotoxic,
antiinflammatory, hypoglycemic, gastric emptying inhibi-
tory, gastroprotective, hepatoprotective, spasmolytic and
spasmogenic, amongst others (Preethi et al., 2006 and
Preethi and Kuttan, 2009). C. officinalis seeds and buta-
nol fraction are selected in order to isolate the more po-
lar compounds as well as evaluation of its biological
activities.
Calendula officinalis Linn. (Asteraceae) is used medici-
nally in Europe, China and India amongst several places
in the world. It is also known as ‘African marigold’ and
is widely cultivated as an ornamental and medicinal
plant (Basch et al., 2006). It has been a subject of several
chemical and pharmacological studies. It is used in tradi-
tional medicine, especially for wound healing, jaundice,
blood purification and as an antispasmodic. Calendula
flowers in European and western Asian folk medicines
are used to treat inflammatory conditions of internal or-
gans, gastrointestinal ulcers and dysmenorrhea and as a
diuretic and diaphoretic in convulsions. In addition, it is
used externally as natural medicine for inflammation of
the oral and pharyngeal mucosa, wounds and burns
MATERIALS AND METHODS
Reagents. NaOH and DMSO were purchased from Wako
Pure Chemical Industries, Ltd (Osaka, Japan). The 3-(4,5-
dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazoliumbromide
(
Tanaka et al., 2010). Previously published results indi-
cated that C. officinalis posses multiple pharmacological
(
MTT) from Sigma (St. Louis, MO), EMEM from Nissui
activities including antiinflammatory and antioedematous
Chemical Co (Osaka, Japan). Other chemicals are of the
highest grade commercially available.
(Ukiya et al., 2006), antioxidant (Matysik et al., 2005),
immunostimulant (Wagner et al., 1985), wound healing
Leach, 2008), hepatoprotective (Lin et al., 2002), antibac-
(
terial and antifungal (Szakie et al., 2005) and antiviral
Plant material. C. officinalis flowers were collected in
April, 2013 from crops grown at Faculty of Pharmacy
fields. The plant was identified by Prof. Ibrahim Mashaly,
Systematic Botany Department, Faculty of Sciences,
Mansoura University. The seeds were separated and dried.
A voucher specimen (No. 1865) is kept in Pharmacognosy
Department, Faculty of Pharmacy, Mansoura University.
(Silva et al., 2007).
Chemical studies have underlined the presence of var-
ious classes of compounds, the main being triterpenes,
triterpene oligoglycosides, flavonoids, coumarines, qui-
nones, volatile oil, carotenoids and amino acids have
been obtained from the flowers (Muley et al., 2009). The
*
Correspondence to: Kuniyoshi Shimizu, Department of Agro-
environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka,
Japan.
E-mail: Japanshimizu@agr.kyushu-u.ac.jp
Extraction and isolation procedures. Dried powdered
seed (0.6kg) was percolated with MeOH at room
Received 25 May 2015
Revised 15 January 2016
Accepted 19 January 2016
Copyright © 2016 John Wiley & Sons, Ltd.

8
36
A. ZAKI ET AL.
temperature. The combined extracts were collected and
evaporated to dryness under reduced pressure at 40°C.
The residue, 90g, was suspended in distilled water and
extracted successively with methylene chloride, EtOAc
and n-butanol. The different extracts were evaporated
under reduced pressure to obtain methylene chloride
fraction (fraction A, 25 g) and EtOAc fraction (fraction B,
cells. So, for the other well plate, 50 μL of MTT reagent
in PBS (250 μg/mL) was added to each well. The plates
were incubated in a humidified atmosphere of 5% of
CO at 37°C for 4h. After the medium was removed,
2
1.0-mL isopropyl alcohol (containing 0.04N HCl) was
added, and the absorbance was measured at 570nm af-
ter overnight keeping in dark.
2
0 g) and n-butanol fraction (fraction C, 15 g); the n-butanol
fraction was selected for further study.
Hyaluronic acid (HA) ELISA assay on (NHDF-Ad).
The isolated compounds were tested for the effect of
the production of HA by the use of HA ELISA assay
using Biotech Trading Partners (Encinitas, California)
according to the manufacturer’s instruction.
Isolation of compounds. Three grams of fraction C was
dissolved totally in the smallest volume of methanol
(
1–2 mL) and then mixed well with about 3-g RP18 silica
gel and left at room temperature to dry and applied onto
the top of a silica gel packed (RP ) glass column (120g,
This kit is an enzyme-linked binding protein assay
that uses a capture molecule known as HA binding pro-
tein (HABP). After growing of the fibroblast cells, they
are incubated in HABP-coated micro well plate. Prop-
erly samples and HA reference solution are added to
this plate, allowing HA present to react with the
immobilized binding protein. After removal of unbound
molecules by washing, HABP conjugated with horse-
radish peroxidase (HRP) solution is added to the
microwells to form complexes with bound HA. Follow-
ing another washing step, a chromogenic substrate of
tetramethylbenzidine and hydrogen peroxide is added
to develop a colored reaction. The intensity of the color
is measured in optical density units with a spectropho-
tometer at 450nm. The higher the intensity of the color,
the higher ability of the sample to produce HA.
1
8
3
3× 3.7cm) pre-packed in water. Isocratic elution was
carried out with MeOH – H O (6.5 : 3.5). The effluent
2
was collected in 10-mL fractions monitored by TLC
using MeOH – H O (8 : 2) as solvent system, and the
2
developed TLCs were heated after spraying with vanillin/
sulfuric acid spray reagent. Similar fractions were pooled to-
gether. After further purification through re-crystallization
from hot methanol (60 °C), two main compounds were
isolated as yellow plates; CS1 (310mg) and CS2 (220mg).
Cell line. A mouse melanoma cell line, B16 and mouse
neuroblastoma cells neuro-2Awere obtained from RIKEN
Cell Bank. The cells were maintained in EMEM supple-
mented with 10% (v/v) fetal bovine serum (FBS) and
0.09mg/mL theophylline. Adult normal human dermal
fibroblasts (NHDF-Ad, Lonza, Tokyo, Japan) were rou-
tinely maintained in 10% FBS in Dulbecco’s modified
Eagle’s medium (DMEM). All of cultivation was con-
ducted at 37°C in a humidified atmosphere of 5% CO2.
Anti-obesity activity. The pancreatic lipase inhibition
activity of both compounds was measured by an in vitro
enzyme reaction, which use 4-methylumbelliferyl oleate
(4-MUO) as the substrate (Mizutani et al., 2010). Each
sample was dissolved in DMSO (400 and 800 μg/mL) and
diluted in 25 μL of buffer solution consisting of 13mM
Tris–HCl, 150mM NaCl and 1.3 mM CaCl (pH =8).
2
Melanin assay in B16 melanoma cell line. This assay was
determined as described previously (Ashour et al.,
These samples were applied to a 96-well plate. DMSO
and Orlistat (final concentration 1 μM) were used as a
negative control and positive control, respectively. Then,
2
2
013). The cells (1mL) were placed in two plates of
4-well plastic culture plates (one plate for determining
5
0 μL of 0.25 mM 4-MUO (dissolved in buffer) was ap-
plied, followed by the addition of 25 μL of lipase enzyme
50 U/mL) to all wells except blank wells as well as sam-
ple blank wells. After plates were incubated for 30min at
5°C, 100 μL of 0.1 M sodium citrate buffer (pH 4.2) was
added to each well to stop the reaction. The amount of
-MUO released by the action of the lipase was measured
melanin and the other for cell viability) at a density of
5
1
×10 cells/well and incubated for 24h in media prior
(
to being treated with the samples. After 24 h, the media
were replaced with 998 μL of fresh media and 2 μL of
the test sample at their maximum solubility (n = 3). At
the same time, negative control (2-μL DMSO) and pos-
itive control, and arbutin at concentration 50mg/mL in
DMSO were tested. The cells were incubated for an ad-
ditional 48h, and then the medium was replaced with
fresh medium containing each sample. After 24h, the
remaining adherent cells were assayed. To determine
the melanin content (for one plate) after removing the
medium and washing the cells with PBS, the cell pellet
was dissolved in 1.0 mL of 1 N NaOH. After overnight
keeping in dark, the crude cell extracts were assayed
by using a microplate reader at 405nm to determine
the melanin content. The results from the cells treated
with the test samples were analyzed as a percentage of
the results from the control culture. On the other hand,
cell viability was determined by using MTT assay which
provides a quantitative measure of the number of viable
cells by determining the amount of formazan crystals
produced by metabolic activity in treated versus control
2
4
with a fluorescence spectrophotometer (FlexStation 3
Microplate Reader, Molecular Devices, CA, USA) at
excitation and emission wavelengths of 355 and 460nm,
respectively.
Adipocyte differentiation inhibition assay. The effects of
the two isolated compounds on adipocyte differentia-
tion were examined as previously described (Zhang
et al., 2012 and Wu et al., 2013). Briefly, 3T3-L1 cells were
seeded into collagen-coated 24-well plates (IWAKI,
5
Tokyo, Japan) at a density of 0.8×10 cells/well. Two days
after confluence, the medium was changed to differentia-
tion medium containing 10% FBS, 10 μg/mL insulin
(Wako, Tokyo Japan), 1-μM dexamethasone (Wako,
Tokyo, Japan), and 0.5mM 3-isobutyl-1-methylxanhine
Copyright © 2016 John Wiley & Sons, Ltd.
Phytother. Res. 30: 835–841 (2016)

OLEANOLIC ACID DERIVATIVES FROM CALENDULA OFFICINALIS SEEDS
837
(
IBMX, Galbiochem, San Diego, CA, USA). At the same
RESULTS AND DISCUSSION
time, compounds or solvent control (DMSO) were added
to the cells. Berberine was used as a positive control
for differentiation inhibition (Huang et al., 2006). After
Identification of isolated compounds
2
days incubation, the medium was replaced with post
Compound CS1 (Fig. 1) was isolated as faint yellow
plates, m.p. 270–272°C. IR (KBr, ʋmax) spectrum showed
characteristic absorption bands for triterpene glycoside at
differentiation medium containing 10% FBS, 10 μg/mL
insulin and corresponding samples. The medium was
th
refreshed every 2days until the 5 day after post differen-
À1
3
445, 2927, 1634, 1457, 1379, 1071, 1043cm and absorp-
tiation medium was used.
À1
tion band at 1732cm ascribable to a carbonyl function
Oil Red-O was used to stain the lipid droplet in the
differentiated 3T3-L1 cells. The cells were washed with
PBS twice, fixed with 10% formalin for 1 h and stained
with 0.36% Oil Red-O (Sigma, St. Louis, MO, USA)
in isopropanol–water solution at room temperature for
1
(
Pavia et al., 2001). The H-NMR spectrum revealed sin-
glet resonances at δ 0.79, 0.82, 0.90, 0.92, 0.94, 1.04 and
1
.1, an olefin proton at δ 5.24 (s) and an oxygenated
methine at δ 3.19 (dd, J=4.2, 11.4); these signals are char-
acteristic to triterpenes of olea-12-ene skeleton.
1
h. After washing with water for 4 times, the cells were
photographed using a Nikon Microscope (TE200) at
0× magnification. Finally, the dye retained in the cells
13
C-NMR spectrum revealed the presence of 48
resonances, two characteristic signals at δ 123.9 and 144.8
assignable for the aglycone of the oleanone skeleton.
Twenty oxygenated carbons (Table 1) were detected;
two of them are because of carboxy groups at δ 176.6
and 178.1, respectively (Agrawal, 1992 & Mahato and
Kundu, 1994).
The DEPT-135 experiment helped in the differentia-
tion of the 48 carbon resonances into 7 methyl, 12 meth-
ylene, 20 methine and 9 quaternary carbons. Within
these resonances 7 methyl, 10 methylene, 5 methine
and 8 quaternary carbons were attributed to the aglycon
moiety with a characteristic olean-12-ene olefin bond at
C-12, and a carboxy group at C-28, corresponding to
oleanolic acid.
1
were dissolved with isopropanol containing 4% Nonidet
P-40 and quantified by reading the absorbance at 570nm
with a microplate reader (Biotek-ELX800, BioTek,
Winooski, Vermont, USA).
The toxicity of the samples to the 3T3-L1 cells was
measured with the MTT method as described previously
(
Tanaka et al., 2014). Confluent 3T3-L1 cells in collagen
coated 24-well plates were treated with samples for 72h.
And then 50 μL of MTT (5 mg/mL) was added to each
well. After incubation for 4 h, the formazan crystals in
the cells were dissolved with isopropanol, and the absor-
bance was read at 570nm.
Upon acid hydrolysis of compound CS1 using 10%
HCl and co-chromatography of the resulted sugar with
the authentic sugar and sugar acid samples, using
Protection against H O -induced neurotoxicity in neuro-2A
2
2
cells. The assay was performed as we previously described
Mira et al., 2013). The concentration of H O that could de-
BuOH–AcOH–H O (5:1:4) as a solvent system: glucose
2
(
2
2
and two acids, glucuronic acid and oleanolic acid, were
liberated.
crease cell viability of neuro-2A cells was first determined
and then the ability of isolated compounds at non cytotoxic
concentration (2.5, 5 and 10 μg/mL) to protect against
3C-NMR spectra showed resonances for three sugar
18 signals) units with anomeric carbons at δ 95.7,
(
H O -induced oxidative damage (150 μM) (Zhao et al.,
2
2
105.3 and 106.4. The up-field shifted anomeric carbon
at δ 95.7 indicated the glycosylation of C-28 carboxy
group (δ 178.1). This conclusion was confirmed by the
correlation between the anomeric proton of this glucose
moiety H-1′ at δ 5.4 (d, J =7.8) and the C-28 at δ 178.1.
The presence of carbon signal at δ 176.6 is assignable
for C-6 of sugar acid: β-D-glucopyranosiduronic acid
moiety. In the HMBC experiment of CS1 (Fig. 2),
long-range correlations were observed between the
2
011) was determined. Catechin at 100μM was used as a
positive control.
Statistical analysis. The results are expressed as means
±
standard deviation (n =3). Data were analyzed for sta-
tistical significance using one-way ANOVA, followed by
Dunnett’s test as a post-hoc test with GraphPad Prism
H-1′′-proton δ 4.3 (d, J= 7.2, δ 106.4) and the C-3
c
5.0 software for Windows (Inc., San Diego, CA, USA).
Figure 1. Chemical structure of CS1 and CS2.
Copyright © 2016 John Wiley & Sons, Ltd.
Phytother. Res. 30: 835–841 (2016)

8
38
A. ZAKI ET AL.
1
3
1
Table 1. C and H-NMR data of CS1 and CS2
CS1
CS2
cpd
#
1
3
a
1
a
13
a
1
a
C
H
C
H
1
2
3
4
5
6
7
8
9
1
39.8
26.9
90.6
40.2
57.0
19.3
33.2
40.7
49.2
37.9
24.0
123.9
144.8
42.9
28.9
24.2
48.0
42.6
47.2
31.5
34.9
34.0
28.5
17.0
16.0
17.7
26.3
178.1
33.5
23.9
95.7
73.9
78.7
71.1
78.3
62.4
106.4
74.8
86.1
72.3
77.3
176.6
105.3
72.8
75.1
70.6
77.3
62.7
1.01, 1.58
1.66, 1.88
3.19, dd, J = 4.2, 11.4
—
39.8
26.9
90.8
40.2
57.1
19.4
34.2
40.6
ND
37.9
24.6
122.5
146.7
43.0
29.3
24.6
49.3
43.5
48.2
31.8
35.3
34.4
28.5
17.0
16.0
18.2
26.5
186.0
34.0
24.4
3.18, dd, J = 4.2, 11.4
—
0.76
0.76
1.41, 1.52
1.59, 1.68
—
—
1.61
0
—
—
1.97, 2.1
br s, 5.24
—
1
1
1.97, 2.1
br s, 5.24
—
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
′
—
—
0.98, 1.07
1.2, 1.4
—
—
2.85, dd, J = 9.6, 13.8
1.19, 1.72
—
2.9, dd, J = 9.6, 13.8
—
1.2, 1.37
1.149, 1.08
s, 1.04
s, 0.828
s, 0.942
s, 0.794
s, 1.14
s, 1.04
s, 0.831
s, 0.938
s, 0.869
s, 1.13
—
—
s, 0.928
s, 0.901
5.4, d, J = 7.8
3.32
3.30
3.30
3.38, m
3.61, 3.81
4.3, d, J = 7.2
3.41, m
3.60
3.56
3.58, m
—
s, 0.946
s, 0.869
′
′
′
′
′
′′
106.4
74.8
86.1
72.3
77.2
176.6
105.3
72.9
75.2
70.6
77.3
62.7
4.3, d, J = 7.8
3.41, m
3.60
3.56
3.58, m
—
′′
′′
′′
′′
′′
′′′
′′′
′′′
′′′
′′′
′′′
4.5, d, J = 7.8
3.6
3.49, dd, J = 3, 9.6
3.78
3.58, m
3.61, 3.81
4.5, d, J = 7.8
3.6
3.49, dd, J = 3, 9.6
3.78
3.58, m
3.61, 3.81
a13
1
3
C and H are measured in CD OD at 150 MHz and 600 MHz, respectively. N.D.: non detectable.
(
δ 90.6), between H-1′′′ δ 4.5 (d, J =7.8, δ 105.3) and
glucopyranosyl-oleanolic acid 3-O-β-D-glucopyranosyl
(1→3)-β-D-glucopyranosiduronic acid. Further confir-
mation was done by comparing the data with those
in the literature (Liang et al., 2010).
c
c
C-3′′ (δ 86.1), which indicated the glycosidation of C-3
with the glucuronic acid with additional glucose linked
to its C-3′′. This linkage was confirmed through the
downfield shift of C-3′′ at δ 86.1. The full assignments
1
13
of H- and C-NMR (Table 1) spectra were facilitated
by comparison with those of oleanolic acid glycosides
Compound CS2 was isolated as faint yellow plates, m.
1
13
p. 262–264 °C. The H- and C-NMR data of this com-
13
(
Gohar et al., 2002) and further confirmed by HSQC,
pound showed close similarity to CS1. The C-NMR
spectra showed the absence of the anomeric carbon at
δ 95.7 and the other oxygenated carbon signals at δ
HMBC (Fig. 2) and COSY spectra. Accordingly,
compound CS1, was concluded to be 28-O-β-D-
Copyright © 2016 John Wiley & Sons, Ltd.
Phytother. Res. 30: 835–841 (2016)

OLEANOLIC ACID DERIVATIVES FROM CALENDULA OFFICINALIS SEEDS
839
First, sample concentration that showed no cell toxic-
ity to NHDF-Ad (normal-human dermal fibroblast from
adult skin) after 48-h incubation with sample was deter-
mined by MTT assay. HA production-promoting effect
on NHDF-Ad was evaluated at concentration of the
samples that showed no cell toxicity. The amount of
HA produced by NHDF-Ad was measured using an
4
ELISA kit. Previously, NHDF-Ad cells (2.0× 10 cells/
well) were cultured in a 96-well plate with 100 μL of
DMEM supplemented with 10% FBS. After 24-h incu-
bation, test samples [0.5 μL/well, DMSO as a control,
ascorbic acid (17.6 μg/mL) as a positive control] were
added to the cells soon after the medium was replaced
with 100 μL of DMEM supplemented with 0.5% FBS.
After 48-h incubation, the HA amount produced by
NHDF-Ad in the supernatant was quantified by using
a human HA ELISA kit (ACEL, Japan). A standard
curve was made in the same ELISA plate (n= 3). On
the other hand, the cell viability of the remaining cells
was measured by MTT assay (n =3). The statistical dif-
ference between the control and each sample was deter-
mined by Student’s t-test.
It was found that compound CS1 at both tested con-
centration has significant stimulating effect for the pro-
duction of HA and at the same time it showed no
toxicity to the cell. However, compound CS2 did not
show any stimulating effect on the production of HA at
both tested concentration. Results are shown in Fig. 4.
Figure 2. HMBCs of CS1 and CS2.
7
3.9, 78.7, 71.1, 78.3 and 62.4, which indicated the liber-
ation of C-28 glycosidation. Therefore, the downfield
shift of C-28 from δ 178.1 to δ 186.1. Furthermore,
1
H-NMR spectrum revealed the disappearance of corre-
sponding resonances anomeric proton at δ 5.4.
Compound CS2 was further confirmed by HSQC,
HMBC and COSY spectra. Accordingly, compound
CS2 was concluded to be oleanolic acid 3-O-β-D-
glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid.
B16 melanoma cell line assay
Compound CS2 showed melanin biosynthesis stimula-
tory effect based on cell viability as shown in Fig. 3.
Compound CS1 did not show any activity at any tested
concentration. Results are shown in Fig. 3.
HA production assay in fibroblast
Scientific studies have shown that HA improves skin hy-
dration, stimulates production of collagen in skin, works
as an antioxidant and free radical scavenger, maintains
skin elasticity, cushions joints and nerve tissues, has an
antibacterial and antiinflammatory activity and main-
tains the fluid in the eye tissues, which may help to pro-
tect against numerous possible eye concerns (Necas
et al., 2005).
Figure 4. Effect of isolated compounds (CS1 and CS2) on the pro-
duction of hyaluronic acid. The values are represented as the mean
±
1
standard deviation (SD), n = 3. Significant difference between
% DMSO and each concentration was determined by Student’s
t-test: *P < 0.05, **P < 0.01.
Figure 3. Inhibitory activity of compounds (CS1 and CS2) on mel-
anin synthesis in B16 melanoma cell. The values are represented as
the mean ± standard deviation (SD), n = 3. Significant difference
from the control value and each compound was determined by
Student’s t-test: *P < 0.05, **P < 0.01.
Figure 5. Inhibition effect of isolated compounds (CS1 and CS2)
on pancreatic lipase. The values are represented as the mean
± standard deviation (SD), n = 3. Significant difference between
1% DMSO and each concentration was determined by Student’s
t-test: *P < 0.05, **P < 0.01.
Copyright © 2016 John Wiley & Sons, Ltd.
Phytother. Res. 30: 835–841 (2016)

8
40
A. ZAKI ET AL.
Figure 6. Inhibition effect of isolated compounds (CS1 and CS2) on 3T3-L1 adipocyte differentiation. A, the lipid droplet formation in 3T3-L1
cells were stained with oil Red-O. B, Relative 3T3-L1 differentiation was calculated from the quantification of stained lipid content by
measuring absorbance at 570 nm. Values are represented as the mean ± standard deviation (SD), n = 3. Significant difference between
1
% DMSO and each concentration was determined by Student’s t-test: *P < 0.05, **P < 0.01.
Anti-obesity activity
Excessive gastrointestinal absorption of fats and adipo-
genesis are two major causes of obesity. To evaluate
the potential anti-obesity effect of both isolated com-
pounds, both the lipase and adipocyte differentiation in-
hibition activity were investigated for them.
For the in vitro lipase assay, the clinically used anti-
obesity drug orlistat (0.5 μg/mL) was used as positive
control and was found to inhibit the activity of pancre-
atic lipase by 88.9% (Fig. 5); however, it was found that
both compounds have no effect on lipase inhibition.
These results are not consistent with De Meloa et al.,
2
010 which showed a significant effect for oleanolic acid
for decreasing the obesity. These results suggest that the
anti obesity effect may need the presence of free hy-
droxyl and carboxyl group of the oleanolic acid moiety.
The mouse 3T3-L1 cell line is a well characterized cell
line for adipogenic assay. 3T3-L1 preadipocyte cells
show fibroblastic phenotype in normal condition and
adipocyte phenotype after treatment with differentia-
tion media. This assay was run parallel with anti-lipase
assay as both compounds were subjected to the adipo-
cyte differentiation assay at the concentration without
significant cytotoxicity. Results showed that both com-
pounds did not show any inhibitory effect, but it showed
that compound CS2 has stimulating effect on lipase at
concentration 800 μg/mL. Results are shown in Fig. 6.
The results of this assay comply with the result of anti-
lipase.
2 2
Figure 7. Neuroprotection against H O -induced neurotoxicity in
neuro-2A cells by isolated compounds. Values are represented as
means ± standard deviations (SD), n = 5. * and ** Significant differ-
2 2
ence from control in (A) and from cell viability of H O (150 μM)
treatment in (B, C, D) at P < 0.05 and P < 0.01, respectively.
1
0-μM concentration. These results confirm that
glycosidation at C-28 decreases the activity, and the pres-
ence of free carboxylic group is necessary for the activity.
CONCLUSION
In our ongoing study to find biologically active com-
pounds from medicinal plants, two compounds were iso-
lated from the butanol faction of C. officinalis seeds
identified as 28-O-β-D-glucopyranosyl-oleanolic acid 3-O-
β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid
Protection against H O -induced neurotoxicity in
2
2
neuro-2A cells
(
CS1) and oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-
H O (150 μM) induced neurotoxicity in neuro-2A cells
β-D-glucopyranosiduronic acid (CS2). Biological evalua-
tion was carried out for these two compounds. It was
found that compound CS2 has a melanin biosynthesis
2
2
and caused 47.5±2.3% decrease in cell viability (Fig. 7).
While compound CS1 did not show any neuroprotec-
tion, compound CS2 showed dose-dependent increase
in cell viability. CS2 could protect neuro-2A cells and in-
creased cell viability by 9.4±7.6 and 20.4± 3.4% at 5 and
stimulatory activity and neuroprotection against H O2
2
induced-neurotoxicity; however, compound CS1 has a
potent stimulatory effect for the production of HA on
Copyright © 2016 John Wiley & Sons, Ltd.
Phytother. Res. 30: 835–841 (2016)

OLEANOLIC ACID DERIVATIVES FROM CALENDULA OFFICINALIS SEEDS
841
normal human dermal fibroblast from adult (NHDF-
Ad). Both compounds did not show any inhibitory effect
on both lipase and adipocyte differentiation that are not
consistent with the published result which showed a sig-
nificant effect for oleanolic acid for decreasing the obe-
sity. These results suggest that the anti obesity effect
may need the presence of free hydroxyl and carboxyl
group of the oleanolic acid moiety. It is worth to mention
that these activities were done for the first time for these
triterpene derivatives.
Conflict of Interest
The authors have declared that there is no conflict of interest.
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Phytother. Res. 30: 835–841 (2016)