Hepatoprotective and cytotoxic activities of Anvillea garcinii and isolation of four new secondary metabolites

Article abstract of DOI:10.1007/s11418-017-1118-1

Anvillea garcinii is a medicinal plant traditionally used for the treatment of dysentery, gastrointestinal troubles, hepatitis, lung disease, colds, digestive problems and pulmonary affections and in liver diseases. Four new sesquiterpene lactones, garcinamines A–D, along with seven known compounds, were isolated from the leaves of A. garcinii. This is the first report of the isolation of amino acid analogues of parthenolide-type sesquiterpene lactones from the family Asteraceae. Total ethanol extract of leaves as well as the chloroform and n-butanol fractions were tested for their hepatoprotective effect using the carbon tetrachloride liver toxicity model. The chloroform fraction, at a dose of 400?mg/kg, demonstrated a significant hepatoprotective effect comparable to silymarin in all serum and tissue parameters. The cytotoxicity of all extracts and compounds were evaluated against five human cancer cell lines: MCF-7, HCT-116, HepG2, Hela and A-549. The results indicated that the chloroform and n-butanol fractions and compounds 3, 4, 7 and 8 displayed significant cytotoxic activity against these cell lines.

Full text of DOI:10.1007/s11418-017-1118-1

J Nat Med
DOI 10.1007/s11418-017-1118-1
ORIGINAL PAPER
Hepatoprotective and cytotoxic activities of Anvillea garcinii
and isolation of four new secondary metabolites
Shagufta Perveen1 Areej Mohammad Al-Taweel1 Hasan Soliman Yusufoglu2
•
•
•
Ghada Ahmed Fawzy1,3 Ahmed Foudah2 Maged Saad Abdel-Kader2,4
•
•
Received: 14 June 2017 / Accepted: 26 July 2017
Ó The Japanese Society of Pharmacognosy and Springer Japan KK 2017
Abstract Anvillea garcinii is a medicinal plant tradition-
ally used for the treatment of dysentery, gastrointestinal
troubles, hepatitis, lung disease, colds, digestive problems
and pulmonary affections and in liver diseases. Four new
sesquiterpene lactones, garcinamines A–D, along with
seven known compounds, were isolated from the leaves of
A. garcinii. This is the first report of the isolation of amino
acid analogues of parthenolide-type sesquiterpene lactones
from the family Asteraceae. Total ethanol extract of leaves
as well as the chloroform and n-butanol fractions were
tested for their hepatoprotective effect using the carbon
tetrachloride liver toxicity model. The chloroform fraction,
at a dose of 400 mg/kg, demonstrated a significant hep-
atoprotective effect comparable to silymarin in all serum
and tissue parameters. The cytotoxicity of all extracts and
compounds were evaluated against five human cancer cell
lines: MCF-7, HCT-116, HepG2, Hela and A-549. The
results indicated that the chloroform and n-butanol
fractions and compounds 3, 4, 7 and 8 displayed significant
cytotoxic activity against these cell lines.
Keywords Anvillea garcinii Á Sesquiterpene lactones Á
Hepatoprotective activity Á Cytotoxicity
Introduction
For centuries, medicinal plants have traditionally been used
for treating liver diseases. Several natural leads with
diverse chemical structures were discovered as potential
hepatoprotective agents. Moreover, the development of
new natural anti-cancer drugs with specificity and higher
potency against different cancer cells has become an
important target in biomedical research. In continuation of
our work on medicinal Saudi plants, we selected Anvillea
garcinii (Burm.f.) DC. (Anvillea garcinii subsp. radiata),
which is a shrub with florescent yellow flowers. The genus
Anvillea belongs to the family Asteraceae, and comprises
four species distributed in the region from North Africa to
Iran, including a number of Middle Eastern countries, such
as Egypt, Palestine and Saudi Arabia [1]. Anvillea garcinii
is widely used by local people for its medicinal properties
and is traditionally used for the treatment of dysentery,
gastrointestinal troubles, hepatitis, lung disease, colds,
digestive problems and pulmonary affections, and in liver
diseases [2]. Anvillea garcinii was proven to have signifi-
cant antidiabetic potential by decreasing blood glucose
levels and maintaining serum lipid concentrations at nor-
mal [3]. Previous phytochemical studies on the various
parts of A. garcinii have led to the isolation of different
biologically active compounds, including germacranolides
[4–8], flavanoids and their glycosides [9, 10]. Germacra-
nolides and their isomers isolated from A. garcinii were
Electronic supplementary material The online version of this
article (doi:10.1007/s11418-017-1118-1) contains supplementary
material, which is available to authorized users.
& Shagufta Perveen
shagufta792000@yahoo.com; shakhan@ksu.edu.sa
1
Department of Pharmacognosy, College of Pharmacy, King
Saud University, P. O. Box 22452, Riyadh 11495, Kingdom
of Saudi Arabia
2
Department of Pharmacognosy, College of Pharmacy, Prince
Sattam Bin Abdulaziz University, P.O. Box 173,
Al-Kharj 11942, Saudi Arabia
3
Department of Pharmacognosy, Faculty of Pharmacy, Cairo
University, Cairo 11562, Egypt
4
Department of Pharmacognosy, Faculty of Pharmacy,
Alexandria University, Alexandria 21215, Egypt
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J Nat Med
shown to possess anti-tumor, anti-HIV and cytotoxic
activities in both in vitro and vivo assays [5, 6]. It has been
reported recently that different extracts of A. garcinii
inhibit the oxidative burst of primary neutrophils in
humans which, together with its high anti-inflammatory
potential, make it a promising candidate for further
medicinal uses and applications [11]. Previous phyto-
chemical work on the Saudi A. garcinii has revealed that
this plant is a rich source of sesquiterpenes. In the present
study, we have isolated four new sesquiterpene lactones
along with seven known compounds from different frac-
tions of the plant extract. In addition, the total ethanol
extract of the leaves as well as the chloroform and n-bu-
tanol fractions were tested for their hepatoprotective effect
against liver toxicity induced by carbon tetrachloride.
Additionally, the cytotoxic activities of A. garcinii extracts
and its isolates were evaluated against five human cancer
cell lines (MCF-7 breast adenocarcinoma, HCT-116 colon
carcinoma, HePG-2 liver hepatocellular carcinoma, Hela
cervical cells and A-549 lung carcinoma).
sesquiterpene lactone attached with an aromatic moiety and
closely resembled the 9a-hydroxyparthenolide, isolated
previously from the chloroform-soluble fraction of the
same plant A. garcinii [8].
The 1H-NMR spectrum showed specific signals of an L-
phenylalanine amino acid group at dH3.46 (1H, m, H-20),
2.92 (2H, d, J = 5.0 Hz, H-30), 7.27 (2H, m, H-50,90), 7.26
(2H, m, H-60,80) and 7.21 (1H, m, H-70). The 13C-NMR
signals of an L-phenylalanine group appeared at dC174.0
(C-10), 63.4 (C-20), 38.2 (C-30), 138.1 (C-40), 130.0 (C-50,
90), 128.7 (C-60, 80), 126.8 (C-70) and confirmed the pres-
ence of an L-phenylalanine amino acid group (Tables 1, 2)
[12]. The missing C-13 methyl signal in NMR and the
appearance of an additional downfield methylene group at
dH 2.91 (1H, J = 10.0 Hz, H-13a) and 2.74 (1H, m, H-13b)
strongly suggested that C-13 is attached to the nitrogen of
L-phenylalanine amino acid. The point of attachment of the
L-phenylalanine group was further confirmed by the
HMBC spectrum, in which H-13a (dH 2.91) showed
2J correlations with the methine carbon (dC 46.8, C-11) and
3J correlations with C-12 (177.6) and C-20 (63.4). Further
confirmation of the position and presence of the L-pheny-
lalanine moiety was made by the acid hydrolysis of com-
pound 1. The a-orientation of the hydroxyl group at C-9
was confirmed by the upfield chemical shift of the C-9 at
dC 70.4 ppm; in the case of 9b-hydroxyparthenolide, the
C-9 b-orieanted hydroxymethine carbon usually appeared
in the range of dC 78–79 ppm in the 13C-NMR spectrum
[4]. In the nuclear Overhauser enhancement (NOE)
experiment on 1, correlations were observed between the
6b-proton, 11b-proton and 9b-proton, which further con-
firmed the a-position of the hydroxyl group at C-9 carbon.
The relative configuration of C-4 and C-5 was determined
by NOE experiments. NOE correlations were observed
between the 5a-H and 7a-H, 7a-H and13a-H2, 6b-H and
9b-H, and 6b-H and 15b-H3. Acid hydrolysis of 1 provided
the L-phenylalanine and 9a-hydroxyparthenolide [8] and
this was confirmed through co-TLC, the 1H-NMR spectrum
and the sign of their optical rotation with standards.
Therefore, compound 1 was unambiguously elucidated as
13-L-phenylalanine-9a-hydroxyparthenolide, and named
garcinamine A.
Results and discussion
Structure elucidation of new compounds (1–4)
Chromatographic separation of the chloroform and n-bu-
tanol fractions of the ethanol extract of A. garcinii, using
silica, Sephadex LH-20, and RP-18 column chromatogra-
phy, yielded four new (1–4) and seven known compounds
(5–11) (Fig. 1). Their structures were elucidated by spec-
tral data analysis, including IR, 1D, 2D NMR, and ESI–
MS.
Garcinamine A (1) obtained as yellow gummy solid
[a]25D = –27.0 (c = 0.10, CH3OH) and the molecular
formula was established as C24H30NO6 at m/z 428.2068
(calcd 428.2073) [M–H]- by negative ESI–MS. The IR
spectrum showed characteristic signals for a hydroxyl
group (3442 cm-1), a c-lactone group (1762 cm-1) and a
double bond (1650 cm-1). The 1H-NMR spectrum showed
a downfield signal at dH 5.48 (d, J = 10.0 Hz) for an
olefinic proton, three hydroxymethine protons at dH 2.51
(m, H-5), 4.02 (m, H-6) and 3.98 (m, H-9) and character-
istic signals at dH 1.59 (s, H-14), 1.19 (s, H-15) were
assigned for two methyl groups, which suggests the pres-
ence of a sesquiterpene lactone ring (Table 1). The 13C-
NMR and DEPT spectra of 1 showed two methyl groups at
dC 16.2 and 17.3, five methylene at dC 22.9, 36.1, 36.8,
45.5 and 38.2, 12 methine at dC 120.7, 66.1, 82.2, 35.5,
70.4, 46.8, 63.4, 130.0 9 2, 128.7 9 2 and 126.8, and five
quaternary carbon signals at dC 61.3, 138.3, 177.6, 174.0
and 138.1 (Table 2). These NMR spectroscopic data sug-
Garcinamine B (2) was obtained as a yellow gummy
solid, [a]25D = -29 (c = 0.15, CH3OH). The positive ion
mode ESI–MS gave a [M ? H]? peak at m/z 382 corre-
1
sponding to the molecular formula C20H32NO6. The H-
and 13C-NMR data were closely similar to compound 1 and
it showed features common to garcinamine A (1) except for
the characteristic signals of an L-valine amino acid group
instead of an L-phenylalanine amino acid moiety attached
1
at C-13 methylene carbon atom. The H- and 13C-NMR
spectra showed signals of an L-valine amino acid group at
dH 2.88 (1H, d, J = 5.0 Hz, H-20), 1.24 (1H, brs, H-30),
gested that compound
1
was
a
parthenolide-type
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J Nat Med
6'
4'
OH
9
OH
5'
4'
3'
3'
1
4
8'
10
5
2
8
7
2'
2'
HN
13
HN
13
1'
COOH
14
COOH
3
6
1'
11
O
O
O
12
O
O
15
2
1
O
O
OH
4'
3'
2'
5'
1'
N
COOH
13
O
O
O
O
5
3 9-α OH
4 9-β OH
O
O
OH
OH
O
O
O
O
O
7 9-α OH
8 9-β OH
6
O
O
OR3
OH
R2O
O
9 R = H, R1 = OCH3, R2 = β-
10 R = H, R1 = OCH3, R2 = β-
11 R = β- -glucose-6-O-α- -rhamnoside, R1 = H, R2 = H, R3 = H
D
-glucoside, R3 = CH3
-glucoside, R3 = H
D
R1
OR
D
L
OH
O
Fig. 1 Structures of compounds 1–11 from the leaves of A. garcinii
0.89 (3H, d, J = 5.0 Hz, H-40), 0.86 (3H, d, J = 5.0 Hz,
H-50) and dC 159.0 (C-10), 68.3 (C-20), 30.9 (C-30), 19.6 (C-
40), 18.8 (C-50) (Tables 1, 2). The methylene protons of
H-13a (d 2.63, m) and H-13b (dH 2.90) showed
2J correlations with the methine carbon (dC 47.6, C-11) and
3J correlations with C-12 (177.6) and methine carbon (dC
68.3, C-20) of the L-valine moiety. Acid hydrolysis of
compound 2 afforded 9a-hydroxyparthenolide [8] and L-
valine amino acid, and this was confirmed through TLC,
NMR and the sign of its optical rotation. Based on 1H- and
13C-NMR and COSY data and correlations, the stereo-
chemistry of compound 2 was assumed to be same as
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J Nat Med
Table 1 1H-NMR data for
compounds 1–4 (DMSO, J in
Hz, d in ppm, 500 MHz)
Position
1
dH
2
dH
3
dH
4
dH
1
5.48 (d, J = 10)
5.48 (d, J = 10.0)
5.48 (d, J = 10.0)
2.03 (m)
2.40 (t, J = 5.0)
1.14 (m)
1.80 (m)
–
5.32 (dd 10.0, 5.0)
2.07 m
2.40 m
1.10 m
2.0 m
2a
2b
3a
3b
4
2.08 (brd, J = 10)
2.03 M
2.39 (brd, J = 10)
2.48 m
1.12 m
1.15 m
1.88 m
1.85 m
–
–
–
5
2.51 m
2.62 m
2.65 (d, J = 10.0)
4.03 (m)
2.03 (m)
1.89 (m)
2.01 (m)
4.0 (m)
–
2.70 (d, J = 10.0)
3.99 (m)
2.40 (m)
1.80 (m)
1.85 (m)
3.96 (m)
–
6
4.02 m
4.0 m
7
2.26 m
2.18 m
8 a
8b
9
2.58 (d, J = 10.0)
2.66 m
2.00 m
2.01 m
3.98 m
3.99 m
10
11
12
13a
13b
14
15
10
–
–
2.51 m
2.50 m
2.70 (m)
–
2.65 (m)
2.10 (m)
3.13 (m)
3.01 (m)
1.63 (s)
1.21 (s)
–
–
–
2.91 (d, J = 10.0)
2.63 m
3.12 (m)
3.0 (m)
1.61 (m)
1.20 (m)
–
2.74 m
2.90 m
1.59 s
1.61 s
1.19 s
1.20 s
–
–
20
3.46 m
2.88 (d, J = 5.0)
3.48 (t, J = 10.0)
2.10 (m)
1.86 (m)
1.82 (m)
1.71 (m)
3.15 (m)
2.70 (m)
–
3.40 (t, J = 10.0)
2.07 (m)
1.85 (m)
1.81 (m)
1.73 (m)
3.11 (m)
2.63 (m)
–
30a
30b
40a
40b
50a
50b
60
70
80
90
2.92 (d, J = 5.0)
1.24 brs
–
–
–
0.89 (d, J = 5.0)
–
–
7.27 m
–
0.86 (d, J = 5.0)
7.26 m
7.21 m
7.26 m
7.27 m
–
–
–
–
–
–
–
–
–
–
compound 1. Thus, the structure of 2 was elucidated as
13-L-valine-9a-hydroxyparthenolide, and named garci-
namine B.
correlations with the methine carbon C-11 (45.3) and
3J HMBC correlations with C-12 (177.2), C-7 (36.6), C-20
(67.5) and C-50 (53.6), which confirmed the position of the
amino acid group at C-13 carbon. Acid hydrolysis of
compound 3 afforded 9a-hydroxyparthenolide and L-pro-
line amino acid, and this was confirmed through TLC,
NMR and the sign of its optical rotation. Therefore, com-
pound 3 was elucidated as 13-L-proline-9a-hydroxy-
parthenolide, and named garcinamine C.
Garcinamine C (3) was isolated as a yellow gum
[a]25D = -20 (c = 0.17, CH3OH). The positive ion mode
ESI-MS gave a molecular ion peak [M ? H]? at m/z 380
1
corresponding to molecular formula C20H30NO6. The H-
and 13C-NMR data were similar to those of 1and 2, and the
only difference was observed in an amino acid moiety at
C-13 carbon. In the 1H-NMR spectrum, the signals of the L-
proline amino acid group appeared at dH 3.48 (t,
J = 10.0 Hz, H-20), 2.10 and 1.86 (each m, H-30a, b), 1.82,
1.71 (each m, H-40a, b), 3.15 and 2.70 (each m, H-50a, b).
The 13C-NMR spectrum gave signals for the L-proline
moiety at dC 174.4 (C-10), 67.5 (C-20), 29.3 (C-30), 23.9 (C-
40) and 53.6 (C-50) (Tables 1, 2). The methylene protons
H-13a (3.12) and H-13b (3.0) showed 2J HMBC
Garcinamine D (4) was isolated as a yellow gum
[a]25D = -22 (c = 0.15, CH3OH). The positive ion
mode ESI-MS gave a quasi-molecular [M ? H]? peak at
m/z 380 corresponding to molecular formula C20H30NO6.
Detailed comparison of the NMR spectra of 4 with those
of 3 indicated that the only differences in chemical shifts
were C-9 oxymethine and C-14 methyl groups. According
to the literature, if the 9-hydroxy group has a-orientation
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J Nat Med
Table 2 13C-NMR data for compounds 1–4 (DMSO, d in ppm,
125 MHz)
Hepatoprotective activity
In the current investigation, hepatotoxicity was induced
using carbon tetrachloride, which converted in the endo-
plasmic reticulum into trichloromethyl free radical (CCl3)
and Cl3COO which, with the aid of oxygen, conjugated
with essential cellular macromolecules, cellular lipids and
proteins to induce lipid peroxidation [15]. Lipid peroxi-
dation forms reactive aldehydes that can form adducts with
proteins. These reactions increase the endoplasmic reticu-
lum’s and other membranes’ permeability to Ca2?, result-
ing in severe disturbances of calcium homeostasis and
consequently necrotic cell death [16]. Treatment of animals
with the hepatotoxic agent carbon tetrachloride resulted in
significant increase of transaminases (AST and ALT) and
alkaline phosphatase (ALP) levels that reflect the func-
tional status of the liver and consequently indicate hepa-
tocyte damage [17]. Severe jaundice was reflected by the
elevated levels of serum bilirubin (Table 3).
Position
1
dC
2
dC
3
dC
4
dC
1
120.7
22.9
120.6
23.4
36.1
61.3
66.4
81.9
36.6
36.8
70.5
137.8
47.6
177.6
46.0
16.5
17.5
159.0
68.3
30.9
19.6
18.8
–
121.1
23.4
36.1
61.6
66.1
82.1
36.6
35.7
70.5
137.6
45.3
177.2
51.4
16.4
17.3
174.4
67.5
29.3
23.9
53.6
–
123.7
23.6
36.4
61.7
65.7
81.8
42.5
38.0
78.3
138.1
46.4
177.0
51.9
11.2
17.3
174.3
67.5
29.2
24.2
53.9
–
2
3
36.1
4
61.3
5
66.1
6
82.2
7
35.5
8
36.8
9
70.4
10
11
12
13
14
15
10
20
30
40
50
60
70
80
90
138.3
46.8
177.6
45.5
16.2
17.3
The use of silymarin at a dose of 10 mg/kg (20.7 lmol/
kg) prior to the administration of CCl4 resulted in a sig-
nificant decrease (p \ 0.001) in the elevated AST, ALT,
gamma-glutamyl trans-peptidase (GGT), ALP and biliru-
bin levels in rats (Table 3). Silymarin acts as an antioxidant
by scavenging pro-oxidant free radicals and by increasing
the intracellular concentration of glutathione. It also
enhances the cellular membrane’s permeability to protect
against xenobiotic injury. Moreover, it promotes the syn-
thesis of ribosomal RNA by stimulating DNA polymerase-I
as well as having a steroid-like action in regulating DNA
transcription and stimulating protein synthesis leading to
regeneration of liver cells [18, 19].
174.0
63.4
38.2
138.1
130.0
128.7
126.8
128.7
130.0
–
–
–
–
–
–
–
–
–
then the C-9 carbon shows the signal at dC 70-72 ppm,
while in the case of the b-oriented 9-hydroxy group, the
C-9 carbon signal appeared at dC 78–79 ppm in the 13C-
NMR spectrum. The downfield 13C-NMR signal of C-9
carbon atom of 4 at dC 78.3 confirmed the b-orientation
of the hydroxyl group at position 9 [4]. The methyl group
at C-10 carbon atom was shifted from dC 16.4 to dC 11.2,
which further confirmed the b-orientation of the hydroxyl
group at C-9 position. Acid hydrolysis of compound 4
afforded 9b-hydroxyparthenolide and L-proline amino
acid, and this was confirmed through TLC, NMR and the
sign of its optical rotation. Therefore, compound 4 was
elucidated as 13-L-proline-9b-hydroxyparthenolide, and
named garcinamine D.
Rats supplemented with the total ethanol extract of A.
garcinii at 400 mg/kg counteracted the toxic effect of CCl4
and resulted in a significantly (p \ 0.01, 0.001) moderate
decrease in the elevated levels of AST, ALT, GGT, ALP
and bilirubin. The total extract at 400 mg/kg also signifi-
cantly reduced the concentration of malonaldehyde
(MDA). The level of total protein was significantly
(p \ 0.001) increased (Table 4). The fractions obtained
after liquid–liquid fractionation were also tested for their
hepatoprotective effect at 2 doses, 200 and 400 mg/kg. The
n-butanol fraction did not show any protective effect at the
two doses tested. However, the chloroform-soluble fraction
at the higher dose showed highly significant (p \ 0.001)
improvement in all the serum parameters, comparable to
the effect of the standard drug silymarin (Table 3). The
effect on restoring non-protein sulfhydryl groups (NP-SH)
(p \ 0.01) was equal to that of silymarin. Restoration of
the normal levels of MDA and total protein was highly
significant (p \ 0.01, 0.001) and close to that produced by
silymarin (Table 4).
The known compounds isolated from the chloroform
and n-butanol fractions were identified as parthenolide-9-
one (5), 9a-hydroxy-1b,10a-epoxyparthenolide (6), 9a-
hydroxyparthenolide (7), 9b-hydroxyparthenolide (8), spi-
nacetin-7-glucoside (9) [5], patuletin-7-glucoside (10) [13]
and rutin (11) [14] by comparing the NMR spectra with
published literatures.
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Table 4 Effect of A. garcinii
leaf extracts on MDA, NP-SH
and total protein in liver tissue
Treatments (n = 6)
Dose mg/kg MDA (nmol/g)
1.06 0.02
NP–SH (nmol/g) Total protein
(g/L)
Normal
4.12 0.16
123.75 3.36
CCl4
7.07 0.20***a 2.72 0.17***a
1.68 0.07***b 3.55 0.08**b
52.69 1.74***a
111.37 1.82***b
59.88 2.76b
72.25 2.17***b
82.23 2.94***b
100.99 2.08***b
54.29 1.35b
Silymarin ? CCl4
10
Total ethanol extract ? CCl4 200
Total ethanol extract ? CCl4 400
Chloroform fraction ? CCl4 200
Chloroform fraction ? CCl4 400
5.79 0.47*b
2.57 0.08b
4.70 0.21***b 2.85 0.20b
3.30 0.22***b 3.67 0.30*b
2.12 0.16***b 3.55 0.09**b
n-butanol fraction ? CCl4
n-butanol fraction ? CCl4
200
400
7.09 0.14b
2.56 0.15b
5.30 0.25***b 2.58 0.07b
63.87 1.18***b
All values represent mean SEM. * p\0.05, ** p\0.01, *** p\0.001; ANOVA, followed by Dunnett’s
multiple comparison test
a
As compared with control group
b
As compared with CCl4 group
Fig. 2 Hepatoprotective effect of A. garcinii leaf extract and fractions
The histopathological results in the liver of rats intoxi-
cated with carbon tetrachloride in the presence or absence
of A. garcinii extracts are depicted in Fig. 2. The
histopathology of the liver of rats treated with A. garcinii
total ethanol extract at 400 mg/kg showed little protection,
with multiple areas of necrosis, vacuolization of hepato-
cytes and fatty changes (Fig. 2e). The best protection was
achieved in the group supplemented with 400 mg/kg body
weight of the chloroform fraction, where only slight
necrosis and vacuolization of hepatocytes was observed
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J Nat Med
(Fig. 2g). Thus, the hepatoprotective effect of the chloro-
form fraction may be related to its phytoconstituents,
exemplified by the isolated sesquiterpene lactones. This is
in agreement with previous reports on the marked biolog-
ical activities of sesquiterpene lactones, including their
hepatoprotective effects [20–22]. The decrease in activity
of the n-butanol fraction may be related to its constituents,
with the amino acids coupled to the sequiterpene lactones.
with the literature: parthenolides have attracted particular
attention owing to their promising potential as anticancer
agents [23]. The cytotoxic effect of 6 on cervical and liver
carcinomas was even stronger than that of the vinblastine
positive standard. Although IC50 values for 6 and 7 were
comparable, 6 was shown to be a better cytotoxic com-
pound, which demonstrates the effect of the stereochem-
istry of 9-hydroxyparthenolide at the C-9 position on
cytotoxic activity. Additionally, it was proved that the
replacement of the 9-hydroxy group in the aglycone 7.
Thus, we could deduce that the glycosylation decreased the
cytotoxic activity of the sesquiterpene lactone.
Cytotoxic activity
The cytotoxic activities of total ethanol extract, fractions
and compounds isolated from A. garcinii were studied using
the chorionic villus sampling method and vinblastine sulfate
as a reference drug. Five human cancer cell lines were used
and the response parameter (IC50) was calculated for each
cell line. From the cytotoxicity results shown in Table 3, the
chloroform and the n-butanol fractions showed moderate to
significant cytotoxic activity. The chloroform fraction was
strongly selective on the hepatocellular carcinoma, with
IC50 of 5.80 lg/ml compared to vinblastine (IC50 3.48 lg/
ml). This further confirmed the strong hepatoprotective
activity of the chloroform fraction. Moreover, the chloro-
form and n-butanol fractions demonstrated significant
activity against colon, liver, cervical and lung carcinomas.
with IC50 values ranging from 10.6 to 19.8 lg/ml). The total
ethanol extract showed relatively weak cytotoxic activity
(IC50 ranging from 37.6 to 94.4 lg/ml), which might be due
to antagonistic action between the compounds (Table 5).
As for the compounds isolated, 6 and 7 demonstrated
significant cytotoxic effect against the five cell lines (IC50
ranging from 2.98 to 12.40 lg/ml). This is in agreement
Concerning the two epimers 3 and 4, the results demon-
strated the higher cytotoxic activity of 4 (IC50 ranging from
2.23 to 13.40 lg/ml), as well as the high selectivity of this
compound on cervical and liver carcinomas. It is worth
noting that the coupling of the sesquiterpene lactone with
amino acids affected the cytotoxic activity variably. On
comparing compounds 1 (coupled to L-phenylalanine), 2
(coupled to L-valine) and 3 (coupled to L-proline), it could be
seen that compound 3 with L-proline amino acid is the
strongest cytotoxic agent. Based on these in vitro results, we
are planning to investigate the in vivo efficacy of active
constituents of A. garcinii concerning cytotoxic activity.
Materials and methods
General
All the instruments used in the study are provided in the
supplementary material.
Table 5 In vitro cytotoxic activity (IC50 values) of A. garcinii extract, fractions and compounds on five human cancer cell lines
Sample conc. (lg/ml)
MCF-7
HCT-116
HepG-2
HeLa
A-549
Ethanol extract
93.90 9.50a
24.50 5.56a
27.70 4.96a
78.30 4.30a
183.0 18.40a
24.20 1.43a
13.40 1.06a
402.0 14.45a
85.32 1.56a
6.55 0.35
37.60 5.72a
10.60 0.89a
11.30 0.81a
14.25 0.93a
12.50 0.86a
11.70 1.54a
3.40 0.14c
211.0 2.56a
58.98 1.25a
3.97 1.52
39.80 12.4a
5.80 0.60b
12.20 1.04a
35.22 1.23a
28.0 1.07a
18.70 2.74a
3.49 0.28
388.0 9.10a
12.33 2.47a
3.01 0.55
61.80 3.55a
12.0 0.79b
48.80 4.35a
21.22 1.43a
24.90 0.07a
6.24 0.49
2.23 0.66a
403.0 10.8a
35.66 1.54a
2.98 0.42a
12.40 1.52b
6.54 0.39
94.40 9.19a
14.70 4.16c
19.80 6.28c
18.33 0.98c
19.80 2.97c
6.20 0.24b
3.77 0.44
178.0 20.4a
125.30 3.12a
3.70 0.24
CHCl3 fraction
n-Butanol fraction
1
2
3
4
5
6
7
8
6.70 1.00
5.54 0.24a
7.60 0.71a
4.97 1.16
Vinblastine sulfate
5.44 0.57
2.58 0.43
3.48 0.22
3.58 0.48
Bold values show strong IC50 of particular compound
* IC50: concentration of extract required to reduce cell survival by 50%
#
Mean of IC50 values standard deviation; mean of three assays
b
a
c
p \ 0.001, p \ 0.01, p \ 0.05 compared to reference drug
123

J Nat Med
Plant material
H2O–MeOH (7:3) was a binary mixture which was sepa-
rated through HPLC with a flow rate 1.0 ml/min using
H2O:MeOH (3:7) as eluent, to afford 10 (14 mg,
Rt16.8 min). Compound 11 (20 mg) was isolated by direct
precipitation of fractions B-3 and B-4.
The leaves of A. garcinii were collected in March 2014
from the new industrial area 17 km south-west of Al-Kharj
city and identified by taxonomist Dr. M. Atiqur Rahman,
College of Pharmacy, Medicinal, Aromatic and Poisonous
Plants Research Center, King Saud University. A voucher
specimen (PSAU-CPH-6-2014) has been deposited in the
herbarium of the College of Pharmacy, Prince Sattam Bin
Abdulaziz University.
Garcinamine A (1)
Yellow gummy solid; [a]25D = –27.2 (c 0.10, CH3OH);
(-) HRESIMS m/z 428.2068 [M-H]- (calcd. for
C24H30NO6, 428.2073); 1H- and 13C-NMR data, see
Tables 1 and 2.
Extraction and isolation
The air-dried leaves (1.0 kg) were powdered and extracted
three times with a mixture of ethanol–water in 8:2 ratio at
30 °C, and then filtered. The filtrate was evaporated in
vacuo to give a dark brown residue (115 g), which was
suspended in water and fractionated with chloroform (35 g)
and n-butanol (65 g). A part of the chloroform fraction
(25 g) was loaded on silica gel column and eluted with n-
hexane, n-hexane–ethyl acetate and with pure ethyl acetate
to yield three sub-fractions, C-1, C-2, and C-3. The sub-
fraction C-1 (1.0 g) which was eluted with n-hexane–ethyl
acetate (9.0:1.0) was further subjected to silica gel columns
eluted with n-hexane–ethyl acetate (8.0:2.0) as eluent to
afford 5 (10 mg). The sub-fraction C-2 (0.5 g) which was
eluted with n-hexane–ethyl acetate (8.0:2.0) was a mixture
of two components. It was further subjected to preparative
TLC over silica gel plates using n-hexane–ethyl acetate
(7.0:3.0) as eluent to afford 6 (28 mg) and 7 (30 mg). The
sub-fraction C-3 was subjected to silica gel columns eluted
with n-hexane–ethyl acetate (7.0:3.0) as eluent to afford 8
(8 mg).
Garcinamine B (2)
Yellow gummy solid; [a]25D = –22.5 (c 0.15, CH3OH);
(?) HRESIMS m/z 382.2224 [M ? H]? (calcd. for
C20H32NO6, 382.2230); 1H- and 13C-NMR data see
Tables 1 and 2.
Garcinamine C (3)
Yellow gummy solid; [a]25D = –29.3 (c 0.15, CH3OH);
(?) HRESIMS m/z 380.2065 [M ? H]? (calcd. for
C20H30NO6, 380.2073); 1H- and 13C-NMR data, see
Tables 1 and 2.
Garcinamine D (4)
Yellow gummy solid; [a]25D = –25.6 (c 0.10, CH3OH);
(?) HRESIMS m/z 380.2048 [M ? H]? (calcd. for
C20H30NO6, 380.2073); 1H- and 13C-NMR data, see
Tables 1 and 2.
A part of the n-butanol fraction (45 g) was subjected to
Sephadex column and eluted with a mixture of water and
methanol (10% methanol ? 100% methanol) as mobile
phases, and pooled in four major sub-fractions B1–B4.
Fraction B-1 (0.5 g) was applied to medium pressure RP-
18 column and eluted with a mixture of water and methanol
(9:1) to yield 1 (10 mg). Fraction B-2 (1.0 g) was subjected
to Sephadex LH-20 column eluted with water and metha-
nol (8:2) to obtain two sub-fractions B-2a and B-2b. Sub-
fraction B-2a (100 mg) was further purified by HPLC (flow
rate 3 ml/min; wavelength 375 nm; CH3OH–0.01%TFA:
H2O, 1:1) to afford 2 (12 mg, Rt 13.4 min). Sub-fraction
B-2b (150 mg) was purified by HPLC (flow rate 1.0 ml/
min; wavelength 375 nm; CH3OH–0.01%TFA: H2O, 1:1)
to afford 3 (12 mg, Rt 12.7 min) and 4 (15 mg, Rt 13.1).
Fraction B-3 (500 mg) was rechromatographed on Sepha-
dex LH-20 (flow rate 1.0 ml/min) and eluted with H2O–
MeOH (7.5:2.5) to afford compound 9 (20.0 mg, Rt
18.5 min). The sub-fraction B-4 (1.5 g) obtained from
Acid hydrolysis of compounds 1–4
Methanol solutions of compounds 1–4 (each 5 mg) con-
taining 1N HCl were refluxed for 1 h. After completion of
the reaction, the mixture was concentrated and diluted with
water and extracted with ethyl acetate. The ethyl acetate
extract was subjected to silica gel column chromatography
and on elution with CHCl3:MeOH (9:1) it gave 9a-hy-
droxyparthenolide from compounds 1, 2 and 3, while 9b-
hydroxyparthenolide was obtained from compound 4. The
water extract was concentrated and subjected to RP-C18
column chromatography (water:methanol 7:3) and the
fractions gave positive ninhydrin tests to confirm the
presence of amino acid. The amino acid was L-phenylala-
nine from compound 1, L-valine from compound 2 and L-
proline from compounds 3 and 4, and these amino acids
were identified from NMR and their signs of optical rota-
tions by comparison with standards [12].
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J Nat Med
Animals
Determination of NP-SH, MDA and total protein
Male Wistar albino rats (150–200 g) of about the same
age (8–10 weeks) were obtained from the Experimental
Animal Care Center, College of Pharmacy, King Saud
University. The animals were housed under optimum
conditions of humidity, temperature and light/dark con-
ditions, and were allowed access to Purina chow (Lab-
Diet, St. Louis, MO, USA) and drinking water ad libitum.
The procedures used in this study were approved by the
Ethical Committee of the College of Pharmacy, Prince
Sattam Bin Abdulaziz University, based on the existing
international regulations for the handling and care of
laboratory animals.
The liver samples were separately cooled in a beaker
immersed in an ice bath. The tissues were homogenized in
0.02 M ethylene-diamine tetra-acetic acid (EDTA) in a Pot-
ter–Elvehjem type C homogenizer (Sigma-Aldrich). Homo-
genates equivalent to 100 mg tissues were used for the
measurements. Non-protein sulfhydryl groups (NP-SH) were
quantified as reported previously, while concentrations of
MDA were determined according to the procedure of Utley
etal. [25].ForTPdetermination,partsofthehomogenatewere
treated with 0.7 ml of Lowry’s solution, mixed and incubated
for 20 min in the dark at room temperature. Diluted Folin
reagent (0.1 ml) were then added and samples were incubated
at room temperature in the dark for 30 min. The absorbance of
the resulting solutions was then measured at 750 nm.
Chemicals
Histopathology
Silymarin, EDTA, Mayer hematoxylin, eosin-phloxine,
Folin reagent, trichloroacetic acid, 5,50-dithio-bis-(2-ni-
trobenzoic acid), 2-thiobarbituric acid and paraffin were
obtained from Sigma-Aldrich, St. Louis, MO, USA. The
solvents used in the investigation were of analytical
grade.
The fixed liver samples were put in cassettes and loaded
into an automated vacuum tissue processor (ASP 300 S,
Leica Biosystems, Nussloch, Germany). The samples were
blocked in paraffin wax and thin sections (3 lm) were cut
using a microtome (TBS SHUR/cut 4500, Triangle
Biomedical Sciences, Durham, NC, USA). The sections
obtained were stained with Mayer’s hematoxylin solution
and counterstained in eosin–phloxine solution [26]. Slides
were examined using Slide Scanner (SCN 400 F Leica
Microsystems, Wetzlar, Germany) for microscope imaging
using objective magnifications of 109 and 409.
Hepatoprotective activity
Male Wistar rats were divided into nine groups of five
animals each. Group I was the control group, taking normal
saline. Groups II–IX were administered a single dose of
CCl4 (1.25 ml/kg body weight). Group II received only
CCl4 treatment, while Group III was administered sily-
marin at a dose of 10 mg/kg per os (20.7 lmol/kg). Groups
IV–IX were treated with two doses: 200 and 400 mg/kg of
the total ethanol extract, CHCl3 and n-butanol fractions,
respectively. In each group, treatment started 5 days before
CCl4 administration and continued till day six. After 24 h
of CCl4 administration, the animals were killed using ether
anesthesia. Blood samples were obtained by heart puncture
and the serum was separated for determining the different
biochemical parameters.
Statistical analysis
Results are expressed as mean standard error (SE) of
mean. Statistical analysis was performed using one-way
analysis of variance (ANOVA). When the F value was
found to be statistically significant (p \ 0.05), further
comparisons between groups were made using Dunnett’s
multiple comparison test. All statistical analyses were
performed using SPSS software v.17.0 (released Aug. 23,
2008), Chicago, IL, USA.
Determination of biochemical enzymatic parameters
AST, ALT, GGT, ALP and bilirubin
Cytotoxicity assay
Cell culture
The biochemical parameters, aspartate aminotransferase
(AST), alanine aminotransferase (ALT), gamma-glutamyl
trans-peptidase (GGT), alkaline phosphatase (ALP) and
total bilirubin were determined according to the previously
reported methods of Edwards and Bouchier [24]. The
enzyme activities were measured using diagnostic strips
(Reflotron, Roche, Basel, Switzerland) and were read on a
ReflotronÒ Plus instrument (Roche).
Human cell lines MCF-7 cells (breast cancer cell line),
HepG-2 (hepatocellular carcinoma), A-549 (human lung
carcinoma), HELA cells (human cervical carcinoma) and
HCT-116 (colon carcinoma) were obtained from The
Holding Company for Biological Products and Vaccines
(VACSERA) Tissue Culture Unit. The cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) with
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J Nat Med
10% heat-inactivated fetal bovine serum, HEPES buffer,
1% L-glutamine and 50 lg/ml gentamycin. The cells were
kept at 37 °C under humidified condition with 5% CO2 and
were subcultured twice a week.
traditionally by local people for the treatment of many
diseases including liver problems. Our results justified the
use of the plant for improving liver aliments. This is the
first report to confirm the use of A. garcinii as a hepato-
protective traditional medicine. The chloroform and the n-
butanol fractions showed moderate to significant cytotoxic
activity. The chloroform fraction was strongly selective on
hepatocellular carcinoma compared to vinblastine refer-
ence drug. Moreover, the chloroform, n-butanol fractions
and compounds 3, 4, 7 and 8 demonstrated significant
activity against colon, hepatocellular, cervical and lung
carcinomas.
Evaluation of cytotoxicity using viability assay
The cells were seeded in 96-well plates at a concentration
of 1 9 104 cells per well in 100 ll of growth medium.
Different concentrations of the test sample were added
after 24 h of seeding in fresh medium. Serial two-fold
dilutions of the tested sample were added to confluent cell
monolayers dispensed into 96-well microtiter plates (Fal-
con, NJ, USA). The microtiter plates were incubated at
37 °C with 5% CO2 for 48 h. Three wells were used for
each concentration of the test sample. Control cells were
incubated without test sample and with or without DMSO.
By the end of the incubation period, media were aspirated
and crystal violet solution (1%) was added to each well for
30 min. The plates were rinsed using distilled water. Gla-
cial acetic acid (30%) was then added to all wells and
mixed thoroughly. Colorimetric evaluation of fixed cells
was carried out by absorbance measurements in an auto-
matic microplate reader (TECAN, Inc.) at 490 nm. Treated
samples were compared with the cell control in the absence
of the tested samples. All experiments were performed in
triplicate. The optical density was measured using the
microplate reader to determine the number of viable cells.
The percentage of viability was calculated as [1 - (ODt/
ODc)] 9 100%, where ODt is the mean optical density of
wells treated with the tested sample and ODc is the mean
optical density of untreated cells, and was illustrated as a
dose–response curve. The 50% inhibitory concentration
(IC50) was estimated from graphic plots of the dose–re-
sponse curve using Graphpad Prism software (San Diego,
CA. USA). The reference antitumor drug used was vin-
blastine sulfate [27].
Acknowledgements This research project was supported by a grant
from the ‘‘Research Center of the Female Scientific and Medical
Colleges’’, Deanship of Scientific Research, King Saud University.
Compliance with ethical standards
Conflict of interest The authors declare they have no conflicts of
interest.
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