Triterpene saponins from Chenopodium quinoa Willd.

Article abstract of DOI:10.1016/j.phytochem.2008.03.001

Twenty triterpene saponins (1-20) have been isolated from different parts of Chenopodium quinoa (flowers, fruits, seed coats, and seeds) and their structures have been elucidated by analysis of chemical and spectroscopic data including 1D- and 2D-NMR. Four compounds (1-4) were identified: 3β-[(O-β-d-glucopyranosyl-(1 → 3)-α-l-arabinopyranosyl)oxy]-23-oxo-olean-12-en-28-oic acid β-d-glucopyranoside (1), 3β-[(O-β-d-glucopyranosyl-(1 → 3)-α-l-arabinopyranosyl)oxy]-27-oxo-olean-12-en-28-oic acid β-d-glucopyranoside (2), 3-O-α-l-arabinopyranosyl serjanic acid 28-O-β-d-glucopyranosyl ester (3), and 3-O-β-d-glucuronopyranosyl serjanic acid 28-O-β-d-glucopyranosyl ester (4). The following known compounds have not previously been reported as saponin constituents from the flowers and the fruits of this plant: two bidesmosides of serjanic acid (5, 6), four bidesmosides of oleanolic acid (7-10), five bidesmosides of phytolaccagenic acid (11-15), four bidesmosides of hederagenin (16-19), and one bidesmoside of 3β,23,30-trihydroxy olean-12-en-28-oic acid (20). The cytotoxicity of these saponins and their aglycones was tested in HeLa cells. Induction of apoptosis in Caco-2 cells by bidesmosidic saponins 1-4 and their aglycones I-III was determined by flow cytometric DNA analysis. The saponins with an aldehyde group were most active. The relationships between structure and cytotoxic activity of saponins and their aglycones are discussed.

Full text of DOI:10.1016/j.phytochem.2008.03.001

Phytochemistry 69 (2008) 1919–1926
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpene saponins from Chenopodium quinoa Willd.
Tiwatt Kuljanabhagavad a,d, Piyanut Thongphasuk b,c, Walee Chamulitrat c, Michael Wink d,
*
a Chemistry Program, Faculty of Science and Technology, Suan Dusit Rajabhat University, Bangkok 10300, Thailand
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Rangsit University, Pathumtani 12000, Thailand
c Internal Medicine IV (Gastroenterology and Infectious Diseases), University Hospital Heidelberg, D-69120 Heidelberg, Germany
d Department of Biology, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty triterpene saponins (1–20) have been isolated from different parts of Chenopodium quinoa
(flowers, fruits, seed coats, and seeds) and their structures have been elucidated by analysis of chemical
Received 26 October 2007
Received in revised form 26 February 2008
Available online 29 April 2008
and spectroscopic data including 1D- and 2D-NMR. Four compounds (1–4) were identified: 3b-[(O-b-
glucopyranosyl-(1 ? 3)-a- -arabinopyranosyl)oxy]-23-oxo-olean-12-en-28-oic acid b- -glucopyranoside
(1), 3b-[(O-b- -glucopyranosyl-(1 ? 3)-a- -arabinopyranosyl)oxy]-27-oxo-olean-12-en-28-oic acid
b- -glucopyranoside (2), 3-O-a- -glucopyranosyl ester (3), and
3-O-b-
D-
L
D
D
L
Keywords:
D
L-arabinopyranosyl serjanic acid 28-O-b-D
Chenopodium quinoa
Bidesmosidic saponins
Cytotoxicity
Apoptosis
Chemotaxonomy
Structure–activity relationship
D-glucuronopyranosyl serjanic acid 28-O-b-D-glucopyranosyl ester (4). The following known
compounds have not previously been reported as saponin constituents from the flowers and the fruits
of this plant: two bidesmosides of serjanic acid (5,6), four bidesmosides of oleanolic acid (7–10), five
bidesmosides of phytolaccagenic acid (11–15), four bidesmosides of hederagenin (16–19), and one bides-
moside of 3b,23,30-trihydroxy olean-12-en-28-oic acid (20). The cytotoxicity of these saponins and their
aglycones was tested in HeLa cells. Induction of apoptosis in Caco-2 cells by bidesmosidic saponins 1–4
and their aglycones I–III was determined by flow cytometric DNA analysis. The saponins with an alde-
hyde group were most active. The relationships between structure and cytotoxic activity of saponins
and their aglycones are discussed.
Ó 2008 Published by Elsevier Ltd.
1. Introduction
michael and Wink, 2001), toxicity to brine shrimps (Meyer et al.,
1990), and against viral diseases, cholesterol-lowering effects,
Chenopodium quinoa Willd. (Chenopodiaceae), commonly
known as quinoa, is a well-known staple food of the Andean com-
munities serving as an alternative food source in other regions
(Brinegar and Goundan, 1993; Delatorre-Herrera, 2003; Dini
et al., 2005). The seed contains high-quality protein and is particu-
larly rich in essential amino acids and its carbohydrate content has
been reported to have a higher nutritional value than that of cere-
als such as corn, oats, wheat, and rice (Ruales and Nair, 1992;
Lindeboon, 2005). More importantly, quinoa seed has more Ca,
Fe, Mn, Mg, Cu, and K than other cereals (Ruales and Nair, 1993;
Konishi et al., 2004). Quinoa seeds are a potential crop for many
animal feed formulations, for direct feeding, and as a human food
(Takao et al., 2005). The seeds are used in the same way as rice
and wheat and are ground into flour to prepare breads, cakes,
and fermented drinks (Ando et al., 2002; Bhargava et al., 2006).
However, the main disadvantage has been the bitterness related
to the saponins present (Brady et al., 2007).
and enhance drug absorption through mucosal membranes. They
act as immunological and absorption adjuvants to enhance anti-
gen-specific antibody and mucosal responses (Estrada et al.,
1998). Saponins from Chenopodium quinoa are a complex mixture
of triterpene glycosides that are derivatives of oleanolic acid,
hederagenin, phytolaccagenic acid, serjanic acid, and 3b,23,30-tri-
hydroxy olean-12-en-28-oic acid, which bear hydroxyl and carbox-
ylate groups at C-3 and C-28, respectively, and are based on
b-amyrin. The major carbohydrates are arabinose, glucose, and gal-
actose; while glucuronic acid and xylose have been found to be less
common (Cuadrado et al., 1995; Dini et al., 2001a,b, 2002; Mizui
et al., 1988, 1990; Madl et al., 2006; Mastebroek et al., 2000;
Woldemichael, 2000; Zhu et al., 2002). This study was centered
on the isolation, structure elucidation, and biological activities of
triterpene saponins and their aglycones in this plant. Herein, we
report the isolation of twenty glycosides of 3b-hydroxy-23-oxo-
olean-12-en-28-oic acid (1), 3b-hydroxy-27-oxo-olean-12-en-28-oic
acid (2), serjanic acid (3–6), oleanolic acid (7–10), phytolaccagenic
acid (11–15), hederagenin (16–19), and 3b,23,30-trihydroxy olean-
12-en-28-oic acid (20). Flowers, fruits, seed coats, and seeds were
investigated. For the first time we report results from flowers and
fruits. A derivative of 3b-hydroxy-27-oxo-olean-12-en-28-oic acid
Previous investigations on saponins isolated from seeds of C.
quinoa have shown that they exert antimicrobial activity (Wolde-
* Corresponding author. Tel.: +49 62 21544880; fax: +49 62 21544884.
E-mail address: wink@uni-hd.de (M. Wink).
0031-9422/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.phytochem.2008.03.001

1920
T. Kuljanabhagavad et al. / Phytochemistry 69 (2008) 1919–1926
is described for the first time in the Chenopodiaceae. The occur-
rence of 3b-hydroxy-27-oxo-olean-12-en-28-oic acid might repre-
sent an interesting intermediate in the biosynthesis of these
substances. Several reports showed that sugar moiety and type of
linkage are also directly related to the structure–activity of sapo-
nins (Takechi et al., 1996; Woldemichael and Wink, 2001; Voutqu-
enne et al., 2002). However, data in the literature for comparative
structure–activity relationship studies in apoptosis induction and
cytotoxicity are limited (Chwalek et al., 2006). The present paper
reports on the cytotoxicity and apoptosis-inducing activity of sap-
onins 1–4 and their aglycones I–III.
sponded to the carbon signals at d 106.54,106.24, and 95.80,
respectively. The sugars were identified as two units of glucose,
and one unit of arabinose by authentic samples and a detailed study
of COSY, HSQC, and HMBC spectra. The b-anomeric configurations
of the glucose and arabinose units were deduced from their
3JH1,H2 coupling constants (7.4–8.0 Hz) (Woldemichael and Wink,
2001). The sequence of the sugar connected to the C-3 of the agly-
cone was deduced from the HMBC correlations of anomeric H-1 (d
4.95) of the arabinose moiety with carbon signal at d 81.98 (C-3)
and 11.72 (C-24), indicating the attachment of arabinose at C-3.
The anomeric H-1 (d 5.30) of the glucose moiety exhibited HMBC
correlation with d 84.01 (C-3) of the arabinose moiety, indicating
connectivity between C-1 and C-3. Subsequently, the glucose sugar
2. Results and discussion
substituted at C-3 of a-L-arabinoside was also inferred from the
analysis of chemical shifts data in the literature (Jiang et al., 2006;
Yoshimitsu et al., 2007). The presence of a second monodesmosidic
moiety at C-28 was deduced by the HMBC interactions of anomeric
H-1 of glucose (d 6.29) with C-28 carbonyl (dC 176.13). The C-23
aldehyde proton (d 9.36) showed HMBC interactions with carbon
signals at d 11.72 (C-24), 53.01 (C-4), and 81.98 (C-3). The 1H
NMR subspectra of individual monosaccharide units were obtained
by using selective irradiation of the easily identifiable anomeric
proton signals, as well as irradiations of other non-overlapping pro-
ton signals in a series of COSY and HSQCAD experiments (Table 2).
Basic hydrolysis of 1 gave the prosapogenin; acid hydrolysis of the
resulting prosapogenin then yielded arabinose. Acid hydrolysis of 1
gave the aglycone 3b-hydroxy-23-oxo-olean-12-en-28-oic acid
along with arabinose and glucose. On the basis of the above
evidence, compound 1 was a new compound, determined as
Preliminary TLC comparison of saponin fractions of the four dif-
ferent plant parts of C. quinoa revealed similar results in each case.
The crude saponins obtained from seed coats (10.7% yield), seeds
(8% yield), flowers (4% yield), and fruits (4% yield) were fractionated
by a combination of silica gel open column chromatography to
afford 20 compounds (1–20) in a pure form as confirmed by HPLC
analysis. Sixteen of these were identified as 3-O-b-
syl-(1 ? 2)-b- -glucopyranosyl-(1 ? 3)-a- -arabinopyranosyl serj-
anic acid 28-O-b- -glucopyranosyl ester (5) (Dini et al., 2001b),
3-O-b- -glucopyranosyl-(1 ? 3)-a- -arabinopyranosyl serjanic acid
28-O-b- -glucopyranosyl ester (6) (Dini et al., 2002), quinoside D
(7) (Mizui et al., 1990), quinoa saponin 7 (8) (Mizui et al., 1990),
3-O-b- -glucopyranosyl-(1 ? 3)-a- -arabinopyranosyl oleanolic
acid 28-O-b- -glucopyranosyl ester (9) (Dini et al., 2001a), chikuse-
D-glucopyrano-
D
L
D
D
L
D
D
L
D
tsusaponin IVa (10) (Mizui et al., 1990), quinoa saponin 4 (11)
(Woldemichael, 2000), quinoa saponin 3 (12) (Woldemichael,
2000), quinoa saponin 5 (13) (Mizui et al., 1988), quinoa saponin
3b-[(O-b-
D
-glucopyranosyl-(1 ? 3)-a-L-arabinopyranosyl)oxy]-23-
oxo-olean-12-en-28-oic acid b-
D-glucopyranoside.
Compound 2 was isolated as a white powder. It gave a positive
Liebermann–Burchard test. The molecular formula of 2 was estab-
lished as C47H74O18Na by positive-mode ESI-MS (m/z 949.5). Its
HR-ESI-MS gave a peak at 949.4671 [M+Na]+ (calculated for
8 (14) (Mizui et al., 1990), 3-O-b-
glucopyranosyl-(1 ? 4)-O-b- -glucopyranosyl
acid 28-O-b- -glucopyranosyl ester (15) (Dini et al., 2001b), quinoa
D
-glucopyranosyl-(1 ? 4)-O-b-
D-
D
phytolaccagenic
D
47H74O18Na, 949.4772). The 1H NMR spectrum of compound 2
saponin 2 (16) (Mizui et al., 1988), Hedera nepalensis saponin F (17)
(Mizui et al., 1988), quinoa saponin 9 (18) (Mizui et al., 1990),
C
showed signals for six methyl groups at d 1.08, 0.97, 0.85, 1.09,
0.74, and 0.84 assigned to Me-23, Me-24, Me-25, Me-26, Me-29,
and Me-30 of the triterpene moiety of the oleanane-type (Seebach-
er et al., 2003), respectively. These signals were correlated with
their corresponding carbon signals at d 32.01, 28.05, 16.22, 16.73,
32.92, and 23.92 in the HSQC spectrum. The 1H and 13C NMR spectra
of 2 exhibited three anomeric protons resonated as doublets at d
4.95 (J = 7.3 Hz), 5.30 (J = 7.8 Hz), and 6.29 (J = 8.0 Hz), which corre-
sponded to the carbon signals at 106.61, 106.01, and 95.74, respec-
tively. The sugars were identified as two units of glucose and one
unit of arabinose by comparison with authentic samples. The b-
anomeric configurations of the glucose and arabinose units were
quinoa saponin 1 (19) (Mizui et al., 1988), and 3-O-b-
osyl-(1 ? 3)-a- -arabinopyranosyl 3b,23,30-trihydroxy olean-12-
en-28-oic acid 28-O-b- -glucopyranosyl ester (20) (Madl et al.,
D-glucopyran-
L
D
2006), by comparison of physical data with literature values and
from spectroscopic evidence. The chemical structures of the identi-
fied saponins 1–20 are depicted in Fig. 1.
Compound 1 was isolated as a white powder. It gave a positive
Liebermann–Burchard test. The molecular formula of 1 was estab-
lished as
[M+Na]+). Its HR-ESI-MS gave
(calculated for C47H74O18Na, 949.4772). The 1H NMR spectrum of
(Table 1) shows signals for six tertiary methyl groups
C
47H74O18Na by positive-mode ESI-MS (m/z 949.5
a
peak at 949.4698 [M+Na]+
3
1
deduced from their JH1,H2 coupling constants (7.4–8.0 Hz) (Table
(d0.80,0.91,0.92,0.98,1.08, and 1.15), an olefinic broad singlet (d
5.40, H-12), and an oxymethine proton at d 3.99, dd, J = 11.8 and
4.0 Hz, H-3. These signals indicated a pentacyclic skeleton in 1.
The 1H NMR spectrum further showed double doublets at d 3.20
(J = 13.0 and 2.6 Hz) attributed to H-18, a characteristic signal of
the oleanane-type skeleton (Seebacher et al., 2003). A singlet for
an aldehyde proton was resonated at d 9.39 in the 1H NMR spec-
trum. The 13C NMR spectrum (Table 1) of compound 1 showed sig-
nals corresponding to 47 carbons, resolved into 6 methyl, 13
methylene, 20 methine, and 8 quaternary carbons. Among them,
17 signals were assigned to the oligosaccharide moiety. The 13C
NMR spectrum of 1 showed olefinic carbon signals at dC 123.40
(C-12) and 143.81 (C-13). The chemical shifts of C-3 (d 81.98) and
C-28 (d 176.13) indicated a bisdesmosidic glycoside (Woldemichael
and Wink, 2001; Sahu and Achari, 2001). The 1H and 13C NMR spec-
tra of 1 exhibited three anomeric protons resonated as doublets at d
4.95 (J = 7.3 Hz), 5.30 (J = 7.8 Hz), and 6.29 (J = 8.0 Hz), which corre-
2) (Bouguet-Bonnet et al., 2002; Woldemichael and Wink, 2001).
Compound 2 exhibited a triplet for the olefinic proton H-12 which
appeared at low field position d 5.45 in the 1H NMR spectrum, and
signals for double bonds at d 123.40 and 143.81 in the 13C NMR
spectrum (Table 1). Two carbonyl signals, resonating at d 176.12
and 209.01 were also shown in the 13C NMR spectrum and assigned
to a 28-glycosyl ester carbonyl and aldehyde group, respectively.
The NMR spectra of 2 contained one singlet at d 2.14, suggesting
that one of the methyl groups of oleanolic acid was replaced by
an aldehyde group. With respect to this type of triterpene, the sig-
nals from C-13 are shifted upfield by 7.3 ppm; the C-12 appeared at
5.1 ppm because of higher deshielding in compound 2. This obser-
vation indicates that the aldehyde function is in close proximity of
the double bond and located at C-14 instead of Me-27. The glycosyl
ester function was proposed to be at C-28 in view of the 13C NMR
spectrum, which showed d values of 23.43, 46.72, and 33.62 for
C-16, C-17, and C-22, respectively. In the HMBC spectrum the pro-

T. Kuljanabhagavad et al. / Phytochemistry 69 (2008) 1919–1926
1921
29
30
20
21
22
28
COO
19
18
I
IV
12
26
11
9
13
17
16
HO
O
HO
O
25
10
COO
14
15
OH
OH
OH
OH
1
8
2
3
27
5
7
HO
HO
HO
HO
4
6
RO
RO
RO
RO
24
23
CHO
II
COOCH3
V
HO
HO
O
COO
COO
COO
OH
OH
OH
OH
O
CHO
CH2OH
COOCH3
III
VI
HO
O
HO
COO
OH
OH
OH
OH
O
HO
HO
RO
RO
CH2OH
CH2OH
COO
VII
HO
O
OH
OH
HO
RO
CH2OH
Compound
Aglycone
I
R
Flower
Fruit
Seed
Seed coat
+
+
+
+
→
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
β-D-Glc(1 3)-α-L-Ara
β-D-Glc(1→3)-α-L-Ara
α-L-Ara
+
+
+
+ +
+ +
+ +
+
+
+ +
+ +
+ +
+ +
+
II
+
+
+
+
III
III
III
III
IV
IV
IV
IV
V
V
V
V
V
β-D-GlcA
+
+
β-D-Glc(1 2)-β-D-Glc(1 3)-α-L-Ara
β-D-Glc(1→3)-α-L-Ara
β-D-Xyl(1 3)-β-D-GlcA
β-D-Glc(1 2)-β-D-Glc(1 3)-α-L-Ara
β-D-Glc(1 3)-α-L-Ara
→
→
+
+
+
+
+
→
→
→
+
+
+
+
→
+
+
+
+
+
+
+ +
+ + + +
+ + + +
+ +
+
+ +
+ + + +
+ + + +
+ +
+ +
+ +
+
β-D-GlcA
+ + + +
+ + +
+ +
+ +
+ +
+
+ + + +
+ + +
+ +
+ +
+ +
+
β-D-Glc(1→3)-α-L-Ara
α-L-Ara
β-D-Glc(1 3)-β-D-Gal
β-D-Glc(1 2)-β-D-Glc(1 3)-α-L-Ara
β-D-Glc(1→4)-β-D-Glc(1→4)-β-D-Glc
β-D-Glc(1→3)-β-D-Gal
α-L-Ara
→
→
→
+
+
VI
VI
VI
VI
VII
+
+
+
+
+ +
+ + +
+
+ +
+ + +
+
+ +
+ +
+ +
+ +
+ + +
+ + + +
β-D-GlcA
→
β-D-Glc(1 3)-α-L-Ara
β-D-Glc(1→3)-α-L-Ara
(+) trace quantities, (+ +) low quantities, (+ + +) moderate quantities, (+ + + +) high quantities
Fig. 1. Structures of saponins 1–20 (aglycones I–VII, 3b-hydroxy-23-oxo-olean-12-en-28-oic acid, I; 3b-hydroxy-27-oxo-olean-12-en-28-oic acid, II; serjanic acid, III;
oleanolic acid, IV; phytolaccagenic acid, V; hederagenin, VI; 3b,23,30-trihydroxy olean-12-en-28-oic acid, VII).
ton signal at d 2.14 showed long-range correlation with the car-
bonyl aldehyde at d 209.01 indicating that Me-27 was oxidized to
CHO. The anomeric H-1 (d 5.30) of the glucose moiety exhibited
HMBC correlation with d 84.32 (C-3) of the arabinose moiety, point-
ing to a connectivity between C-1 and C-3. Subsequently, the glu-
prosapogenin; acid hydrolysis of the resulting prosapogenin
then yielded arabinose. Acid hydrolysis of 2 gave the aglycone 3b-
hydroxy-27-oxo-olean-12-en-28-oic acid and the arabinose and
glucose. Thus the structure of 2 proved to be the new compound
3b-[(O-b-
D-glucopyranosyl-(1 ? 3)-a-
L-arabinopyranosyl)oxy]-27-
cose sugar substituted at C-3 of a-
L
-arabinoside was also inferred
oxo-olean-12-en-28-oic acid b-D-glucopyranoside.
from the analysis of chemical shifts data in the literature (Jiang
et al., 2006; Yoshimitsu et al., 2007). The shift values of C-3 at d
88.38 and C-28 at d 176.12 indicated that 2 is 3,28-bisdesmoside
(Woldemichael and Wink, 2001). Basic hydrolysis of 2 gave the
Compound 3 was isolated as a white powder. It gave a positive
Liebermann–Burchard test. The molecular formula of 3 was estab-
lished as C42H66O14Na by positive-mode ESI-MS (m/z 817.5). Its
HR-ESI-MS gave a peak at 817.4352 [M+Na]+ (calculated for

1922
T. Kuljanabhagavad et al. / Phytochemistry 69 (2008) 1919–1926
Table 1
13C NMR spectroscopic assignments (d) of the aglycone and monosaccharide moieties of saponins 1–4a
Aglycone
Monosaccharide
C
1
2
3
4
C
1
2
3
4
C-3
1
2
3
4
Arabinose
106.54
71.92
84.01
69.22
Arabinose
106.61
72.10
84.32
69.34
Arabinose
105.93
73.89
75.49
70.45
Glucuronic acid
107.55
80.01
76.60
75.22
1
2
3
4
5
6
7
8
36.32
23.29
81.98
53.01
46.82
18.63
30.62
38.45
46.02
34.32
24.14
123.40
143.81
40.21
26.20
21.50
48.11
40.02
44.53
28.72
32.01
30.42
210.74
11.72
13.17
15.15
23.89
176.13
31.12
21.84
38.49
26.38
88.38
39.40
55.87
18.32
35.72
41.91
49.74
37.40
24.14
123.40
143.81
59.14
21.33
23.43
46.72
46.71
42.22
43.63
30.62
33.62
32.01
28.05
16.22
16.73
209.01
176.12
32.92
23.92
38.52
26.51
87.22
39.53
56.01
18.47
33.53
39.72
47.51
37.01
23.33
123.12
144.22
42.04
28.25
23.91
46.23
42.62
43.34
44.22
30.74
34.55
28.42
17.54
15.45
16.92
26.04
177.24
28.52
180.26
51.78
38.51
26.50
87.21
39.53
56.02
18.41
33.52
39.74
47.55
37.06
23.37
123.12
144.24
42.15
28.12
23.84
46.15
42.52
43.25
44.12
30.65
34.42
28.34
17.86
15.42
16.89
26.15
177.24
28.39
180.26
51.77
5
6
67.49
67.12
67.68
76.21
172.34
Glucose
106.24
74.78
78.41
71.13
78.29
62.34
Glucose
95.80
74.84
78.30
72.10
78.61
62.52
Glucose
106.01
76.45
78.21
71.73
78.03
62.71
Glucose
95.74
74.00
78.78
71.11
78.13
69.58
1
2
3
4
5
6
C-28
1
2
3
4
5
6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
–COOCH3
Glucose
95.89
75.01
80.21
71.57
79.67
62.78
Glucose
95.71
74.12
79.30
71.12
78.84
62.21
a
Chemical shifts were referenced according to the highest field signals of pyridine-d5 (8.71 ppm for 1H and 149.9 ppm for 13C).
two cases was at d 177.24–180.26, confirming the presence of a
methyl ester at this position. Acid hydrolysis of 3 afforded serjanic
acid, glucose, and arabinose. The sugars were identified by compar-
ison with authentic samples. The NMR data comparison between 3
with 6, which was reported by Dini et al. (2002), indicated that
both 1H and 13C NMR signals were in good agreement with those
of serjanic acid. The sequence of the sugar connected to the C-3
of the aglycone was deduced from the HMBC correlations of ano-
meric H-1 (d 4.90) of the arabinose moiety with a carbon signal
at d 87.22 (C-3), indicating the attachment of arabinose at C-3.
The presence of a second monodesmosidic moiety at C-28 was de-
duced by the HMBC interactions of anomeric H-1 of glucose (d
6.29) with the C-28 carbonyl (dC 180.26). Thus, the structure of 3
Table 2
1H MR chemical shift data (dH, J, Hz) of the monosaccharide moieties of saponins 1–4a
1
2
3
4
C-3
1
2
3
4
Arabinose
4.95 d(7.3)
4.59
Arabinose
4.95 d(7.3)
4.59
Arabinose
4.90 d(7.7)
4.59
Glucuronic acid
5.14 d(7.0)
4.13
4.08
4.08
4.08
4.35
4.41
4.40
4.41
4.61
5
6
3.62, 4.21
3.62, 4.21
3.62, 4.21
4.67
5.14 d(7.0)
Glucose
5.30 d(7.8)
4.02
Glucose
5.30 d(7.8)
4.00
1
2
3
4
5
6
C-28
1
2
4.24
4.22
4.23
4.21
was shown to be the new compound 3-O-a-L-arabinopyranosyl
4.23
4.22
serjanic acid 28-O-b- -glucopyranosyl ester.
D
4.37, 4.29
Glucose
6.29 d(8.0)
4.17
4.52
Compound 4 was isolated as a white powder. It also gave a po-
sitive Liebermann–Burchard test. The molecular formula of 4 was
established as C43H66O16Na by positive-mode ESI-MS (m/z 861.5).
Its HR-ESI-MS gave a peak at 861.4243 [M+Na]+ (calculated for
Glucose
6.29 d(8.0)
4.16
Glucose
6.29 d(8.0)
4.17
Glucose
6.29 d(8.0)
4.22
3
4
5
3.97
3.96
3.97
4.04
4.38
4.35
4.38
4.36
C43H66O16Na, 861.4249). The presence of a peak at m/z 501
4.28
4.27
4.28
4.28
6
4.42, 2H
4.42, 2H
4.42, 2H
4.44, 2H
[M+H–176–162–Na]+ corresponds most probably to loss of one
glucuronic acid and one glucose molecule from m/z 861. The ion
at m/z 686 [M+H–176]+ corresponds to a loss of glucuronic acid
in the positive-mode ESI-MS. On acid hydrolysis, 4 afforded serjan-
ic acid and sugars that were identified as glucuronic acid and glu-
cose by comparing with authentic samples. In addition, the
anomeric 13C signals at d 107.55 and 95.71 and their corresponding
protons at d 5.14 (1H, d, J = 7.0 Hz) and 6.29 (1H, d, J = 8.0 Hz),
respectively, were strongly indicative of two hexoses. The COSY
and HSQCAD spectra allowed identification of the spin systems of
the two monosaccharide residues (Table 2). The linkage sites of the
monosaccharide moieties were first inferred from the hydrolysis
a
Chemical shifts were referenced according to the highest field signals of pyri-
dine-d5 (8.71 ppm for 1H and 149.9 ppm for 13C).
C42H66O14Na, 817.4351). The presence of a peak at m/z 501 [M+H–
162–132–Na]+ corresponds most probably to the loss of one
glucose and one arabinose molecule from m/z 817. The ion at m/z
656 [M+H–162]+ corresponds to a loss of glucose in the positive-
mode ESI-MS. On acidic hydrolysis, the aglycone moiety of
compound 3 gave signals in the 1H and 13C NMR spectra, which
corresponded to serjanic acid (Table 1). The signal for C-30 in

T. Kuljanabhagavad et al. / Phytochemistry 69 (2008) 1919–1926
1923
experiment. This was indicative of C-28 esterification of the glu-
cose moiety and the C-3 linkage of glucuronic acid. Confirmation
of this came from the HMBC spectrum by which JCH correlations
were observed between C-1 (d 107.55) and H-3 (d 3.40) of glucu-
ronic acid and between C-28 (d 177.24) and H-1 (d 6.29) of glucose.
The relative stereochemistry of both monosaccharides was deter-
mined as b-D-glucuronopyranose and b-D-glucopyranose on the ba-
sis of the characteristic J1,2 coupling constants (Woldemichael and
Wink, 2001). Thus the structure of 4 proved to be the new com-
To elucidate whether cell growth inhibitory effects of bidesmos-
idic saponins and their aglycones were associated with apoptosis,
we performed DNA analysis for hypoploid sub-G1 peaks in cells
treated with 100 lg/ml bidesmosidic saponins 1–4 and their agly-
cones I–III for 24 h. Apoptosis levels induced by saponins 1–4 were
13.18%, 13.18%, 25.50%, and 26.40%, respectively, and those in-
duced by their aglycones I–III were 51.40%, 51.40%, and 50.23%,
respectively. Observed apoptotic effects on Caco-2 cells were in
agreement with the cytotoxicity assay (Table 3). The bidesmosidic
saponins 1–4 have an IC50 ꢀ 100 lg/ml or 100 lM and showed
weak apoptosis at ꢀ13–26%; while the aglycones I–III with a lower
IC50 ꢀ 25–50 lg/ml showed higher apoptosis at ꢀ50%. Our com-
pounds exhibited relative high IC50 ꢀ 100 lM, which were compa-
rable to that reported for silybin (Varghese et al., 2005). However,
silybin even with relatively high in vitro IC50 has been shown to be
effective in inhibiting tumor formation in vivo (Varghese et al.,
2005). On the other hand, some natural products such as an alka-
loid emetine can be highly cytotoxic exhibited IC50’s of 0.17 and
0.52 lM in jurkat T-cells and healthy human PBMC, respectively
(Möller and Wink, 2007). Bidesmosidic saponins can not interfere
with biomembrane and are therefore usually nontoxic, except if
they have further functional groups. Monodesmosidic saponins
can dive into biomembrane with their lipophilics side chain and
complex cholesterol, whereas their hydrophilic sugar chain binds
to extracellular glycoproteins and glycolipids. Aglycones are not
active on biomembrane, but can be active towards other targets,
e.g. proteins if they have reactive functional groups such as alde-
hyde function. Our results indicate significant differences in struc-
tures–activity relationship of the isolated bidesmosidic saponins
depending on nature and position of functional group within struc-
ture of the aglycones.
3
pound 3-O-b-D-glucuronopyranosyl serjanic acid 28-O-b-D-gluco-
pyranosyl ester.
Cytotoxic activities of isolated compounds 1–4 and their agly-
cones: 3b-hydroxy-23-oxo-olean-12-en-28-oic acid (I), 3b-hydro-
xy-27-oxo-olean-12-en-28-oic acid (II), and serjanic acid (III)
were evaluated in HeLa cells employing the MTT assay (Sladowski
et al., 1993). Cytotoxicity of the isolated compounds, expressed as
IC50 is summarized in Table 3.
The cytotoxic effects of saponins 1 and 2 were very similar
(IC50 > 100 lg/ml). These compounds have same molecular weight
and both bear the 1 ? 3 linkage of sugars at C-3 and one glucose
unit at C-28; the aglycones of saponins 1 and 2 are different how-
ever, being 3b-hydroxy-23-oxo-olean-12-en-28-oic acid (I) and
3b-hydroxy-27-oxo-olean-12-en-28-oic acid (II), both having the
same IC50 of 25.4 lg/ml. It appears that the CHO groups at C-23
or C-27 are correlated with the obtained increased cytotoxicity.
They can form Schiff’s bases with amino groups of protons and
DNA bases. A similar result was observed by Woldemichael and
Wink (2001) in a study of the structure–activity relationships of
saponins and their prosapogenins. Hederagenin, with an IC50 of
15–23 lg/ml has shown to be more potent than oleanolic acid with
an IC50 of 62–99 lg/ml, suggesting that the substitution of a
hydroxymethylene group at C-23 enhances the activity of the ole-
anolic acid. Indeed, a number of monodesmosidic saponins of plant
3. Conclusion
origin have shown potent cytotoxic activity, such as 3-O-a-
rhamnopyranosyl-(1 ? 2)-a- -arabinopyranosyl hederagenin (IC50
6.3–8.7 lg/ml), no activity has been observed when a sugar is at-
tached at C-28, as in the case of b- -Glup-(1 ? 6)-b- -Glup (Hung
L-
L
An interesting feature of the saponins isolated from this plant is
the presence of the aldehyde group at the C-23 and C-27 of the cor-
responding aglycones of saponins 1 and 2, respectively. These
sapogenins were reported for the first time in aerial parts of Clima-
coptera obtusifolia (Chenopodiaceae) (Yeskaliyeva et al., 2006) and
of Fagonia cretica (Zygophyllaceae) (Khalik et al., 2000). It is inter-
esting to note that the reported serjanic acid also occurs in Phyto-
laccaceae (Caryophyllales) (Balsevich et al., 2006) and Sapindaceae
(Sapindales) (Larhsini et al., 2003). Moreover, two new saponins
with aglycones, 3b-hydroxy-23-oxo-olean-12-en-28-oic acid (I)
and 3b-hydroxy-27-oxo-olean-12-en-28-oic acid (II), both having
an aldehyde function in the b-amyrin skeleton, have been found
in the families Caryophyllaceae (Caryophyllales) and Zygophylla-
ceae (Sapindales), (Yeskaliyeva et al., 2006; Khalik et al., 2000).
This metabolite might represent an intermediate in Chenopodium
saponin oxidative biosynthetic steps leading from a methyl group
to the corresponding aldehyde function. The following aglycone
moieties identified in C. quinoa are representative of oxidative
products at C-23 from alcohol to aldehyde and C-27 from methyl
to aldehyde: 3b-hydroxy-23-oxo-olean-12-en-28-oic acid (I), and
3b-hydroxy-27-oxo-olean-12-en-28-oic acid (II, Fig. 1). Phy-
tolaccagenic acid (V) may originate from serjanic acid (III) by sub-
sequent oxidative enzymatic steps involving the formation of the
corresponding alcohol substituted at C-23. Additionally, hederage-
nin (VI) might originate from oleanolic acid (IV) by a selective oxi-
dative demethylation at C-23 and, in turn, hederagenin can be a
substrate for the production of 3b,23,30-trihydroxy olean-12-en-
28-oic acid (VII), following a mechanism involving a stereochemi-
cally specific enzyme able to insert one hydroxyl group into the
30b position of the triterpene skeleton. Although these mecha-
nisms must be demonstrated, the suggested hypothesis can give
D
D
et al., 2005). Moreover, the presence of a free carboxylic acid at
C-28 seems to be essential for cytotoxicity (Bang et al., 2005). How-
ever, cytotoxic effects of aglycones I and II, both with an IC50 of
25.4 lg/ml, were closely comparable to that of hederagenin (VI).
The cytotoxic effects of bidesmosidic saponins 3 and 4 were
very similar (IC50 > 100 lg/ml). These compounds have the same
sapogenin (III; serjanic acid, IC50 50 lg/ml), but a different sugar
unit at C-3. Compound 3 contains a sugar at C-3, arabinose, but 4
has glucuronic acid in this position. Both compounds have glucose
linked to C-28 and the same aglycone. Oleanolic acid has been
shown to be non-cytotoxic (IC50 > 50 lg/ml) in HL-60 cells (Mimaki
et al., 1999). This indicates that the substitution of the 30-O-
methyl of spergulagenic acid does not increase cytotoxicity of the
aglycone, as compared with oleanolic acid (IV).
Table 3
Cytotoxicity of saponins 1–4 and their sapogenins I–III in HeLa cells as compared to
apoptosis induce in Caco-2 cells
Compound
IC50 (lg/ml)
Sub-G1 population (%)
1
2
3
4
I
>100
>100
>100
>100
25.4
25.4
50.5
13.18
13.18
25.50
26.40
51.40
51.40
50.23
II
III
Serial compound dilutions (0.195–100 lg/ml) were incubated with HeLa cells for
24 h and cell viability was evaluated by MTT reduction to the blue-colored for-
mazan in living cells. The compounds activity was compared with the untreated
samples (control). All values were means of triplicates.

1924
T. Kuljanabhagavad et al. / Phytochemistry 69 (2008) 1919–1926
an idea of the biosynthesis of these substances. Most of the identi-
fied saponins from C. quinoa have arabinose, glucose, galactose, and
glucuronic acid as the first sugar at C-3 and, when present, glucose
at C-28. This suggests a high enzymatic selectivity for the sugar po-
sition independent of the sapogenins involved.
were partitioned between EtOAc (to yield 3 g and 2 g) and n-BuOH
(to yield 16 g and 4 g), respectively.
A batch of 140 g of powdered fruits was extracted with MeOH
by exhaustive maceration (3 Â 2.5 l) to yield 9 g of each residue.
The methanolic extracts of fruits were dissolved in H2O. The H2O
extract was partitioned between EtOAc and n-BuOH to yield EtOAc
0.5 g and n-BuOH 4 g. Ten grams of n-BuOH of seed coats, ten
grams of n-BuOH of seeds, four grams of n-BuOH of flowers, and
four grams of n-BuOH of fruits were dissolved in n-BuOH saturated
with water and submitted to a column chromatography (column
400 Â 35 mm) with 40–60 lm of silica gel, Merck. Ten milliliter
fractions were collected and checked by TLC on silica gel (Merck),
developed with CHCl3–EtOH–H2O (4:2:0.4 v/v/v), respectively.
Chromatograms were sprayed with Liebermann–Burchard reagent
and heated to 120 °C in an oven for 3 min. Approximately, 400 frac-
tions at 10 ml each, were collected for each plant part by fraction
collector eluted with CHCl3–MeOH–H2O (7.5:2.3:0.2 ? 4:5:1 v/v/
v) used as solvent system. Fractions showing similar profiles were
combined. Column chromatography gave 12 fractions for each
plant part, each fraction containing two to four major saponins.
Single saponins were separated from each fraction by silica gel
Lichrospher RP-18 eluted with acetonitrile–water (20–64%). In this
way 20 (1–20) pure compounds were isolated. Individual saponins
were separated from the fractions by means of reversed-phase
chromatography on a 400 Â 25 mm, 40–63 lm, Lichrospher 100,
RP-18 column (Merck), eluted with aqueous CH3CN. Fraction I
eluted with 20% CH3CN afforded compound 1 (8 mg) and com-
pound 2 (10 mg); further elution with 25% CH3CN gave compound
3 (8 mg) and 4 (6 mg). Fraction II eluted with 25% CH3CN yielded
compound 5 (5 mg) and 6 (12 mg). After elution with 30% CH3CN,
compound 7 (15 mg) and compound 8 (2 mg) were obtained. Frac-
tion III eluted with 30% CH3CN gave compound 9 (63 mg) and com-
pound 10 (5 mg); 30% CH3CN allowed compound 11 (9 mg) to be
obtained. Fraction IV eluted with 25% CH3CN gave compound 12
(6 mg), and elution with 30% CH3CN gave compound 13 (8 mg),
compound 14 (12 mg), compound 15 (10 mg), compound 16
(7 mg), compound 17 (3 mg), compound 18 (4 mg), compound 19
(4 mg), and then compound 20 (12 mg).
4. Experimental
4.1. General experimental procedures
1H and 13C NMR were measured on a Varian 500 MHz AC NMR
spectrometer at the operating frequencies of 500.13 and
125.67 MHz, respectively. The saponins were examined as solutions
in pyridine-d5 in 5-mm tubes at 25 °C. Two-dimensional (2D) NMR
experiments (H,H DQF-COSY; H,H TOSY; H,C HSQC; H,C HSQCAD;
H,C HMBC) were carried out on all compounds using the pulse se-
quences from the Varian user library. On the basis of 2D-NMR anal-
yses, assignments of 1H and 13C signals were obtained. High
performance liquid chromatography was carried out on a Knauer
Eurochrom 2000 HPLC system with a Lichrospher 100, RP-18
(Merck), column of 250 Â 4 mm or 250 Â 8 mm i.d. Peaks were de-
tected at 206 nm with a linear gradient of 20–64% CH3CN in 0.1% tri-
fluoroacetic acid in water at flow rate 1 ml/min during 30 min
(Woldemichael and Wink, 2001). The LC-ESI-MS system consisted
of an HPLC from Latek Laborgeräte GmbH (Heidelberg, Germany)
coupled with a Micromass VG Quattro II mass spectrometer from
Waters (Manchester, United Kingdom). ESI-MS was operated under
Waters MassLynx 4.0 Software. Mass spectrometric detection of so-
dium adducts of triterpene glycosides was performed in the positive
ion mode over the range of 400–1400 m/z and chromatograms were
processed using Waters MassLynx 4.0 software. HR-ESI was mea-
sured on a Bruker ApexQe FT-ICR-Instrument (Bruker Daltonik
GmbH, Bremen). Column chromatography was performed on silica
gel (Kieselgel 70–230 mesh and 230–400 mesh, Merck), and thin
layer chromatography (TLC) was performed on pre-coated silica
gel 60 F254 (0.25 mm, Merck). Spray reagents were for the saponins:
Liebermann–Burchard reagent and 10% H2SO4 in methanol reagent
and heated at 120 °C in an oven for 3 min; for the sugars: aniline
phthalate reagent and heated at 100 °C in an oven for 5 min. For
cytotoxicity using a Microplate Reader (Tecan Ultra, Tecan Deutsch-
land GmbH, Germany). All reagents used were of analytical grades.
4.4. Basic hydrolysis of saponins 1–4
Each individual pure compound (2 mg) was heated at reflux
with 10 ml of 5% KOH solution in water for 2 h. The cooled reaction
mixture was adjusted to pH 6 by 1 N HCl and then extracted with
EtOAc (3 Â 10 ml). The saponins were submitted to acid hydrolysis
with 2 N HCl in 50% aqueous methanol for 2 h. The aglycones in the
EtOAc layer were evaporated to dryness, 1.46 mg of each pure
aglycone was obtained (73% yield), dissolved in pyridine-d5 in a
3-mm tube, and analyzed by 1H NMR and 13C NMR. The aqueous
layer was evaporated to dryness and analyzed by TLC on silica
gel in the solvent CHCl3–MeOH–H2O (70:40:5 v/v/v). Glucuronic
acid, Rf 0.15, and arabinose, Rf 0.40, were identified by comparison
with an authentic samples.
4.2. Plant material
Powdered seed coats, powdered seeds, and powdered flowers of
C. quinoa were obtained from Avelup (Temuco, Chile). Entire fruits
of C. quinoa were obtained from three-month-old plants grown in
greenhouses at the Botanical Garden, University of Heidelberg,
Germany. Plants were harvested in mid-June 2005 after the begin-
ning of the fruiting season and transported to the laboratory. The
fruits were ground and air-dried, finely powdered and then used
for extraction. All samples were stored at À20 °C until analysis.
4.3. Extraction and isolation
4.5. Acid hydrolysis of saponins 1–4
A total of 300 g of powdered seeds were defatted at room tem-
perature with petroleum ether and extracted with MeOH by
exhaustive maceration (3 Â 2.5 l) to yield 32 g of residue which
was successively dissolved in water and partitioned with EtOAc
and n-BuOH to yield 2.5 g and 14 g of concentrate, respectively.
Also, 300 g of powdered seed coat, and 200 g of powdered flowers
were extracted with MeOH by exhaustive maceration (3 Â 2.5 l) to
yield 36 g, and 12 g of each residue, respectively. The methanolic
extracts of seed coats (36 g) and flowers (12 g) were dissolved in
H2O, respectively. The H2O extracts of each different plant part
The individual pure compounds (2 mg) were hydrolyzed with
10 ml of 2 N HCl in 50% aqueous methanol under reflux for 2 h at
100 °C, and thereafter sugars and aglycones were separated and
the aqueous solution was extracted with EtOAc (3 Â 10 ml). The
aglycones in the EtOAc layer were evaporated to dryness, 1.5 mg
of each pure aglycone was obtained (75% yield), dissolved in pyri-
dine-d5 in a 3-mm tube, and analyzed by 1H NMR and 13C NMR.
The aqueous layer was evaporated to dryness and analyzed by
TLC. The presence of glucuronic acid, glucose and arabinose was
established by comparison with authentic samples. The TLC on sil-

T. Kuljanabhagavad et al. / Phytochemistry 69 (2008) 1919–1926
1925
ica gel in the solvent system CHCl3–MeOH–H2O (70:40:5 v/v/v) re-
4.9. Statistical analysis
sulted in the Rf 0.15, 0.26, and 0.40 respectively.
To obtain a quantity of the aglycone of saponin 1,2 and 3, two
grams of the n-BuOH fraction of seed coats was hydrolyzed with
400 ml of the acidic solution underreflux for 6 h at 100 °C, and there-
after sugars and aglycones were separated and the aqueous solution
was extracted with EtOAc three times (3 Â 100 ml). The aglycones in
the EtOAc layer were evaporated to dryness (1420 mg), applied to
silica gel column chromatography with CHCl3–MeOH (9:1 v/v) as a
solvent system to give 3b-hydroxy-23-oxo-olean-12-en-28-oic acid
(I; 8 mg) and 3b-hydroxy-27-oxo-olean-12-en-28-oic acid (II;
15 mg). The mixtures of phytolaccagenic acid and serjanic acid (frac-
tions 17–49; 240 mg) was rechromatographed on silica gel with
CHCl3–MeOH (9:1 v/v) and the residue was recrystallized from
CH2Cl2–n-hexane giving serjanic acid (III; 20 mg) and phytolacca-
genic acid (V; 45 mg). All of the aglycones were characterized by
comparison of their 1H and 13C NMR spectral with published data
(Bandara et al., 1990). The substances were used as standard com-
pounds for biological activity tests.
Statistical analysis was performed by Student’s t test. P-values
less than 0.05 was considered to be statistically significant. The
IC50 values were calculated using Excel based program.
Acknowledgements
This study was financially supported by Suan Dusit Rajabhat
University. We are most grateful to Dr. Ingrid von Baer for collec-
tion and identification of the plant material, Dr. Jeannette Ndaya
Tshibangu for technical assistance with HPLC, Dr. Melanie Distl
for technical assistance with LC–MS, Dr. Walter Kramer for NOESY
technique NMR analysis, Mr. Tobias Timmermann for technical
assistance in recording NMR spectra, and Mr. Florian Herrmann
for his valuable suggestion for the cytotoxic assays. We also thank
the anonymous reviewers for their valuable comments.
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4.6. Cell culture and treatment protocols
The experiment was performed according to the method re-
ported by Thongphasuk et al. (2008). Briefly, the human colon ade-
nocarcinoma cell line Caco-2 cells were cultured in DMEM
containing 10% FCS, 1% P/S, 1% L-glutamine, and 1% nonessential
amino acids in a 5%-CO2 incubator at 37 °C. For cytotoxicity stud-
ies, cells were seeded at 50 Â 103 cells per well in 24-well plates.
The medium was replaced with serum-free medium containing
various concentrations of selected saponins or 0.1% DMSO control.
After 24 h of treatment, cells were harvested.
4.7. DNA analysis of apoptosis by flow cytometry
The harvested cells were washed with PBS and then pelleted by
centrifugation. Cells were fixed with cold 80% ethanol and kept at
4 °C overnight. After washing with PBS and centrifugation, cells
were treated with 100 lg/ml RNase A and incubated at 37 °C for
30 min. Following addition of propidium iodide (PI, 50 lg/ml),
DNA contents were analyzed by a BD FACSCalibur with CellQuest
software (Becton Dickinson) for acquisition and data analysis. Cells
gated as % sub-G1 represent cell populations exhibiting apoptosis.
TM
4.8. Cytotoxic activity
Dini, I., Schettino, O., Simioli, T., Dini, A., 2001a. Studies on the constituents of
Chenopodium quinoa seeds: isolation and characterization of new triterpene
saponins. J. Agric. Food Chem. 49, 741–746.
Cytotoxic effects were measured in vitro on HeLa (cervix adeno-
carcinoma) cell line, using MTT assay (Sladowski et al., 1993) with
some modification. The cytotoxicity tests were carried out in 96-
well plates, using 1 Â 104 cells per well in all cases, which were al-
lowed to adhere to the cell culture plate for 48 h before treatment.
The supernatant was removed and 100 ll of serially diluted com-
pounds (0.195–100 mg/ml), 100 ll [Dulbecco’s Modified Eagle’s
Medium (DMEM) 500 ml, penicillin–streptomycin 5 ml, NEA 10%
5 ml, FBS 5 ml] were added to each well. After 24 h of incubation,
the culture medium was carefully removed, and 20 ll of 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
solution (5 mg/ml) was added to each well. The plates were incu-
bated at 37 °C for 4 h. The medium was then removed and the
resulting formazan crystals were dissolved with 100 lL DMSO.
Each sample was prepared in triplicate. The final DMSO concentra-
tion was adjusted to <0.1%. The plate was mixed on a microshaker
for 10 min and then the samples was measured at 570 nm using
Microplate Reader (Tecan). For each compound, the percent viabil-
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