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Identification of Flavonoids and Bufadienolides and Cytotoxic Effects of

Kalanchoe daigremontiana Extracts on Human Cancer Cell Lines

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DOI: 10.1055/a-1099-9786

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Identification of Flavonoids and

Bufadienolides and Cytotoxic Effects
of Kalanchoe daigremontiana Extracts
on Human Cancer Cell Lines

DOI 10.1055/a-1099-9786
Planta Med

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 Original Papers
Identification of Flavonoids and Bufadienolides and Cytotoxic Effects
of Kalanchoe daigremontiana Extracts on Human Cancer Cell Lines
Authors
Justyna Stefanowicz-Hajduk 1, Monika Asztemborska 2, Mirosława Krauze-Baranowska 3, Sylwia Godlewska 3,
Magdalena Gucwa 1, Barbara Moniuszko-Szajwaj 4, Anna Stochmal 4, J. Renata Ochocka 1
Affiliations
1 Department of Biology and Pharmaceutical Botany,
Medical University of Gdańsk, Gdańsk, Poland
2 Institute of Physical Chemistry of the Polish Academy of
Sciences, Warsaw, Poland
3 Department of Pharmacognosy, Medical University of
Gdańsk, Gdańsk, Poland
4 Department of Biochemistry and Crop Quality, Institute of
Soil Science and Plant Cultivation, State Research Institute,
Puławy, Poland
Key words
Kalanchoe daigremontiana, bufadienolides, bersaldegenin,
flavonoids, HPLC, RTCA
received August 22, 2019
revised December 30, 2019
accepted January 10, 2020
Bibliography
DOI https://doi.org/10.1055/a-1099-9786
published online | Planta Med © Georg Thieme Verlag KG
Stuttgart · New York | ISSN 0032‑0943
Correspondence
Dr. Justyna Stefanowicz-Hajduk
Department of Biology and Pharmaceutical Botany,
Medical University of Gdańsk
Al. Hallera 107, 80-416 Gdańsk, Poland
Phone: + 48 5 83 49 14 09, Fax: + 48 5 83 49 13 29
justyna.stefanowicz-hajduk@gumed.edu.pl
Supporting information available online at
http://www.thieme-connect.de/products
Introduction
Genus Kalanchoe (Bryophyllum) belonging to the Crassulaceae
family is widely distributed in tropical and subtropical areas of
Africa, Asia, and America. The genus consists of approximately
150 species of plants that have been used for the local treatment
of many diseases (e.g., periodontal and skin injuries, ear infec-
tions, coughs, gastric ulcers, and arthritis) [1–6]. Crude extracts
Stefanowicz-Hajduk J et al. Identification of Flavonoids … Planta Med
AB STR AC T
Kalanchoe species are well-known medicinal plants used in
traditional medicine as anti-inflammatory and analgesic rem-
edies. Recently, it has been reported that Kalanchoe plants
have cytotoxic properties; however, data on traditional use of
these plants in tumor treatment are extremely limited. Kalan-
choe daigremontiana is one of the most popular species culti-
vated in Europe, and it is used, among other things, as a rem-
edy in treating skin injuries and wounds. Studies on the bio-
logical activity of this species are scarce, and there is a lack of
data on the cytotoxic activity of K. daigremontiana extracts on
epithelial cancer cells in the literature. In our present study,
we analyzed the phytochemical composition of K. daigre-
Electronic reprint for personal use
montiana ethanol extract and fractions–water and dichloro-
methane–by the HPLC‑DAD‑ESI‑MS method and estimated
cytotoxic activity of the extracts on human adenocarcinoma
(HeLa), ovarian (SKOV-3), breast (MCF-7), and melanoma
(A375) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide) assay, real-time cell analyzer
(RTCA), and flow cytometry. We identified 6 bufadienolide
compounds and 19 flavonoids, mostly kaempferol, quercetin,
isorhamnetin, and myricetin glycosides, of which only 3 flavo-
noids have been identified in K. daigremontiana to date. Other
flavonoids that were characterized in our study have not yet
been found in this plant. The ethanol extract and water frac-
tion of K. daigremontiana did not show significant cytotoxic
activity on the tested cell lines. In contrast, the dichlorometh-
ane fraction showed the strongest activity against all cell lines
with IC50 values of ≤ 10 µg/mL. The results indicated that this
activity is mainly due to the presence of bersaldegenin-1,3,5-
orthoacetate.
or juice of Kalanchoe plant species also exhibit anticancer proper-
ties; however, in the field of ethnomedicine, only K. brasiliensis has
been described in the literature as a remedy for treating human
prostate cancer [7].
Kalanchoe daigremontiana (Bryophyllum daigremontianum)
Raym.-Hamet & H. Perrier is one of the more well-known plant
species of Kalanchoe. The plant comes from Madagascar, and it is
named “Mother of Thousands”. In Europe, the species is culti-
 Original Papers
▶ Table 1 Chromatographic data (tR, UV, and MS) of bufadienolides identified in the dichloromethane extract of Kalanchoe daigremontiana.
Peak number tR min UV λmax
B1 30.22 299
B2 32.98 294
B3 35.28 300
B4 37.37 299
B5 41.89 299
B6 42.48 299
*tR values can be interchangeable
vated mainly for its ornamental value and medicinal applications
in the household treatment of skin irritation and wounds [2, 8].
Kalanchoe daigremontiana has anti-inflammatory, antimicro-
bial, antioxidant, and cytotoxic activities [9–12]. However, the ef-
fect of extracts of this plant on cancer cells was tested only on hu-
man lymphoblastic leukemia T cells (H9 and J45 cell lines) with es-
timation of inhibitory concentration values [11].
The different activities of K. daigremontiana depend on its phy-
tochemical composition, and its analysis has shown that flavo-
noids, phenolic acids, and bufadienolides are the main metabo-
lites [8, 11, 13–15]. To date, only 4 flavonoid compounds of K. dai-
gremontiana have been reported in the literature [8, 16].
Electronic reprint for personal use
In the present study, we conducted a qualitative phytochemi-
cal analysis of the composition of K. daigremontiana extract and
2 fractions–dichloromethane and water obtained from the plant
leaves–together with estimation of their cytotoxic activities on
4 human cancer cell lines–cervical adenocarcinoma (HeLa), breast
(MCF-7), ovarian (SKOV-3), and malignant melanoma (A375). We
also evaluated the activity of one of K. daigremontiana bufadieno-
lide compounds (bersaldegenin-1,3,5-orthoacetate) that can be
responsible for the cytotoxic effect of the plant. To the best of
our knowledge, no study has reported the cytotoxic activity of
K. daigremontiana extracts and this compound against these types
of tumor cells.
Results and Discussion
The HPLC‑DAD‑ESI‑MS method revealed significant differences
between the chemical composition of dichloromethane extract
and 2 other extracts (water and ethanol). The dichloromethane
extract was rich in bufadienolides and contained 6 compounds
(B-1 to B-6) identified according to the obtained ultraviolet (UV)
and electrospray ionization mass spectrometry (ESI‑MS) spectra
(▶ Table 1) [2, 14]. All compounds are well-known constituents
of K. daigremontiana leaves [14]. Among them, only 1 bufadieno-
lide (methyl daigremonate, B-5) was also detected as a compo-
nent of ethanol extract.
In both water and ethanol extracts, 19 compounds belonging
to flavonoids (F-1 to F-19) (▶ Table 2) were identified. The pres-
ence of only 1 compound F-9 (quercetin 3-O-glucoside) was con-
firmed by comparison of its tR values with that of the standard
compound. This flavonoid compound was previously described in
the flowers of K. blossfeldiana [1, 17]. All other flavonoids were
m/z [M + H]+ Compound
473 bryophyllin A
475 bryophyllin C*
475 bersaldegenin-3-orthoacetate*
487 daigremontianin
503 methyl daigremonate
457 bersaldegenin-1,3,5-orthoacetate
identified only on the basis of the obtained UV and ESI‑MS spectra
(▶ Table 2) and their comparison with literature data [1, 8, 17, 18].
Most of these compounds belong to the derivatives of kaempfer-
ol, namely compounds F-2, F-12, F-15, F-16, F-18, and F-19. Three
compounds were classified as quercetin derivatives (F-1, F-9, and F-
14), 3 as myricetin (F-3, F-4, and F-6), 3 as isorhamnetin (F-8, F-13,
and F-17), and 1 as patuletin or 8-methoxyquercetin (F-10). In con-
trast to the obtained UV spectra of kaempferol glycosides, in the UV
spectra of derivatives of quercetin, isorhamnetin, and patuletin (or
8-methoxyquercetin), apart from maximum absorption I (between
252 and 256 nm), a shoulder was present (between 264 and
267 nm), thus confirming the presence of the o-dihydroxy group in
the side phenyl group [18]. Among the separated and identified
compounds by HPLC‑DAD‑ESI‑MS, 3 were previously reported to be
isolated from the leaves of K. daigremontiana, namely kaempferol 3-
O-xylosyl-rhamnoside-7-O-glucopyranoside [8] (F-2), quercetin 3-
O-xylosyl-rhamnoside [17] (F-14) and kaempferol 3-O-xylosyl-
rhamnoside [8] (F-18). Moreover, in the analyzed plant material, an-
other compound, namely kaempferol 3-O-pentosyl-rhamnoside,
was identified as it contains arabinose (F-16) instead of xylose moi-
ety (F-18). According to the elution order of both compounds F-16
and F-18, which possess the same molecular weight ([M + H]+ at
565+ m/z) and described by Nielsen, et al. [17], the peak eluted with
lower tR value of 35.16 min was assigned to kaempferol 3-O-arabi-
nosyl-rhamnoside. Considering the literature data on the presence
of kaempferol and isorhamnetin glycosides containing rhamnose
and/or acetyl-rhamnose in the Kalanchoe species [1], the following
flavonoids were identified as kaempferol 3,7-O-dirhamnoside
(kaempferitrin) (F-12), kaempferol O-hexoside-acetyl-rhamnoside
(F-19), isorhamnetin O-hexoside-rhamnoside (F-8), and isorhamne-
tin O-pentoside-rhamnoside (F-17). With regard to the presence in
the ESI‑MS spectra of compound F-13, molecular ions were detected
at 479 m/z [M + H]+ and 477 m/z [M – H]− and fragment ion at 317
m/z [Ag + H]+; accordingly, this compound was identified as iso-
rhamnetin O-hexoside. To date, only isorhamnetin 3-O-rhamnoside
has been identified in the leaves of Bryophyllum pinnatum from the
Crassulaceae family [1]. By analyzing the obtained UV and MS spec-
tra of compound F-1 eluted with tR value of 16.56 min, it was found
that this compound might be quercetin triglycoside, with a structure
similar to that of kaempferol triglyceride (F-2) eluted approximately
3 min further (▶ Table 2). Considering the values of m/z of molecular
ions [M – H]− observed in the MS spectra of compounds F-3, F-4, and
F-6 at 625 m/z, 479 m/z, and 463 m/z, respectively, their preliminary
Stefanowicz-Hajduk J et al. Identification of Flavonoids … Planta Med
 ▶ Table 2 Chromatographic data (tR, UV, and MS) of compounds–flavonoids identified in the water and ethanol extracts of Kalanchoe daigremon-
tiana.

Peak number tR min UV λ max m/z [M + H]+/[M + H]−/[Ag + H]+ Compound [lit.]
F-1 16.56 242 sh, 267, 301 sh, 352 –/741−/– quercetin O-triglycoside (pentoside-rhamnoside-
hexoside)
F-2 19.74 263,318 sh, 338 747+/726−/– kaempferol 3-O-xylosyl-rhamnoside-7-O-glucoside
(daigremontrioside) [8]
F-3 21.62 265, 294 sh, 350 –/625−/– myricetin O-diglycoside (hexoside-rhamnoside)
F-4 21.85 263, 300 sh, 350 –/479−/– myricetin O-hexoside
−
F-5 22.87 244 sh, 272, 349 sh –/513 /– unknown isoflavone
F-6 25.20 262, 300 sh,349 –/463−/– myricetin O-rhamnoside
F-7 25.75 263, 296 sh, 346 – unknown flavonoid
+ +
F-8 26.25 252, 267 sh, 296 sh, 352 641 /–/317 isorhamnetin O-diglycoside (hexoside-rhamnoside)
F-9 26.76 256, 265 sh, 300 sh, 350 465+/–/303+ quercetin 3-O-glucoside [1, 17]
F-10 29.67 253, 264 sh, 301 sh, 350 465+/–/333+ patuletin (or 8-methoxyquercetin) O-pentoside [1]
−
F-11 30.27 241 sh, 265, 354 sh –/471 /– unknown isoflavone
F-12 30.73 264, 295 sh, 346 579+/–/287+ kaempferol 3,7-O-dirhamnoside (kaempferitrin) [1]
F-13 31.37 253, 266 sh, 297 sh, 350 479+/477−/317+ isorhamnetin O-hexoside
+ +
F-14 37.78 255, 264 sh, 347 581 /–/303 quercetin 3-O-xylosyl-rhamnoside [8]
F-15 34.61 264, 295 sh, 345 475+/–/287+ kaempferol O-acetyl-rhamnoside
519−(A)
F-16 35.16 264, 299 sh, 345 565+/563−/287+ kaempferol 3-O-arabinosyl-rhamnoside [17]
F-17 35.52 253, 267 sh, 350 593−/–/595+ isorhamnetin O-diglycoside (pentoside-rhamnoside)

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+ − +
F-18 35.94 263, 295 sh, 318 sh, 341 565 /563 /287 kaempferol 3-O-xylosyl-rhamnoside [8]
F-19 40.78 262, 341 637+/635−/287+ kaempferol O-diglycoside (hexoside-acetyl-
rhamnoside) [1]

Abbreviations: sh–shoulder [18]

structures have been described as myricetin O-hexoside-rhamno-
side, myricetin O-hexoside, and myricetin O-rhamnoside, respec-
tively. Myricetin derivatives have been found in species of the genus
Kalanchoe such as K. marmorata [1]. The UV spectra of the next
2 compounds F-5 and F-11 were similar and characteristic for isofla-
vones, having an absorption maximum I of approximately 270 nm
and 2 shoulders at 244 and 340 nm, respectively [18]. However, to
date, the presence of isoflavones has not been observed in the Kalan-
choe taxa. Isoflavones are described as components of some Sedum
plants, which also belong to the Crassulaceae family [19]. In our an-
alyzed extracts, the presence of kaempferol 3-p-coumaroylarabino-
side (bryophylloside) isolated from K. daigremontiana by Karsten and
Müller-Stoll [8, 16] was not confirmed.
To estimate the cytotoxic effect of K. daigremontiana extract,
fractions, and bersaldegenin-1,3,5-orthoacetate on different can-
cer cell lines, we used MTT assay and RTCA system. Bersaldege-
nin-1,3,5-orthoacetate was selected for our study as the predom-
inant bufadienolide compound in leaves of K. daigremontiana [20].
The cells were treated with the extract or fractions or bersaldege-
nin-1,3,5-orthoacetate for 24 h, and the obtained results are pre-
sented in ▶ Table 3. The extract showed anticancer activity
against the HeLa, SKOV-3, and A-375 cell lines with IC50 values of
approximately 100 µg/mL; the IC50 value for MCF-7 cells was in a
range of 43.58–48.28 µg/mL, according to the RTCA data. Of the
2 obtained fractions, the dichloromethane fraction exhibited sig-
nificant activity on all the tested cell lines with IC50 values ≤ 10 µg/
mL. In contrast, the water fraction did not show any significant ef-
fect, and IC50 values for this fraction were above or approximately
100 µg/mL for all the cancer cell lines. Bersaldegenin-1,3,5-or-
thoacetate showed the strongest anticancer activity with IC50 val-
ues < 1.5 µg/mL. The dose-response curves leading to the IC50 de-
terminations of the extracts and compound are provided as Sup-
porting Information (Fig. S1–S6).
From the results of RTCA and MTT assay, the dichloromethane
fraction and bersaldegenin-1,3,5-orthoacetate were additionally
tested on all cell lines to detect the presence of apoptotic and
dead cell populations after 2 h or 24 h of treatment. The flow cy-
tometry analysis showed that this fraction exhibited a strong ne-
crotic effect on all cell lines and this effect was dose-dependent in
both treatments. The activities of the fraction were similar after
2 h and 24 h. A significant increase in the dead cell population
was observed. In contrast, the population of early apoptotic cells
was not larger than that in the control samples (▶ Fig. 1 and 2).
Next, a compound of the dichloromethane fraction (bersaldege-
nin-1,3,5-orthoacetate) also led to an increase in the dead cell
population after 24 h of the treatment; however, the contribution
of apoptotic cells was much larger than in the experiment with the
fraction (▶ Fig. 2). The mode of action of the bufadienolide com-
pound and dichloromethane fraction differed, which suggests
that mechanisms of these 2 actions may be different in the cells.

Stefanowicz-Hajduk J et al. Identification of Flavonoids … Planta Med

 Original Papers

Evaluation of molecular way of cell death induced by bersaldege-

R is the coefficient of determination; *The results are presented as mean values (with the 95 % confidence intervals) from three independent experiments performed twice with RTCA (n = 6) and 6 times with the MTT
▶ Table 3 The IC50 (µg/mL) values of the extract, dichloromethane and water fractions, and bersaldegenin-1,3,5-orthoacetate of Kalanchoe daigremontiana for different cell lines based on the MTT assay and

15.46 −03 (15.00–15.92−03);

nin-1,3,5-orthoacetate needs further studies; however, previous

4.55−03 (4.29–4.81−03);

7.64−03 (7.62–7.66−03);

7.49−03 (7.43–7.55−03);

7.72−03 (7.64–7.80−03);
tests carried out on different bufadienolides indicate that increase
of intracellular Ca2+ concentration, suppressing vascular endothe-

0.98a (17.01 nM)

0.94a (5.01 nM)

0.93a (8.40 nM)

0.95a (8.24 nM)

0.92a (8.49 nM)

Vinblastine**

lial growth factor receptor 2 (VEGFR-2) or down-regulation of the

protein level of Hsp90, may be involved in signaling pathways
RTCA*

[21–23]. Another authors indicated G2/M cell cycle arrest and in-
duction of caspase-dependent pathway of apoptosis in cells after
treatment with bufadienolide compounds [24, 25].
Bersaldegenin orthoacetate

The available data related to the anticancer properties of Ka-

lanchoe plants suggest that the cytotoxic activity of the dichloro-
a

0.77 (0.72–0.82); 0.96a

1.38 (1.24–1.52); 0.98a

0.99 (0.97–1.01); 0.83a

0.09 (0.08–0.10); 0.95a

0.50 (0.44–0.56); 0.85

methane fraction is partly dependent on the presence of bufadie-

nolides. Some bufadienolides isolated from the leaves of K. pinna-
ta and K. daigremontiana × tubiflora showed antitumor-promoting
(1.09 µM)

(1.68 µM)

(3.02 µM)

(2.17 µM)

(0.19 µM)
activity on Raji cells (Burkittʼs lymphoma) [26]. In addition, bryo-
RTCA*

phyllin B present in K. pinnata also exhibited cytotoxic activity on

the KB cell line [27]. Bufadienolide glycosides (kalantuboside A
and B, bryotoxin C, bersaldegenin-1,3,5-orthoacetate, and bersal-
a

73.65 (69.89–77.41); 0.80a

92.02 (87.28–96.76); 0.84

117.73 (107.75–127.71);

181.73 (176.38–187.08);

degenin-1-acetate) from K. tubiflora showed strong cytotoxicity

100.42 (98.95–101.89);

against lung adenocarcinoma A549, oral adenosquamous carci-

noma Cal-27, melanoma A2058, and promyelocytic leukemia HL-
60 cell lines [24]. Kalanchosides A–C from K. gracilis also exhibited
a cytotoxic effect on some human cancer cell lines (e.g., ovarian,
RTCA*
Aqueous fraction

0.92a

0.97a

0.97a

prostate, lung, epidermoid, and nasopharyngeal) [28]. Although

there are no data on traditional applications of bufadienolides or
bufadienolide-rich preparations, many studies have shown the
Electronic reprint for personal use

> 100.0

> 100.0

> 100.0

> 100.0

> 50.0
MTT*

potential use of these compounds for anticancer therapy. How-

assay (n = 18); **Vinblastine sulfate was used as a positive control; ***HaCaT line was used as a non-tumor control

ever, it should be noted that bufadienolides are toxic to normal

and cancer cells. Uncontrolled use of these compounds is associ-
10.27 (9.72–10.82);

ated with a high risk of dangerous side effects, especially cardio-

9.07 (7.34–10.80);
8.53 (7.47–9.59);

9.31 (9.13–9.49);

9.10 (9.09–9.10);

toxicity due to their narrow therapeutic index [29, 30]. One of the
solutions of using these compounds in therapy is generation of
chemical derivatives that are both therapeutically effective and
RTCA*
Dichloromethane fraction

0.95a

0.89a

0.96a

0.81a

0.95a

less toxic [25, 31]. Another way to reduce toxicity of bufadieno-

lides and bufadienolide-rich extracts may be preparation of modi-
fied liposomes or nanocapsules with prolonged retention time
6.42 (6.18–6.66)

5.42 (5.31–5.53)

8.02 (7.93–8.11)

7.72 (7.41–8.03)

7.39 (7.23–7.55)

[32, 33]. Alvarado-Palacios, et al. applied nanoencapsulation of

the aquoethanolic extract from K. daigremontiana and showed se-
MTT*

lective effect on normal and cancer breast cell lines [33].

In our study, we used ethanol extract from K. daigremontiana,
which showed, together with bufadienolide fraction and bersalde-
101.49 (99.37–103.61);

8.38 (7.73–9.03); 0.92a

genin-1,3,5-orthoacetate, strong effects on the human non-tu-

45.93 (43.58–48.28);

mor keratinocytes. Traditional external and internal applications

111.43 (104.74–

103.31 (101.04–
118.12); 0.86a

105.58); 0.83a

of extracts from the species of Kalanchoe refer to powdered,

crushed leaves, leaf paste, boiled juice, or water extracts rather
RTCA*

than to ethanol preparations [2]. Due to hydrophobic properties

0.83a

0.90a

of bufadienolides, the quantitative content of these compounds

in water extracts is much lower than in ethanol extracts (con-
firmed also by our preliminary, unpublished data). This may ex-
8.29 (8.28–

plain the ethnomedicinal uses of water extracts or crude leaves

Extract

> 100.0

> 100.0

> 100.0
> 50.0
MTT*

with the relatively safer content of bufadienolides in comparison

8.30)

with ethanol and concentrated extracts.

In conclusion, this study introduces cytotoxic effects of ethanol
extract, water and dichloromethane fractions, and bersaldegenin-
HaCaT***
Cell line

SKOV-3

1,3,5-orthoacetate obtained from Kalanchoe daigremontiana on

MCF-7
RTCA.

A-375
HeLa

a 2

different human cancer cell lines. The strongest activity of the di-
chloromethane fraction on the cell lines was due to the presence

Stefanowicz-Hajduk J et al. Identification of Flavonoids … Planta Med

 ▶ Fig. 1 The estimation of early, late, and dead cell populations of HeLa, SKOV-3, MCF-7, A375, and HaCaT cells after 2 h of treatment with the
Electronic reprint for personal use
dichloromethane fraction of Kalanchoe daigremontiana (a) and hydrogen peroxide (a positive control) (b). The apoptotic/necrotic cells were stained
with annexin V and 7-AAD and analyzed by flow cytometry. Each sample was run in triplicate. Error bars represent standard deviations. Significant
differences relative to the control are marked with an asterisk (p < 0.05).

of bufadienolides, whereas flavonoids (mostly kaempferol, quer-

cetin, isorhamnetin, and myricetin glycosides) were identified in
the ethanol and water extracts that showed no significant effects
on the tumor cells. Our study shows that bufadienolides are com-
pounds with high anticancer potential; however, cell death signal-
ing pathways require further investigation.
Materials and Methods
Plant material
Leaves of K. daigremontiana were purchased from a commercial
garden source (Garden Center Justyna, Gdańsk, Poland). The bo-
tanical and genetic identification of plant species was performed
by the authors with the help of A&A Biotechnology Company (Po-

Stefanowicz-Hajduk J et al. Identification of Flavonoids … Planta Med

 Original Papers
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▶ Fig. 2 The estimation of early, late, and dead cell populations of HeLa, SKOV-3, MCF-7, A375, and HaCaT cells after 24 h of treatment with the
dichloromethane fraction of Kalanchoe daigremontiana (a) and bersaldegenin-1,3,5-orthoacetate (b). The apoptotic/necrotic cells were stained
with annexin V and 7-AAD and analyzed by flow cytometry. Each sample was run in triplicate. Error bars represent standard deviations. Significant
differences relative to the control are marked with an asterisk (p < 0.05).

land). A voucher specimen (No. 21 761) was deposited in the Her-
barium of the Medical University of Gdańsk (GDMA Herbarium).
The extract and fractions of K. daigremontiana were prepared
from fresh leaves (100 g), which were macerated and stirred with
95 % ethanol (0.5 L) for 24 h at room temperature. The extract was
filtered, concentrated under reduced pressure at 40 °C, and
lyophilized. To obtain fractions, the concentrated extract was
3 times shaken well with water and dichloromethane. The ob-
tained fractions were divided, concentrated under reduced pres-
sure, and lyophilized. The extract and 2 fractions were dissolved
in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL for
cytotoxicity tests.
Bersaldegenin-1,3,5-orthoacetate was isolated and provided
by the authors from the Institute of Puławy. The compound was
dissolved in DMSO at a concentration of 20 mg/mL for cytotoxic-
ity tests.
Extraction and isolation of bersaldegenin-1,3,5-
orthoacetate
The method of isolation and structure elucidation of bersaldege-
nin-1,3,5-orthoacetate was described by Moniuszko-Szajwaj, et al.

Stefanowicz-Hajduk J et al. Identification of Flavonoids … Planta Med

[14]. In short: the ground roots of K. daigremontiana were ex-
tracted with water at room temperature, and aqueous extract
was concentrated under reduced pressure. This extract was puri-
fied by a vacuum liquid chromatography on a LiChroprep RP-18
column with 80 % aqueous methanol, and a fraction containing
bufadienolides was obtained. The bufadienolide-rich fraction was
then partitioned between water and butanol to afford n-BuOH
extract, which was applied on a Sephadex LH-20 column
(950 × 22 mm, 25–100 µm), eluted with MeOH, and afforded sub-
fractions, which were separated on a Lichroprep RP-18 column
(32 × 320 mm, 40–63 µm), eluted with a linear gradient of 15–
40 % aqueous MeOH in a 0.1 % HCOOH. Individual bufadienolides
were purified by a reversed-phase semi-preparative HPLC isocrati-
cally on a C18 column (Kromasil 100–5-C18, 250 × 10 mm, 5 µm),
using aqueous methanol solutions of different concentrations
containing 0.1 % HCOOH. The one from the isolated compounds
was bersaldegenin-1,3,5-orthoacetate. Its structure elucidation
was performed using ESI‑MS and NMR spectral data analyses, op-
tical rotation, circular dichroism, and IR spectroscopy.
HPLC and ESI‑MS analysis of the ethanol extract, water
and dichloromethane fractions of K. daigremontiana
HPLC systems
The HPLC system (Shimadzu) consisted of 2 LC-20AD pumps, a
degasser DGU-20A5, a semi-micro mixer, a controller CBM-20A,
a thermostat CTO-20AC, an autosampler SIL 20 ACXR, a nitrogen
generator, and LCMS-2020 mass spectrometer with ESI ionization.
Data were acquired and processed by LabSolution software (ver-
sion 1.2).
The mobile phase consisted of A [water : formic acid (100 : 0.1,
V/V)] and B [water : acetonitrile : formic acid (50 : 50 : 0.1, V/V/V)].
The separation was performed using Kinetex C-18 column
(100 mm × 4.6 mm, 2.6 µm) according to the gradient program:
0 min: 12 % B in A; 10 min: 20 % B in A; 35 min: 43 % B; 60 min:
100 % B in A. Column temperature was 20 °C, and the flow rate
was 0.8 mL · min−1. Injection volume was 5 µL for the dichloro-
methane extract and 10 µL for water and ethanol extracts.
ESI‑MS conditions
Mass spectra were acquired using Shimadzu LCMS 2020 in posi-
tive (PI) and negative (NI) ion modes. A full-scan (range m/z 200–
800) and selected ion monitoring (SIM) technique for monitoring
specific signals was used. The ESI‑MS detector parameters were as
follows: detector voltage 1.6 kV and ionization potential 4.5 kV
and 3.5 kV for PI and NI modes, respectively; nebulizing gas (N2)
flow of 1.5 L · min−1; desolvation line and block temperatures of
250 °C and 200 °C, respectively; and drying gas flow (N2) of
15 L · min−1.
Cell cultures
The human cervical adenocarcinoma (HeLa S3), ovarian cancer
(SKOV-3), breast cancer (MCF-7), malignant melanoma (A375)
cell lines, and keratinocytes (HaCaT–non-tumor control cells)
were obtained from the American Type Culture Collection (ATCC).
The HeLa S3, MCF-7, A375, and HaCaT cell lines were cultured in
Dulbeccoʼs Modified Eagleʼs Medium (DMEM). The SKOV-3 line
was cultured in McCoyʼs Medium. Both media were supplemented
Stefanowicz-Hajduk J et al. Identification of Flavonoids … Planta Med
with 100 units/mL of penicillin, 100 µg/mL of streptomycin, and
10 % (v/v) fetal bovine serum (FBS). The cells were incubated at
37 °C and 5 % CO2.
MTT assay
The MTT assay was used to estimate the viability of all cell lines
after treating the cells with the plant extract or fractions. The cells
(HeLa S3, SKOV-3, MCF-7, A375, and HaCaT) were seeded in 96-
well plates at a density of 5 × 103 cells/well and treated for 24 h
with the K. daigremontiana extract or fractions at concentrations
of 2.0–150.0 µg/mL. The DMSO concentration used in the control
sample was 0.75 % (v/v). Subsequently, the cells were incubated
with MTT (0.5 mg/mL), and the absorbance values of formazan
solution were measured on a microtiter plate reader (Epoch). The
data were analyzed in GraFit software v.7 and are expressed as
IC50 mean values (with the 95 % confidence intervals) of 3 inde-
pendent experiments (in 6 repetitions).
Real-Time xCELLigence system
The xCELLigence Real-Time Cell Analyzer Dual Plate instrument
(RTCA DP) was used to estimate the cytotoxic effects of the
extract, fractions and bersaldegenin-1,3,5-orthoacetate on the
tested cell lines. The cells were seeded at a density of 2 × 104
cells/well into E-plate 16. After 24 h, the extracts or bersaldege-
nin-1,3,5-orthoacetate were added at concentrations of 2.0–
150.0 µg/mL and 0.01–20.0 µg/mL, respectively for the next
Electronic reprint for personal use
24 h. The DMSO concentration used in a control sample was
0.75 % (v/v). Vinblastine sulfate dissolved in sterile water was used
as a positive control in the concentration range of 0.9−03–
18.0−03 µg/mL. The cell index (CI) values generated by RTCA ana-
lyzer and taken at different frequencies (10, 25, and 50 kHz) were
used to obtain IC50 values of the extracts and bersaldegenin-
1,3,5-orthoacetate, and were measured every 15 min from the
addition of the extracts/compound to the cells to the end of the
experiment (100 measuring points for every experiment). IC50 val-
ues were calculated using the RTCA software v.1.2.1. All the ex-
periments were independently repeated 3 times in duplicate.
Annexin V and dead cell assay
The Muse Annexin V and Dead Cell Assay Kit was used to deter-
mine the percentage of early apoptotic, late apoptotic, and dead
HeLa S3, SKOV-3, MCF-7, A375, and HaCaT cells after treatment
with the dichloromethane fraction and bersaldegenin-1,3,5-or-
thoacetate. Cells of all cell lines were seeded at a density of
1 × 105 cells/well in 12-well plates and incubated for 24 h. Then,
the fraction or bersaldegenin-1,3,5-orthoacetate were added at
a concentration range of 5.0–100.0 µg/mL and 2.0–50.0 µg/mL,
respectively. DMSO concentration for this analysis did not exceed
0.5 % (v/v). Hydrogen peroxide was used as a positive control.
After 2 h or 24 h, the cells were harvested and stained with re-
agents [annexin V and 7-aminoactinomycin (7-AAD)] from the
kit and analyzed by the Muse Cell Analyzer. The experiment was
performed independently in triplicate.
Statistical analysis
Statistical data were analyzed using the STATISTICA 12.0 software
package. The data are expressed as mean values ± SD. For com-
 Original Papers
parison studies, Studentʼs t-test was performed. The statistical
significance was set at p < 0.05.
Supporting Information
The dose-response curves leading to the IC50 determinations of
the extracts and compound are provided as Supporting Informa-
tion.
Acknowledgements
The authors would like to thank the Medical University of Gdańsk for the
financial support of the study (statutory funds).
Conflict of Interest
The authors declare that they have no conflict of interest.
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