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Bufadienolides of Kalanchoe species: an overview
of chemical structure, biological activity and prospects
for pharmacological use
Joanna Kolodziejczyk-Czepas .Anna Stochmal
Received: 15 January 2017 / Accepted: 26 July 2017 / Published online: 2 August 2017
ÓThe Author(s) 2017. This article is an open access publication
Abstract Toad venom is regarded as the main
source of bufadienolides; however, synthesis of these
substances takes also place in a variety of other animal
and plant organisms, including ethnomedicinal plants
of the Kalanchoe genus. Chemically, bufadienolides
are a group of polyhydroxy C-24 steroids and their
glycosides, containing a six-membered lactone (a-
pyrone) ring at the C-17bposition. From the pharma-
cological point of view, bufadienolides might be a
promising group of steroid hormones with cardioac-
tive properties and anticancer activity. Most of the
literature concerns bufadienolides of animal origin;
however, the medicinal use of these compounds
remains limited by their narrow therapeutic index
and the risk of development of cardiotoxic effects. On
the other hand, plants such as Kalanchoe are also a
source of bufadienolides. Kalanchoe pinnata (life
plant, air plant, cathedral bells), Kalanchoe daigre-
montiana (mother of thousands) and other Kalanchoe
species are valuable herbs in traditional medicine of
Asia and Africa. The present review focuses on the
available data on chemical structures of 31 com-
pounds, biological properties and prospects for ther-
apeutic use of bufadienolides from Kalanchoe species.
Furthermore, it presents some new investigational
trends in research on curative uses of these substances.
Keywords Bufadienolide Kalanchoe
Cytotoxicity Cancer therapy Ethnomedicine
Introduction
Bufadienolides are a group of polyhydroxy C-24
steroids and their glycosides. The first described
bufadienolide was scillaren A, identified in Egyptian
squill (Scilla maritima) (Stoll et al. 1933). The term
‘‘bufadienolides’’ originates from the genus Bufo—
toads, which venom (a skin secretion) contains these
compounds. Both animals (toads, snakes) and plants
(Crassulaceae and Hyacinthaceae, in particular) syn-
thesize bufadienolides, while the bufadienolide
orthoesters were found only in several plant species:
Kalanchoe daigremontiana Raym.-Hamet & H. Per-
rier, Kalanchoe tubiflora (Harv.) Raym.-Hamet, the
hybrid Kalanchoe daigremontiana 9tubiflora,Kalan-
choe pinnata (Lam.) Pers., as well as in Melianthus
comosus Vahl and Bersama abyssinica Fresen
(Melianthaceae family).
The range of biological properties of bufadieno-
lides includes cytotoxic, antitumor and cardiotonic
activities (Gao et al. 2011), however, uncontrolled
J. Kolodziejczyk-Czepas (&)
Department of General Biochemistry, Faculty of Biology
and Environmental Protection, University of Lodz,
Pomorska 141/143, 90-236 Lodz, Poland
e-mail: joannak@biol.uni.lodz.pl
A. Stochmal
Department of Biochemistry, Institute of Soil Science and
Plant Cultivation, State Research Institute, Czartoryskich
8, 24-100 Pulawy, Poland
123
Phytochem Rev (2017) 16:1155–1171
DOI 10.1007/s11101-017-9525-1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
administration of these substances may induce the
occurrence of side effects (Puschett et al. 2010). Most
of the literature concerning the chemical characteris-
tics, biological properties and possible therapeutic
effects of bufadienolides includes data derived from
studies on substances of animal origin. Chemistry and
biological activities of bufadienolides synthesized by
Kalanchoe plants are less known. Members of the
Kalanchoe genus (Crassulaceae) are native for sub-
tropical and tropical regions of Asia, Africa and
America as well as for Australia and Madagascar. In
Europe, K. pinnata and K. daigremontiana are mainly
grown as house ornamental plants; however, their
remedial properties are also known. Furthermore, both
these and other Kalanchoe species are popular
medicinal herbs in different regions of the world
(Table 1). Traditional recommendations for using
these plants include a wide range of diseases, includ-
ing gastric ulcers, kidney stones, rheumatoid arthritis,
bacterial and viral infections, skin diseases, cold as
well as other disorders (e.g. Fu
¨rer et al. 2016; Kawade
et al. 2014; Pattewar 2012; Rajsekhar et al. 2016).
Ethnomedicinal uses of Kalanchoe-derived prepara-
tions are mostly based on internal or external admin-
istration of crude extracts or plant juice. There is no
data on traditional uses of purified bufadienolides or
semi-purified bufadienolide-rich preparations. How-
ever, available findings suggest that therapeutic
activities (anti-cancer action, in particular) of Kalan-
choe-derived medicines may be partly dependent on
the presence of bufadienolides. Studies on these
compounds, originated from various sources, revealed
their anti-inflammatory, anti-cancer, anti-viral and
other beneficial activities (Kamboj et al. 2013).
Different research groups demonstrated anti-cancer
properties of bufadienolides synthetized by Kalanchoe
plants (e.g. Deng et al. 2014; Huang et al. 2013;Wu
et al. 2006; Yamagishi et al. 1989). Daigremontianin
and bersaldegenin-1,3,5-orthoacetate are listed in
literature as sedative substances and natural adaman-
tane derivatives (‘‘trioxaadamantanes’’) that may
possess anti-influenza activity (Wanka et al. 2013).
Additionally, the analysis of existing ethnomedicinal
evidence (e.g. Botha 2016; Lans 2006;Su
¨sskind et al.
2012), followed by studies with contemporary (bio)-
chemical and other scientific methods, may provide
new data on safety or possible risk of using of
Kalanchoe bufadienolide-containing extracts and
preparations in humans.
This work reviews the available data on Kalanchoe
species as a source of bufadienolides. Chemistry,
biological activities and prospects for possibility of
therapeutic use of Kalanchoe plant-derived bufadieno-
lides have been presented. Some information on
possible side effects of bufadienolides have been also
included. The current review comprises data (to May,
2017) from journals recorded in international data-
bases (Medline/Pubmed, Scopus, ScienceDirect/Else-
vier, Springer Link/ICM) and other scientific journals,
non-indexed in these databases.
Bufadienolide structures and their concentration
in Kalanchoe plants
The Kalanchoe species are succulent plants. Their
aerial parts were reported to contain not only steroid
compounds, but also some flavonoids, phenolic acids,
anthocyanins, alkaloids, saponins and tannins (El
Abdellaoui et al. 2010; Chowdhury et al. 2011). The
polyhydroxy C-24 structure of bufadienolides is based
on a six-membered lactone (a-pyrone) ring, located at
position C-17b. Some of these compounds have been
isolated from Kalanchoe plants, and their structures
have been established by spectral techniques. The
available structures of these compounds are presented
in the Figs. 1,2,3and 4. Supratman et al. (2000) have
identified two compounds including bryophyllin A (5)
and bryophyllin C (7) from K. pinnata, and in 2001
five next compounds: bersaldegenin-3-acetate (4),
bersaldegenin-1,3,5-orthoacetate (2), daigremontianin
(9), bersaldegenin-1-acetate (3) and methyl daigre-
monate (30) in study on bufadienolides of K. daigre-
montiana 9tubiflora (Fig. 1). Eight bufadienolides
were identified by Wu et al. (2006), in the extract of
aerial parts of K. gracilis, which included kalancho-
sides A (12), B (13) and C (14), thesiuside (31),
hellebrigenin (10), hellebrigenin-3-acetate (11), bryo-
phyllins A (5) and B (6). The systematic names of
these compounds are shown in the Table 2. From roots
of K. daigremontiana, eight new bufadienolides
named as kalandaigremoside A (15), B (16), C (17),
D(18), E (19), F (20), G (21) and H (22) were isolated
and characterized by Moniuszko-Szajwaj et al. (2016).
The presence of daigredorigenin-3-O-acetate (8)in
K. daigremontiana was reported as early as in the 80s
of the twentieth century (Wagner et al. 1985). Next
compounds were isolated in 2008 by Kuo et al. from
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Table 1 Kalanchoe species in ethnomedicine (a compilation of data)
Species (with English names) Traditional uses and geographical region and/or country
Kalanchoe crenata (Andrews) Haw. (Never-die) Medicinal plant, used during pregnancy by Anyi-Ndenye women (Eastern Ivory Coast, Africa)
(Malan and Neuba 2011)
Leaves are recommended to heal umbilical cord wounds in newborns (Mabira Central Forest
Reserve, Uganda) (Tugume et al. 2016)
Kalanchoe daigremontiana Raym.-Hamet & H. Perrier, syn. Bryophyllum
daigremontianum Raym.-Hamet & H. Perrier. (Mother of Thousands)
One of the most frequently prescribed anthroposophic medications, administered against
psychic agitation, restlessness, and anxiety—studies conducted at Hospital Havelhoehe,
Germany (Su
¨sskind et al. 2012)
Kalanchoe densiflora Rolfe For the treatment of wounds (Samburu of Mt. Nyiru, South Turkana, Kenya) (Bussmann 2006)
Kalanchoe germanae Raym.-Hamet ex Raadts (Air plant) Removal of ganglion—the pound leaves are used on ganglion area (Kenya) (Kipkore et al.
2014)
Kalanchoe glaucescens Britten Leaves are used to treat cough (Mabira Central Forest Reserve, Uganda) (Tugume et al. 2016)
Kalanchoe gracilis Hance, syn. Kalanchoe ceratophylla Haw. To cure injuries, pain, fever and inflammation (Taiwan) (Lai et al. 2010)
Kalanchoe laciniata L. (Christmastree plant) Juice from the leaves is used externally for joint pain (Southern India) (Karuppuswamy 2007)
Powdered leaves are administered to alleviate cough, to cure colds and inflammation and for
healing of boils and wounds (Southern India, Malaysia)
Headache (Philippines)
Crushed leaves are applied externally to decrease body temperature and to heal ulcers
(Cambodia, Laos, Vietnam)
To cure wounds, inflammation and diabetes (India) (Deb and Dash 2013)
Kalanchoe lanceolata (Forsk.) Pers. Anti-malarial remedy (Kenya) (Njoroge and Bussmann 2006)
The leaf juice is administered during dysentery (India) (Bapuji and Ratnam 2009)
Kalanchoe marmorata Bak. Boiled juice is used as eye drops for treatment of eye infections (eastern Ethiopia) (Belayneh
and Bussa 2014)
Kalanchoe petitiana A. Rich. Leaf juice is applied on the fractured for bone setting (Ethiopia) (Ragunathan and Abay 2009)
Phytochem Rev (2017) 16:1155–1171 1157
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Table 1 continued
Species (with English names) Traditional uses and geographical region and/or country
Kalanchoe pinnata (Lam.) Pers., syn. Bryophyllum pinnatum Lam., Bryophyllum
calycinum Salisb. (Life plant, air plant, love plant, Canterbury bells, Cathedral
bells)
In the treatment of urinary bladder stones (India, Trinidad and Tobago) (Lans 2006; Sen
et al. 2008)
Leaf extract is used to cure amoebic dysentery (North Bengal) (Mitra and Mukherjee
2010)
Wounds, bruises, swellings and insect bite (Himalaya) (Hussain and Hore 2007)
Diarrhea (India) (Dash and Padhy 2006)
Antibacterial and anti-inflammatory remedy (Vietnam) (Nguyen et al. 2004)
Internally: to cure acute and chronic bronchitis, pneumonia and others respiratory tract
infections, fever; externally: to treat dermatomycosis (Nigieria) (Okwu and Nnamdi
2011)
Leaves are recommended for treatment of cough in adults and children (Kibale National
Park, Uganda) (Namukobe et al. 2011)
Inflammation, dermatosis, skin problems, wound healing, arthritis, asthma, bruises,
diabetes, infections, tumours and ulcers—worlwide (Quazi Majaz et al. 2011a,b)
Paste from macerated leaves is used externally for muscle and joint pain (Bangaldesh)
(Tumpa et al. 2014)
Preparations from leaves are used to treat digestive disorders (India) (Barukial and
Sarmah 2011)
Decoction from leaves is administered to remove kidney stones (Bangladesh) (Afroz
et al. 2013)
Leaves are chewed with salt as a remedy for dissolving of gall bladder stones
(Bangladesh) (Rahmatullah et al. 2011)
Herbal preparation from roots and leaves is administered to women for recovering after
childbirth (West Java) (Sihotang 2011)
Leaf paste is applied externally to treat scorpion bite (India) (Vaidyanathan et al. 2013)
Leaf juice is recommended to treat cholera, diarrhea and dysentery (Bangladesh) (Khan
et al. 2015)
Leaves are used to treat urinary problems, incl. kidney and gall bladder stones
(Bangladesh) (Bhowmik et al. 2014)
Raw laves are chewed with sugar to treat dysentery and diarrhea; leaf juice is
recommended to cure jaundice; leaf paste is used externally to heal skin infections and
pimples (Bangladesh) (Das and Choudhury 2012)
Kalanchoe tubiflora Raym.-Hamet, syn. Bryophyllum delagoense (Eckl. & Zeyh.)
Druce (Chandelier plant)
One of the most common medicinal plants used for wound healing (Brazil) (Hsieh et al.
2012)
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R1R2R3R4R5R6
3H2H CHO OAc OH OH
4H2H CHO OH OAc OH
8H2HCH
3OH OAc OH
10 H2H CHO H OH OH
11 H2H CHO H OAc OH
12 H2H CHO H OH
13 H2H CHO H OH
14 O H CHO H OH
15 H2HCH
2OH OH OH OH
16 H2HCH
2OAc OH OH OH
17 H2OH CH2OH H ORha OH
18 H2OH CH2OAc H OH OH
19 OOH CH
2OH H OH OH
20 OOH CH
2OAc H OH OH
21 OOH CH
2OAc OH OH OH
22 OOH CH
2OH OAc OH OH
26 OOH CHO H OH
27 OOH CHO H OH
Fig. 1 Structures of the
compounds 3,4,8,10–22,
26–29 and 31
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the extract of K. hybrid: kalanhybrin A (23), B (24)
and C (25). The only one new compound was isolated
from K. tomentosa and it was 3b-(40,60-dideoxy-b-
arabino-hexopyranosyloxy)-2b-acetoxy-5b,14b-dihy-
droxy-19-oxobufa-20,22-dienolide (1) (Rasoanaivo
et al. 1993). Few years ago, two bufadienolide
glycosides, i.e. kalantuboside A (26) and kalantu-
boside B (27) were found in the extract from the whole
plant of K. tubiflora (Huang et al. 2013). Furthermore,
it has been also established that K. lanceolata (Forssk.)
Pers. synthesizes 5-O-acetylhellebrigenin glycosides,
i.e. lanceotoxin A (5-O-acetylhellebrigenin 3-O-a-L-
rhamnoate) (28) and lanceotoxin B (5-O-acetylhelle-
brigenin 3-O-a-L-rhamnopyranoside) (29) (Anderson
et al. 1984). In the flower heads, leaves and stems
extract of K. tubiflora and in the roots of the hybrid K.
tubiflora 9pinnata, three bryotoxin A, B and C were
detected. In the extract from flower heads, leaves and
stems of the hybrid, K. daigremontiana 9pinnata,
only bryotoxins B and C were found. No bryotoxins
were detected in extract from Kalanchoe fedtschenkoi
Raym.-Hamet & H. Perrier (McKenzie et al. 1987).
Quantification of bufadienolides in leaves of K.
pinnata grown in Brazil and Germany (Oufir et al.
2015) revealed that bryophyllin A (5), bersaldegenin-
3-acetate (4), bersaldegenin-1,3,5-orthoacetate (2) and
bersaldegenin-1-acetate (3) are main bufadienolide
components of these plant organs. In plants grown in
Brazil, the total bufadienolide concentrations ranged
from 16.28 to 40.50 mg/100 g of dry weight. The total
content of bufadienolides in plant material from
Germany was lower and attained from 3.78 to
12.49 mg/100 g of dry weight. Additional analyses
of other species indicated that in leaves of K.
daigremontiana and in stems of K. tubiflora, bersalde-
genin-1,3,5-orthoacetate (4) was the predominant
bufadienolide compound. Contrary to K. pinnata, the
leaves of K. tubiflora contained very low amounts of
bryophyllin A (5), bersaldegenin-3-acetate (4), ber-
saldegenin-1,3,5-orthoacetate (2) and bersaldegenin-
1-acetate (3).
On the other hand, there are significant gaps in the
available literature on the presence of bufadienolides
in Kalanchoe species and their distribution in different
organs of these plants. Our preliminary studies
(unpublished data) suggested that the total content of
bufadienolides varied in different plant parts. While
bufadienolides content per gram of dried stems and
roots K. daigremontiana was 65 and 395 lg, respec-
tively, there was no occurrence of these compounds in
the leaves. This distribution in the plant was quite
unusual and probably reflected physiological and
ecological function of these compounds. It is assumed
that bufadienolides, similarly to other secondary
metabolites, are involved in chemical plant protection
against pathogenic microorganisms and herbivores.
They are also recognized as precursors of hormonal
substances and participate in the formation of mem-
branous structures.
Pharmacological actions of bufadienolides
of various origins
The therapeutic effects of bufadienolide-containing
preparations have been known from the ancient times.
A bufadienolide-rich plant Scilla maritima was used
by Egyptians to cure heart diseases. Bufadienolides
28 H2H CHO H OAc
29 H2H CHO H OAc
31 H2H CHO H OAc
Fig. 1 continued
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are also the principal bioactive ingredient of a
traditional Chinese drug Ch’an Su, containing the
skin secretions of toads such as Bufo gargarizans
Cantor and Bufo melanostictus Schneider. Currently,
the most investigated pharmacological activities of
bufadienolides of various origins are cardiotonic and
anticancer properties. Other physiological actions of
bufadienolides include blood pressure stimulating,
antiangiogenic, antiviral, immunomodulatory and
antibacterial activities (Gao et al. 2011; Kamboj
et al. 2013; Wei et al. 2017). Biological activity of
Kalanchoe-derived bufadienolides is a relatively new
R1R2R3
2H2HCHO
5H2OH CHO
7H2OH CH2OH
9OOHCHO
Fig. 2 Structures of
compounds 2,5,7and 9
R1R2R3
23 CHO OH OAC
24 CHO OAC OH
25 CH3OH OAC
Fig. 3 Structures of the
compounds 23,24 and 25
630
Fig. 4 Structures of the
compounds 6and 30
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123
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issue. For that reason, a number of reports directly
related to molecular background of pharmacological
action of bufadienolides isolated from Kalanchoe
plants is limited. Contemporary literature mostly
provides data on pharmacological actions and possible
therapeutic significance of animal bufadienolides,
however, some information on compounds that are
also present Kalanchoe species is available.
The main molecular mechanism of pharmacolog-
ical action of bufadienolides and their derivatives
involves the induction of a local increase of Na
?
as a
result of inhibition of a carrier enzyme: Na
?
/K
?
-
ATPase (EC 3.6.1.37; the sodium pump), commonly
described as a ‘‘digitalis-like’’ effect. Na
?
/K
?
-
ATPase is responsible for maintaining of electro-
chemical gradient of Na
?
and K
?
through the cell
membrane. The keeping of low Na
?
and high K
?
intracellular concentrations and membrane potential is
critical for excitability of nerves and muscle cells
(including cardiomyocytes) as well as for the sec-
ondary active transport. Bufadienolides have the
ability to alter myocardial ion balance resulting in an
increase of intracellular Ca
2?
concentration ([Ca
2?
]i)
via a backward-running of Na
?
/Ca
2?
exchanger, and
as a consequence, leading to contractions of cardiac
and arterial myocytes (Melero et al. 2000; Schoner and
Scheiner-Bobis 2007). Additionally, studies on
numerous cell lines confirmed the anticancer proper-
ties of different bufadienolides (Kamboj et al. 2013)
and provided some information on anticancer mech-
anisms and selective toxicity of bufadienolides
towards malignant cells. Studies on human liver
microsomes (HLMs) indicated that hydroxylation
and dehydrogenation might be the major metabolic
pathways of bufadienolides (Han et al. 2016). Molec-
ular mechanisms of anticancer activities of hellebrin
and its aglycone hellebrigenin (compounds that were
also found in Kalanchoe plants) were described by
Moreno et al. (2013). According to those authors, both
compounds are able to bind to the alpha subunits of the
Na
?
/K
?
-ATPase and display similar growth inhibi-
tory effects in different cancer cell lines, i.e. A549
(lung cancer), U373 (glioblastoma astrocytoma),
Hs683 (glioma), T98G (glioblastoma), MCF-7 (breast
adenocarcionoma), SKMEL-28 (melanoma), PC-3
(prostate cancer) and HT-29 (colorectal cancer). For
hellebrin, the growth inhibitory concentrations at 50%
(IC
50
) were estimated as 6–58 nM, while the IC
50
for
hellebrigenin ranged from 3 to 42 nM. Other
experiments (Yuan et al. 2016) conducted on human
glioblastoma U-87 cell line and a pancreatic SW1990
cancer cell line demonstrated that gamabufotalin and
arenobufagin (bufadienolides of animal origin) pos-
sessed selective cytotoxic activity against tumour cells
rather than normal cells (peripheral blood mononu-
clear cells, PBMCs). Both bufadienolides (at the final
concentrations of 1.6, 8, 40, 200 and 1000 ng/ml)
displayed dose-dependent anticancer effects, when
compared to control (untreated) cells. For gamabufo-
talin, IC
50
values were 16.8 ±6.5 and 8.1 ±1.5 ng/
ml in the U-87 and SW1990 cells, respectively.
Arenobufagin action was characterized by IC
50
10.3 ±3.3 and 9.9 ±2.2 ng/ml, in the U-87 and
SW1990 cells, respectively. Moreover, the authors
suggested that gamabufotalin might be a promising
candidate for using as an adjuvant therapeutic agent.
This opinion was based on data originated from
experiments on PBMCs treated with bufadienolides at
nontoxic concentrations, which resulted in a modula-
tion of fractions of CD4 ?CD25 ?Foxp3 ?regu-
lator T (Treg) cells in mitogen-activated PBMCs. In
pathophysiology of cancer and haematologic malig-
nancies, Treg cells were found to play a critical role in
development of tumour immunotolerance by sup-
pressing the host response to tumour immunity. Thus,
by decreasing the amount and activity of these cells,
gamabufotalin may enhance the efficiency of conven-
tional anticancer drugs (Yuan et al. 2016). Studies of
Zhang et al. (2016) revealed that the treatment of A549
cell line with gamabufotalin (5–500 nM) significantly
reduced viability of the cells, when compared to the
control (untreated A549 cells). For the 48 h-treatment,
the IC
50
value was 48.4 ±2.5 nM. Moreover, no
cytotoxicity was found in analogous experiments on
human normal lung cell line (HLF cells). Molecular
mechanisms of cytotoxic action of the bufadienolide
involved the G2/M cell cycle arrest and induction of
apoptosis in A549 cells. In vivo, gamabufotalin (10 or
20 mg/kg of body weight) was able to down-regulate
the protein level of Hsp90 in tumor tissues of the
xenograft mice, when compared to control animals
(treated with phosphate-buffered saline) (Zhang et al.,
2016). Furthermore, in studies of other scientists (Yu
et al. 2014), gamabufotalin (10, 50 and 100 nM)
suppressed the expression of cyclooxygenase 2 (COX-
2) in lung cancer cells, in comparison to the dimethyl
sulfoxide (DMSO) vehicle control group. Biochemi-
cal mechanisms of this anti-inflammatory action of
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gamabufotalin involve the inhibition of the phospho-
rylation of inhibitor of nuclear factor kappa-B (IjB),
which prevents the translocation of nuclear factor
kappa B (NF-jB) to nucleus and, in consequence,
halts the recruitment of NF-jB and p300 on COX-2
promoter (Yu et al. 2014). As another potential
mechanism of antitumour activity of gamabufotalin,
the inhibition of the vascular endothelial growth factor
(VEGF)-induced angiogenesis by suppressing vascu-
lar endothelial growth factor receptor 2 (VEGFR-2)
signaling pathway has been also suggested (Tang et al.
2016).
Recently published data (Bachmann et al. 2017)
suggest that the bufadienolide-enriched fraction from
K. pinnata leaf juice (containing bersaldegenin-1-
acetate, bryophyllin A, bersaldegenin-3-acetate, ber-
saldegenin-1,3,5-orthoacetate as well as two uniden-
tified compounds: flavonoid (m/z 581, [M ?H]
?
,
303 (aglycone)) and bufadienolide m/z 477
([M ?H]
?
) display biological activity that might be
useful in the treatment of overactive bladder. The
examined fraction (0.01–1 mg/ml) had the inhibitory
effect on detrusor contractility in vitro. The inhibition
was dose-dependent, and no such effects were found
for flavonoid fraction isolated from the leaf juice.
Biological activity of Kalanchoe species-derived
bufadienolides
Anticancer effects
The existing evidence of anticancer properties of
bufadienolides originates mostly from research on
compounds isolated from animal sources, particularly
of toad venom (Takai et al. 2012). However, reports
indicating on the chemopreventive effects of Kalan-
choe bufadienlides are also available. Bryophyllin B,
isolated from Bryophyllum pinnatum (Lam.) Oken (K.
pinnata) was shown to be a potent cytotoxic agent
against the KB cell line, with the ED
50
value\80 ng/
ml (Yamagishi et al. 1989). Studies on 8 bufadieno-
lides, including kalanchosides A–C, isolated from the
aerial parts of K. gracilis Hance revealed considerable
cytotoxic/anticancer activities of all isolated com-
pounds against several human tumour cell lines such
as nasopharyngeal (KB) and its MDR variant (KB-
VIN), lung (A549), ovarian (1A9), prostate (PC-3),
ileocecal (HCT-8), and epidermoid (A431) cells.
Mostly, effectiveness of the examined bufadienolides
was higher than the effect of etoposide (a reference
cytostatic/anticancer drug) and attained the nanomolar
range of their concentrations (Wu et al. 2006).
Furthermore, bryophyllin B was able to inhibit the
replication of HIV in H9 lymphocytes, at the ED
50
value of \0.25 lg/ml and therapeutic index of
[6.27 lg/ml. Additionally, Huang et al. (2013)
demonstrated that bufadienolide glycosides isolated
from K. tubiflora displayed strong cytotoxicity against
four human cancer cell lines: A549, Cal-27 (oral
adenosquamous carcinoma), A2058 (melanoma) and
HL-60 (promyelocytic leukemia). Bufadienolide
effects were assessed in comparison with positive
controls, i.e. mitomycin-C and cycloheximide, while
0.05% DMSO-treated samples were used as vehicle
controls. IC
50
values for the examined glycosides
ranged from 0.01 to 10.66 lM. For mitomycin-C, IC
50
ranged from 4.63 to 9.34 lM, while IC
50
for cyclo-
heximide was detectable only in experiments on HL-
60 cells and attained 40.60 lM (Huang et al. 2013).
The cytotoxic effect against tumour cell lines was also
found in experiments with bufadienolides isolated
from the crude methanol extract of K. hybrida Desf. ex
Steud. Anticancer activity of the isolated compounds
(4 and 20 lg/ml) was evaluated in experimental
models of three cancer cell lines, i.e. MCF-7, NCI-
H460 (large cell lung cancer), and SF-268 (anaplastic
astrocytoma), using actinomycin D (10 mM) and
DMSO (0.3%) as positive and vehicle controls,
respectively. The strongest cytotoxic effects (even
up to 100% of growth inhibition) towards the exam-
ined cells were found for bersaldegenin 3-acetate and
daigredorigenin 3-acetate (Kuo et al. 2008).
Cardiotonic effects
The cardiac glycoside-like effects of a bufadienolide
compound, extracted from K. daigremontiana, were
demonstrated by Scholtysik et al. (1986). During the
studies on animals, the authors observed pharmaco-
logical effects similar to those evoked by digitalis
glycosides. The IC
50
for Na
?
/K
?
-ATPase activity
in vitro was estimated as 1.4 910
-7
M, while for
ouabain (a reference compound) this parameter was
2910
-7
M. Intravenous infusion of the examined
bufadienolide to guinea-pigs with a rate of 20 lg/kg/
min resulted in ventricular arrhythmias and death after
accumulated doses of about 760 and 860 lg/kg of
Phytochem Rev (2017) 16:1155–1171 1163
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Table 2 The systematic names of compounds 1–31
No. Name Systematic name Species and plant organs References
13b-(40,60-dideoxy-b-arabino-hexopyranosyloxy)-2b-acetoxy-5b,14b-dihydroxy-19-
oxobufa-20,22-dienolide
K. tomentosa (leaves) Rasoanaivo et al.
(1993)
2 Bersaldegenin-1,3,5-
orthoacetate =Melianthugenin
(1b,3b,5b)-1,3,5-[(1R)-ethylidynetris(oxy)]-14-hydroxy-19-oxobufa-20,22-dienolide K. daigremontiana 9
tubiflora (leaves)
Supratman et al.
(2001a,b)
3 Bersaldegenin-1-acetate 3-acetoxy-1,5,14-trihydroxy-19-oxobufa-20,22-dienolide K. daigremontiana 9
tubiflora (leaves)
Supratman et al.
(2001a,b)
4 Bersaldegenin-3-acetate (1b,3b,5b)-3-(acetyloxy)-1,5,14-trihydroxy-19-oxobufa-20,22-dienolide K. daigremontiana 9
tubiflora (leaves)
Supratman et al.
(2001a,b)
5 Bryophyllin A =Bryotoxin C [1b(R),3b,5b,11a]-1,3,5-ethylidynetris(oxy)-11,14-dihydroxy-19-oxo-bufa-20,22-
dienolide
K. pinnata (leaves) Supratman et al.
(2000)
6 Bryophyllin B (1b,3b,5b,8n,9n,10n,11a,19R)-1-acetoxy-3,5,14,19-tetrahydroxy-11,19-epoxybufa-
20,22-dienolide
K. gracilis (aerial parts) Wu al et. (2006)
7 Bryophyllin C [1b(R),3b,5b,11a]-1,3,5-ethylidynetris (oxy)-11,14,19-trihydroxybufa-20,22-dienolide K. pinnata (leaves) Supratman et al.
(2000)
8 Daigredorigenin-3-O-acetate 3-(acetyloxy)-1,5,14-trihydroxy-, (1b,3b,5b)-bufa-20,22-dienolide K. daigremontiana
(aerial parts and roots)
Wagner et al.
(1985)
9 Daigremontianin (1b,3b,5b,11a)-1,3,5-ethylidynetris(oxy)-11,14-dihydroxy-12,19-dioxobufa-20,22-
dienolide
K. daigremontiana 9
tubiflora (leaves)
Supratman et al.
(2001a,b)
10 Hellebrigenin (3b,5b)-3,5,14-trihydroxy-19-oxobufa-20,22-dienolide K. gracilis (aerial parts) Wu et al. (2006)
11 Hellebrigenin-3-acetate (3b,5b)-3-acetoxy-5,14-dihydroxy-19-oxobufa-20,22-dienolide K. gracilis (aerial parts) Wu et al. (2006)
12 Kalanchoside A (3b,5b)-3-[(6-deoxy-a-D-glucopyranosyl)oxy]-5,14-dihydroxy-19-oxobufa-20,22-
dienolide
K. gracilis (aerial parts) Wu et al. (2006)
13 Kalanchoside B (3b,5b)-3-[(6-deoxy-a-L-galactopyranosyl)oxy]-5,14-dihydroxy-19-oxobufa-20,22-
dienolide
K. gracilis (aerial parts) Wu et al. (2006)
14 Kalanchoside C 12-oxohellebrigenin-3-O-4,6-dideoxy-a-ribo-hexopyranoside K. gracilis (aerial parts) Wu et al. (2006)
15 Kalandaigremoside A 1b,3b,5b,14b,19-pentahydroxybufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
16 Kalandaigremoside B 19-(acetyloxy)-1b,3b,5b,14-tetrahydroxybufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
17 Kalandaigremoside C 3b-(O-a-L-rhamnopyranosyl)-5b,11a,14,19-tetrahydroxybufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
18 Kalandaigremoside D 19-(acetyloxy)-3b,5b,11a,14-tetrahydroxybufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
1164 Phytochem Rev (2017) 16:1155–1171
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Table 2 continued
No. Name Systematic name Species and plant organs References
19 Kalandaigremoside E 3b,5b,11a,14b,19-pentahydroxy-12-oxo-bufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
20 Kalandaigremoside F 19-(acetyloxy)-3b,5b,11a,14b-tetrahydroxy-12-oxo-bufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
21 Kalandaigremoside G 19-(acetyloxy)-1b,3b,5b,11a,14b-pentahydroxy-12-oxo-bufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
22 Kalandaigremoside H 1b-(acetyloxy)-3b,5b,11a,14b,19-pentahydroxy-12-oxo-bufa-20,22-dienolide K. daigremontiana
(roots)
Moniuszko-
Szajwaj et al.
(2016)
23 Kalanhybrin A Chol-22-ene-19,24-dial, 3-(acetyloxy)-14,21-epoxy-1,5,22-trihydroxy-21-methoxy-,
(1b,3b,5b,14b,21S,22E)-
K. hybrida (whole plant) Kuo et al. (2008)
24 Kalanhybrin B Chol-22-ene-19,24-dial, 1-(acetyloxy)-14,21-epoxy-3,5,22-trihydroxy-21-methoxy-,
(1b,3b,5b,14b,21S,22E)-
K. hybrida (whole plant) Kuo et al. (2008)
25 Kalanhybrin C Chol-22-en-24-al, 3-(acetyloxy)-14,21-epoxy-1,5,22-trihydroxy-21-methoxy-,
(1b,3b,5b,14b,21S,22E)-
K. hybrida (whole plant) Kuo et al. (2008)
26 Kalantuboside A Bufa-20,22-dienolide, 3-[(3-O-acetyl-4,6-dideoxy-a-L-ribo-hexopyranosyl)oxy]-
5,11,14-trihydroxy-12,19-dioxo-, (3b,5b,11a)-
K. tubiflora (whole
plant)
Huang et al.
(2013)
27 Kalantuboside B Bufa-20,22-dienolide, 3-[(4,6-dideoxy-a-L-ribo-hexopyranosyl)oxy]-5,11,14-
trihydroxy-12,19-dioxo-, (3b,5b,11a)-
K. tubiflora (whole
plant)
Huang et al.
(2013)
28 Lanceotoxin A [(3S,5S,8R,9S,10S,13R,14S,,17R)-5-acetyloxy-10-formyl-14-hydroxy-13-methyl-17-
(6-oxopyran-3-yl)-2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1H-
cyclopenta[a]phenanthren-3-yl],(2R,3R,4S,5S)-2,3,4,5-tetrahydroxyhexanoate,C
K. lanceolate (whole
plant)
Anderson et al.
(1984)
29 Lanceotoxin B [(3S,5S,8R,9S,10S,13R,14S,17R)-10-formyl-14-hydroxy-13-methyl-17-(6-oxopyran-
3-yl)-3-[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy-
2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-5-yl]
acetate
K. lanceolata (whole
plant)
Anderson et al.
(1984)
30 Methyl daigremonate Methyl[1b,3b,5b,11a,12a]-(22E)-1,3,5-ethylidynetris(oxy)-14,21-epoxy-11,12-
dihydroxy-19-oxo-5b,14b-chola-20,22-dien-24-oate
K. daigremontiana 9
tubiflora (leaves)
Supratman et al.
(2001a,b)
31 Thesiuside 5-O-acetylhellebrigenin 3-O-b-D-glucopyranoside K. gracilis (aerial parts) Wu et al. (2006)
Phytochem Rev (2017) 16:1155–1171 1165
123
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body weight, respectively. However, in general, the
examined bufadienolide was less toxic than ouabain.
Anti-viral activity
Bufadienolides isolated from leaves of K. pinnata and
K. daigremontiana 9tubiflora are able to inhibit the
activation of Epstein-Barr virus early antigen (EBV-
EA) in Raji cells, induced by 12-O-tetradecanoylphor-
bol-13-acetate. Bryophyllin A had the strongest
inhibitory effect (IC
50
=0.4 lM), while compounds
lacking the orthoacetate moiety such as bryophyllin C
and bersaldegenin-3-acetate possessed significantly
lower activities (IC
50
=1.6 and 3 lM, respectively),
when compared to control samples (untreated with the
bufadienolide) (Supratman et al. 2001a,b).
Inhibition of serine proteinases
So far, research on bufadienolides and enzyme
interactions has been focused only on the inhibition
of ATP-ase activity. The issue of inhibition of other
groups of enzymatic proteins by bufadienolides has
appeared in the literature within last 2 years. In 2015,
Shibao and co-authors published results from studies
on a serine proteinase inhibitor, isolated from Rhinella
schneideri (Schneider’s toad) poison. The inhibitor
was identified as lithocholic acid, a biosynthetic
precursor of bufadienolide. In spite of the fact that
the study was conducted on animal-derived prepara-
tion, it should be mentioned as the first report
confirming that bufadienolide-type compound might
suppress the enzymatic activity of serine proteinase.
Thus, the influence of Kalanchoe-derived bufadieno-
lides on enzymatic properties of serine proteinases still
is very poorly evidenced. Inhibitory action of bufa-
dienolide-rich fraction from K. daigremontiana on
enzymatic activity of thrombin has been recently
described by Kolodziejczyk-Czepas et al. (2017).
Native (untreated with the examined fraction) throm-
bin was used as a control sample. A serine proteinase
enzyme—thrombin (plasma coagulation factor II), is
responsible for the formation of fibrin clot, and thus,
for the prevention of uncontrolled blood loss after
injury of blood vessel. In the above in vitro study,
bufadienolide-rich fraction inhibited enzymatic activ-
ity of thrombin with IC
50
=2.79 lg/ml. The efficacy
of a reference compound (direct inhibitor of throm-
bin)—argatroban (anti-thrombotic drug) was charac-
terized by IC
50
=0.78 lg/ml. On the other hand,
analysis of kinetic parameters of the reaction indicated
that K. daigremontiana fraction contains compounds
with diverse inhibitory mechanisms, when compared
to argatroban. Components of the investigated frac-
tions were uncompetitive inhibitors of thrombin. In
silico studies on interactions of the most common
compounds, identified in the examined bufadienolide-
rich fraction to crystal structure of thrombin were also
conducted. The obtained results indicated that for the
inhibitory effect of K. daigremontiana fraction, most
likely the presence of compounds such as bersalde-
genin-1,3,5-orthoacetate, bersaldegenin-1-acetate,
bersaldegenin, hovetrichoside C, deigredorigenin-3-
acetate is responsible.
Bufadienolides as antioxidants?
Due to hydrophobic, steroid structure of bufadieno-
lide-type compounds, antioxidant properties of those
substances are considered to be weak. However,
existing evidence indicated that bufadienolides pos-
sess some antioxidant potential. Moreover, this group
of compounds may be a base for development of new
derivatives with enhanced antioxidant properties and
decreased toxicity (obtained by chemical alterations of
the pyrone moiety) (Aucamp 2014). Recent studies on
bufadienolide-rich fraction of Kalanchoe daigremon-
tiana roots demonstrated that its DPPH
scavenging
ability was characterized by EC
50
=21.80 lg/ml
(Kolodziejczyk-Czepas et al. 2016). Under the same
experimental conditions, for the reference compounds,
i.e. Trolox and (-)-epicatechin, EC
50
values were 4.64
and 3.30 lg/ml, respectively. It should be emphasized
that earlier results, obtained by other authors in
analogous experiments on different Kalanchoe plants
indicated on significantly lower antioxidant efficacy of
extracts, originated from different organs of Kalan-
choe species (Sharker et al. 2012; Quazi Majaz et al.
2011a,b). Furthermore, antioxidant action of the
mentioned bufadienolide-rich extract of Kalanchoe
daigremontiana was also confirmed using an experi-
mental model of blood plasma exposed to peroxyni-
trite-induced oxidative stress (Kolodziejczyk-Czepas
et al. 2016).
1166 Phytochem Rev (2017) 16:1155–1171
123
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Other biological actions of Kalanchoe
bufadienolides
Toxic (insecticidal) action of daigremontianin and
bersaldegenin-1,3,5-orthoacetate, isolated from the
leaves of K. daigremontiana, was demonstrated using
an experimental system of larvae of Bombyx mori
(Maharani et al. 2008).
The risk of side effects and prospects
for pharmacological use of bufadienolides
occurring in Kalanchoe species
During the last 10 years, a growing interest in the
evaluation of the metabolome of Kalanchoe plants and
biological activities of Kalanchoe-derived extracts
and substances, including bufadienolides has been
observed. For instance, from the total amount of 483
publications containing the name ‘‘Kalanchoe’’ avail-
able in the Medline/Pubmed database, over 150
records derive from the last 10 years (data from 30
March, 2017; search criteria ‘‘Kalanchoe’’). On the
other hand, contrary to in vivo studies, confirming
pharmacological effects of composed extracts isolated
from different Kalanchoe species, physiological
effects of bufadienolides extracted from these plants
and their safety have been poorly described. The vast
majority of reports on pharmacological activity of
different Kalanchoe-based drugs still derive from
traditional medicine and concern preparations based
on crude extracts. However, studies on standardized
preparations from Kalanchoe species also are avail-
able. According to data from a service of the U.S.
National Institutes of Health ‘‘ClinicalTrials.gov’’
(https://clinicaltrials.gov/ct2/home, data from 30
March, 2017; search criteria ‘‘Kalanchoe’’ or
‘‘ Bryophyllum’’), four clinical studies on Bryophyllum
pinnatum/K. pinnata have been recorded. Further-
more, after using a word ‘‘bufadienolide’’ four another
results have been appeared, while a combination of
‘‘ Kalanchoe’’ and ‘‘bufadienolide’’ have not provided
any results. No information on animal or clinical
studies on therapeutic effects of bufadienolides iso-
lated from Kalanchoe was found in Medline/Pubmed,
Scopus, ScienceDirect/Elsevier and Springer Link/
ICM databases (data from 30 March, 2017). Biological
activities of bufadienolides that are synthetized by
these plants are very promising from a
pharmacological point of view, however, they have
been mostly studied in vitro. Therefore, nowadays,
only a preliminary indication of the most promising
prospects for pharmaceutical uses of bufadienolides is
possible.
The therapeutic use of most bufadienolides is
limited by to a narrow therapeutic index and risk of
development of cardiotoxicity (Cheng 2001; Pamnani
et al. 1994). A risk of toxicity of bufadienolide-
containing plant extracts is inadequately evaluated.
For example, no toxicity of the bufadienolide-rich K.
daigremontiana fraction on blood platelets was found
in vitro (Kolodziejczyk-Czepas et al. 2016). On the
other hand, studies in South Africa indicated that
ingestion of cumulative neurotoxic various plant-
derived bufadienolides such as cotyledoside, tyledo-
sides, orbicusides and lanceotoxins is a potential risk
to humans (Botha 2016). Hence, numerous investiga-
tions have been developed to generate chemical and
biotransformed bufadienolide derivatives or ana-
logues with effective therapeutic action and consider-
ably reduced toxicity. The in vitro biotransformations
of natural bufadienolides have been conducted in
various systems—in plant cell suspension cultures,
fungi and bacteria (Gao et al. 2011). Some of these
modified bufadienolides were able to selectively kill
malignant cells. Studies of Daniel et al. (2003) showed
this preferential cytotoxic action towards malignant
cells for both a natural cardioactive bufadienolide—
hellebrin (0.1–100 lM) as well as for its three
derivatives (100 lM), lacked the cardioactive proper-
ties. Medium for the controls was supplemented with
corresponding amounts of the used bufadienolide
vehicle. While normal peripheral blood mononuclear
cells were affected to a minimal extent, the examined
substances induced the caspase-dependent pathway
and initiated apoptosis in Jurkat T lymphoblasts. Since
the therapeutic use of bufadienolides in anti-cancer
therapy is limited by their influence on heart physi-
ology, a considerable potential of using in cancer
therapy may have compounds possessing a tumour-
specific cytotoxicity with simultaneous lack of cardiac
activity (Daniel et al. 2003). Current research of
bufadienolides (hellebrigenin, among others) also
covers some pharmacokinetic aspects of their interac-
tions with human serum albumin (HSA), the main
carrier of various drugs. In vitro and in silico analyses
indicated that the binding affinity for HSA of various
bufadienolides is considerably related to differences in
Phytochem Rev (2017) 16:1155–1171 1167
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
their structures. The presence of C=O bond at the C12
position decreased the binding affinity for HSA, while
other polar groups increased the bufadienolide affinity
to HSA. In particular, the presence of 11-OH or
16-OAc groups may be important for anchoring
bufadienolides within site I of the HSA pocket. The
11-OH or 16-OAc-mediated interactions of bufa-
dienolide and HSA involve the hydrogen bonding
(H-bonding) with protein Tyr150 or Lys199 groups,
respectively (Zhou et al. 2015).
Another way of the enhancing the therapeutic effect
and reducing the toxicity of anticancer drugs such as
bufadienolides may be preparation of long-circulating,
poloxamer-modified liposomes. According to Hu et al.
(2011), these liposomes have significantly prolonged
retention time, when compared to bufadienolide
solutions and unmodified liposomes. The LD
50
value
of modified liposomes was about 3.5 times higher than
the LD
50
recorded for bufadienolide solution (i.e. 4.48
and 1.28 mg/kg, respectively). The use of bufadieno-
lide liposomes resulted in a considerable increase of
anti-tumour efficiency both in mice bearing H22 liver
cancer cells and Lewis pulmonary cancer cells (2.15
and 2.96 times, respectively), compared to the anti-
cancer effects observed in animals treated with
bufadienolide solution. Promising results have been
obtained by Mexican scientists (Alvarado-Palacios
et al. 2015) in experiments on using nanocapsules
containing the aquoethanolic extract from K. daigre-
montiana as selective anticancer agents. The nanocap-
suled extract was characterized by higher cytotoxic
efficacy (IC
50
=48.53 lg/ml) towards MDA-MB-
231 metastatic breast cancer cell line, when compared
to the non-encapsulated aquoethanolic extract
(IC
50
=61.29 lg/ml). Moreover, studies on non-
cancerous breast cell line MCF 10A revealed no
cytotoxic effect of the nanocapsules containing the
aquoethanolic extract of K. daigremontiana (at con-
centrations B200 lg/ml), whereas the non-encapsu-
lated extract displayed significant cytotoxic effect
(IC
50
=100.2 lg/ml).
Conclusions
Ethnomedicinal plants of the Kalanchoe genus may be
regarded as a new source of bufadienolides, since
synthesis of these substances has been confirmed for
these species. At present, however, toad venom
remains the main source of these compounds. On the
other hand, a growing number of reports have
confirmed that Kalanchoe-derived bufadienolides dis-
play a wide range of biological actions, including
cardiotonic, anticancer, anti-viral and other properties.
Despite these promising findings, the therapeutic use
of Kalanchoe plants is considerably limited by the lack
of clinical evidence. Therefore, further studies on
medicinal applications of bufadienolides and extracts
of Kalanchoe species origin are required.
Acknowledgements This work was supported by Grants
506/1136 (from University of Lodz, Poland) and (2012/05/B/
NZ9/00812 from the National Science Centre, Poland). We
thank Łukasz Pecio for the preparing structures of compounds.
Additionally, the authors would to thank Prof. Pawel Nowak for
helpful suggestions, and Prof. Beata Olas for reading the
preliminary outline of this work in 2013.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unre-
stricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Com-
mons license, and indicate if changes were made.
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