Natural compounds discovered in Helleborus sp. (Ranunculaceae) with important medical potential,

Full text

Central European Journal of Biology
* E-mail: maria.maior@ubbcluj.ro
Review Article
1Institute of Technology, Babeş-Bolyai University,
400294 Cluj-Napoca, Romania
2Department of Molecular Biology and Biotechnology,
Faculty of Biology, Babeş-Bolyai University,
400084 Cluj-Napoca, Romania
Maria Cornelia Maior1,*, Cristina Dobrotă1,2
Natural compounds with important medical
potential found in
Helleborus
sp.
1. Introduction
Current studies regarding species of Helleborus are
opening new ways to cure diseases using natural
compounds. Plants belonging to this group have long
been used in traditional medicine to treat various
conditions, such as edema, arthritis and ulcer. In the
Balkan area, Helleborus extracts have been used for
a long time in traditional medicine as painkillers or as
anti-inammatory remedies and in veterinary medicine
against infectious diseases. Several active compounds
including cardioactive glycosides, sterol saponosides,
ecdysteroids and γ-lactones have recently been isolated
from plants of this genus and shown to exert antioxidant,
anti-inammatory and antimicrobial effects. Anti-diabetic
and antitumoral properties were suggested, but further
investigations are required. Due to the scarcity of clinical
trials, there are few published reports on target-organ
toxicity or side effects. This review summarizes the latest
literature on the pharmacological, toxicological, and
clinical studies of Helleborus and its active compounds.
Advancing our understanding of the secondary
metabolite biosynthesis and ability to detect cell lines
with high level of active principles may positively impact
human health at a worldwide scale.
2. Biology and traditional use of
Helleborus spp.
Helleborus spp. (Ranunculaceae), or hellebore, is a
perennial herb native to Europe and Asia. The genus
comprises around 20 species. The underground parts
– rhizomes - containing starch granules and oleosomes
Cent. Eur. J. Biol. • 8(3) • 2013 • 272-285
DOI: 10.2478/s11535-013-0129-x
272
Received 12 June 2012; Accepted 29 November 2012
Keywords: Helleborus sp. • Phytochemicals • Secondary metabolites • Active compounds • Therapeutic potential • Anticancer properties
• Cytotoxicity • Immunomodulation
Abstract: Helleborus (family Ranunculaceae) are well-known as ornamental plants, but less known for their therapeutic benets. Over the
past few years, Helleborus sp. has become a subject of interest for phytochemistry, pharmacology and other medical research
areas. On the basis of their usefulness in traditional medicine, it was assumed that their biochemical prole could be a source
of metabolites with the potential to overcome critical medical issues. There are studies involving natural extracts from these
species which demonstrate that Helleborus plants are a valuable source of chemical compounds with great medical potential.
Some phytochemicals produced by these species have been separated and identied a few decades ago: hellebrin, degluco-
hellebrin, 20-hydroxyecdysone and protoanemonin. Lately, many other active compounds have been reported and considered
as promising remedies for severe diseases such as cancer, ulcer, diabetes and also for common medical problems such as
toothache, eczema, low immunity and arthritis. This paper is an overview of the Helleborus genus focusing on some recently-
discovered compounds and their potential for nding new drugs and useful biochemicals derived from these species.
© Versita Sp. z o.o.

M.C. Maior, C. Dobrotă
[1,2] accumulate the largest amount of metabolites. Most
of the hellebores are acaulescent (leaves and ower
stalks emerge from the rhizomes), only a few species
being caulescent. Leaves are pedate or palmate. The
color of the owers are given by the sepals which
remain on the ower long after fertilization; the petals
are reduced at the base of sepals. Hellebores bloom in
late winter or early spring, between February and April.
Roots, rhizomes and leaves are still used in traditional
medicine to treat humans and animals. Rhizome
extracts in particular have been reported as possessing
a broad spectrum of pharmacological and therapeutic
effects such as cardiotonic, abortive and sedative, as
well as having antioxidative, anti-inammatory and
antimicrobial activities.
There are old archives that mention species of
Helleborus in the Roman Empire and ancient Greece
[3,4]. Helleborus niger was used from antiquity until
Byzantine times to strengthen the heart and has a
positive effect on arteries and nerves [5]. Paracelsus
was prescribing to his patients “an elixirium for a long
life” made by dried leaves of H. niger [6]. There are
16 species of Ranunculaceae found in Renaissance
herbals that were used to treat malaria, 3 of them being
hellebores (H. foetidus, H. niger and H. viridis) [7]. In
Mt. Pelion, Greece, H. cyclophyllus is still used to treat
toothache. A decoction made of the entire plant or only
a piece from the underground part, held temporarily
in the mouth, could numb the entire mouth and stop
tooth pain. A tea made from leaves of H. cyclophyllus
was used by speakers to strengthen their voice [4]. In
Italy, roots of H. foetidus and H. bocconei were used
in the same way as in Greece to treat tooth pains [8]
and in Turkey, roots of H. orientalis were used for the
same purpose [9]. In the Montenegro region, eczema,
skin redness and itching [10] were treated using roots
of H. odorus. Peeled root of H. foetidus introduced into
the vagina, provoked hemorrhage followed by abortion
[8]. In the Italian Apennines, people are still using aerial
parts of H. foetidus to clean house chimneys, stoves,
wood ovens and as oil lamp wicks [11]. Even though
there are many species of Helleborus known as having
active principles, H. niger is the most widely used in
traditional medicine for a wide range of symptoms and
diseases [12]. In European folk medicine, H. niger was
used in expelling thick slime, as a remedy for sore joints,
as an emetic and laxant and for preparing sneezing
powders [13]. Alkaloids, glycosides and saponosides
found in species of Helleborus are considered to
be the constituents responsible for central nervous
system activity [14]. Tea from hellebore leaves, having
a sedative effect, was used in Danish folk medicine to
treat epilepsy and convulsions [15].
In traditional veterinary practice, different species
of Helleborus were used to treat infectious and
inammatory diseases. In Italy, H. foetidus and H. viridis
were used to treat many kinds of disease in pigs, cows,
sheep, mules and donkeys. Pneumonia was diagnosed
by inserting a piece of root in a subcutaneous cut or
in an incision on the ear of the pigs, with the animals
declared ill if the cuts swelled during the next day
[8,16]. In Sicily, H. bocconei subsp. siculus was used
to diagnose and to cure pneumonia in cattle [17,18].
H. odorus was used in a similar way in Serbia, with a
part of the stem being inserted in a hole in a sick sheep’s
ears [19]. Chronic inammatory diseases in pigs and
sheep [20] and infectious diseases [21] were treated
based on the anti-inammatory and antibacterial effects
of H. purpurascens roots, used as a transcutaneous
implantation. A decoction of H. foetidus was used to
clean wounds of animals when no better option was
available [11]. Crushed leaves of H. niger were used in
veterinary medicine in Pakistan as an antihelmintic [22].
H. foetidus is reported as having anti-insect
activity [23]. Many insects avoid hellebores, but some
species are using specic chemical compounds
contained in the plants to defend themselves against
predators. A furostanol saponin found in leaves of
H. foetidus and H. viridis was also found in the
hemolimph of Monophadnus sp. larvae at a more
than 200 fold higher concentration compared to plant
cells. It seems likely that this compound is involved in
the chemical defence of Monophadnus larvae against
potential insect predators [24].
3. Toxicity of Helleborus sp.
The use of H. orientalis extracts is mentioned in Homer’s
Odyssey [25]. Helleborus species are considered to
be toxic, but usually the poisonings are related to an
incorrect dosage [26]. Toxic features of hellebores are
determined mostly by the aglycons of cardiac steroids
but protoanemonin - a toxic γ–lactone, is also a toxic
compound.
Poisoning by cardioactive steroids is primarily
localized to the digestive system with severe
gastrointestinal irritation, vomiting and diarrhea [25,27].
Acute toxicity effects on the central nervous system
include lethargy, confusion and weakness that are not
caused by hemodynamic changes. Chronic toxicity
due to hellebore poisoning is difcult to diagnose and
less obvious. It could determine anorexia, weight loss,
neuropsychiatric disorders (confusion, drowsiness,
headaches, delirium, and hallucinations) and visual
disturbances. Cardiac manifestations of this kind of
273

Natural compounds with important medical
potential found in
Helleborus
sp.
poisoning include a multitude of cardiac dysrhythmias
[25].
Poisoning with protoanemonin occurs mostly in
animals and starts with salivation, vomiting, inammation
of the mouth and throat, abdominal pain that can be
followed by severe ulcerations of the mouth and damage
to the digestive and urinary systems. A severe poisoning
will present colored diarrhea and dark or blood stained
urine, unsteady gait, dizziness, impaired or lost vision.
Although fatal poisoning is rare, when it occurs death
is preceded by convulsions [28,29]. Protoanemonin has
also a toxic effect as a sub-epidermal vesicant, caused
by inactivation of enzymes containing SH groups [30,31].
4. Chemical analysis of Helleborus sp.
The rst reports of a general chemical composition of
hellebores date from 1943 when Karrer isolated for the
rst time from H. niger a cardiac glycoside named hellebrin
[32]. A few classes of compounds from Helleborus spp.
were identied and reported starting with the 1970s
[33,34], including: cardioactive glycosides (hellebrin,
degluco-hellebrin), steroidal saponins, ecdysteroids and
γ-lactones (protoanemonin).
4.1 Cardioactive glycosides
In plants, cardioactive glycosides were discovered in
angiosperms, both in monocotyledons and dicotyledons.
The therapeutic action of cardioactive glycosides
depends on the structure of the aglycone, and on the
type and number of sugar units attached. Two types of
aglycone are known: cardenolides (digitoxigenin from
Digitalis purpurea - C23 compounds), and bufadienolides
(hellebrigenin from Helleborus niger - C24 structures).
The lactone ring is ve-membered in cardenolides and
six-membered in the bufadienolides [35]. Cardenolides
are common substances and there are a few genera
that yield these compounds: Strophanthus, Convallaria
and Digitalis [35]. The bufadienolides are found in
Helleborus, Cotyledon, Kalanchoe, Scylla, Bowiea,
Homeria, Moraea, Bersama, Melianthus and Thesium.
Animal sources of bufadienolides include reies
(Photinus spp.), snakes (Rhabdophis spp.) and toads
(Bufo spp.) [36].
Hellebrin, shown in Figure 1, is the most familiar
cardioactive glycoside of Helleborus, being isolated and
quantied in roots and rhizomes of H. purpurascens
[37]. The structure of hellebrin was determined in 1995
by Muhr et al. using 2D NMR techniques [38]. Its ratio
was monitored in the whole plant, compared to degluco-
hellebrin [34]. This fraction was calculated also in
cultivated H. odorus and H. viridis over the annual cycle,
and the results showed that concentration of hellebrin
is higher in H. purpurascens compared to other species
[34].
4.2 Steroidal saponins
Saponins are steroid or triterpene glycosides widely
distributed in the plant and marine animal kingdoms
and include a large number of biologically active
compounds. In 1976, a medicine proposed to treat
ulcers and containing the main sapogenins from roots
and rhizomes of Helleborus spp. was registered under a
U.S. patent (O.I. Bruchköbel, 1976, Medicine containing
the main sapogenin from Helleborus, U.S. Patent
3,956,491). This class of natural compounds has a wide
structural diversity which may explain their multiple
ranges of bioactivity reported so far [39]. Some of the
steroidal saponins have cancer-related activity, as well
as immunomodulating, antihepatotoxic, antiviral, and
antifungal activities. Saponins have important action
Figure 1. Hellebrin.
O
O
O H
O H
O
O
O
O
O H
H O
O
O H
O H
HO
HO
Figure 2. Saponins skeletons found in Helleborus sp.
O
F u ro st an
O
O
S p ir o s ta n
274

M.C. Maior, C. Dobrotă
on the cardiovascular, central nervous and endocrine
systems [40-42]. Saponins reported in Helleborus spp.
have furostan and spirostan skeleton structures, shown
in Figure 2.
4.3 Ecdysones
Ecdysones are plant derived sterols, classed
as triterpenoids, with a very polar structure [35].
Phytoecdysteroids have been reported to occur in
over 100 terrestrial plant families representing ferns,
gymnosperms and angiosperms [43]. Ecdysteroids
can be found also in insect and crustacean families
and play a defensive role against predators or are
involved in plant metabolism [26,43]. Plant cells
synthesize phytoecdysteroids from mevalonic acid
in the mevalonate pathway, using acetyl-CoA as a
precursor. The largest concentration of ecdysteroids
was found in tissues involved in the defense mechanism
of the plants. Ecdysteroids are used by plants as a
protective mechanism against phytophagous insects.
Phytoecdysteroids can mimic the activity of moulting
hormones in insects and can disrupt moulting, perturbing
normal insect development [44]. When ingested by
non-adapted insects they lead to weight loss, moulting
disruption and/or mortality [45].
Some of the pharmacological effects of
phytoecdysteroids are summarized below: adaptogenic
and antidepresive, hepatoprotective (related to
phospholipid metabolism), chemopreventive (in
cancer treatment), effective in the control of diabetes,
and antimicrobial effects against some fungi and
bacteria species [43]. A well-known ecdysteroid,
20-hydroxyecdysone, found frequently in Helleborus
spp. is shown in Figure 3.
4.4 Protoanemonin
Protoanemonin (Figure 4) is a toxic γ-lactone of
hydroxy-penta-2,4-dienic acid [13]. Frohne and
Pfander described protoanemonin as a volatile, oily
and irritant substance with high afnity for SH groups
[31]. Protoanemonin is considered a biologically active
compound having antimicrobial [46,47], fungicidal
[48], and antimutagenic effects [49,50]. However,
Dickens reported brosarcoma development when
2 mg protoanemonin was injected repeatedly in rats
[51]. When drying the plant, protoanemonin comes
into contact with air and dimerizes to anemonin, a
non-stable compound which is hydrolyzed to a non-
toxic carboxylic acid [52]. Anemonin is also fungicidal
(less potent than protoanemonin) [48] and both of them
(anemonin and protoanemonin) have a sedative effect
[53]. Anemonin has also antispasmodic properties
[54]. Anemonin inhibits cellular tyrosinase activity and
affects protein and mRNA levels in human melanocytes
inhibiting melanin synthesis. That is why, anemonin
may be used as a cosmetic agent for hypopigmentary
purposes [55]. Anemonin is able to inhibit nitric oxide
(NO) production by modulating the expression of iNOS
(inducible nitric oxide synthase) [56,57]. This could
explain its anti-inammatory effects [57,58]. It was also
reported that anemonin inhibits endothelin-1 (ET-1)
[56,57] and intercellular adhesion molecule-1 (ICAM-1)
in RIMEC (rat intestinal microvascular endothelial cells)
preventing intestinal microvascular dysfunction [57]. It
was suggested that anemonin can be used for treating
various other diseases, including cardiovascular
diseases and arthritis when ET-1 and NO activation are
involved in mediation of pathogenesis [57].
Figure 4. Degradation pathway of protoanemonin.
O
O
H
2
C
P ro to a n em o n in
OO
O
O
C H
2
H
2
C
H
H
H
H
A ne m o nin
O H
H O
D im e r iz at io n
H y dr o ly si s
O
O
O
O
Figure 3. 20-Hydroxyecdysone
O H
O H
O H
O H
O
HO
H O
H
H
275

Natural compounds with important medical
potential found in
Helleborus
sp.
4.5 Current chemical analysis on Helleborus sp.
Over the past few years, different Helleborus species
have become the subject of phytochemical studies due to
their putative potential in producing valuable secondary
metabolites and due to the development of analytical
methods that offered the opportunity to perform faster
biochemical characterization of plant extracts. In order
to decipher their biochemical content, different chemical
analysis techniques were used: chemical transformations;
analytical methods (chromatography): TLC (Thin Layer
Chromatography), HPLC-MS (High Performance
Liquid Chromatography - Mass Spectrometry), RP-
HPLC (Reverse Phase - High Performance Liquid
Chromatography) GC (Gas Chromatography) and
various spectroscopic methods using 1D and 2D NMR
techniques: COSY (Correlated SpectroscopY), TOCSY
(Total Correlation SpectroscopY), ROESY (Rotating
frame nuclear Overhauser Effect SpectroscopY),
HMQC (Heteronuclear Multiple Quantum Correlation),
HMBC (Heteronuclear Multiple Bond Correlation). New
chemical compounds were identied and described
in Helleborus spp. and many of them seem to have
pharmacological activity and promising potential for
medical research. Chemical analysis advances on
Helleborus spp. through the last years are shown in
Table 1.
Species Tissue source Compounds Reference
H. odorus ssp. laxus,
H. viridis ssp. viridis
Roots and
rhizomes
hellebrin, degluco-hellebrin, hellebrigenin, bufatetraenolide, spirostane-
type steroids, β-ecdysterone, 5β-hydroxyecdysterone [59]
H. purpurascens Roots 1 new steroidal saponin, hellebrin,
β-ecdysterone, 5α-hydroxyecdysterone [60]
H. torquatus Seeds 3 new bufadienolides, 2 new ecdysteroids [61]
H. atrorubens Leaves 8 avonoids, 7 phenolic acids [62]
H. orientalis Rhizomes 1 novel polyoxygenated spirostanol glycoside [63]
H. orientalis Rhizomes 1 new bufadienolide rhamnoside, 2 bufadienolide glycosides, 5 new
spirostanol saponins [64]c
H. orientalis Rhizomes 2 new bisdesmosidic furostanol saponins,
2 new furospirostanol saponins [65]c
H. orientalis Rhizomes 2 new polyoxygenated spirostanol bisdesmosides, 1 new
polyoxygenated spirostanol trisdesmoside [66]c
H. viridis Leaves 2 new furostanol saponins, 3 new quercetin glycosides [67]
H. orientalis Roots a furostanol saponin, a phytoecdysteroid [68]
H. foetidus Leaves 1 new caffeoylated quercetin glycoside, 1 steroidal saponin, anemonin,
2 phenol glycosides [69]
H. caucasicus Roots and
rhizomes 2 sapogenins [70]
H. caucasicus Roots and
rhizomes
4 new polyhydroxylated and polyunsaturated furostanol glycosides
(caucasicosides) [71]
H. caucasicus Leaves 20-hydroxyecdysone, 1 spirostan [72]
H. bocconei ssp.
intermedius Roots 2 new furostanol saponins, 1 furospirostanol saponin, ecdysterone,
5β-hydroxyecdysterone [73]c
H. orientalis Rhizomes 8 new furostanol glycosides, 2 known furostanol glycosides [74]c
H. thibetanus Rhizomes 2 new bufadienolides, 2 bufadienolides [75]c
H. thibetanus Rhizomes 2 new bufadienolide glycosides, 2 ecdysteroids, 6 bufadienolide [76]
H. viridis Leaves 3 new steroidal saponins [77]
H. caucasicus Leaves 9 new furostanol glycosides, 9 furostanol glycosides, 1 bufadienolide,
1 ecdysteroid [78]
H. niger Leaves 1 new phenyllactic acid, 1 new avonol glycoside, 1 kaempferol
glycoside [79]
Table 1. Progress made over the last years on chemical analysis of Helleborus sp. c Authors that tested the compounds described in their papers
for cytotoxicity.
276

M.C. Maior, C. Dobrotă
4.6 Hellethionins
A new family of thionins was discovered in the roots of
H. purpurascens and named hellethionins. Hellethionins
are part of group of α/β thionins, small-sized multiple-
cystine peptides, that were found in endosperms
of grains, in seeds and in parasitic plants [80,81].
These are considered to be excellent candidates as
biocontrol agents of plant pathogens [82]. In vitro
experimental studies suggest that thionins are toxic to
bacteria, fungi, and yeasts due to their interaction with
membrane phospholipids and their capacity to form
ion channels. Possible roles in the defense of plants
against pathogens, directly at the membrane level,
are assumed. Also, hellethionins are thought to have a
function in cell death [83]. All thionins have the property
to refold correctly into the native structure, making them
robust scaffolds that can be exploited for screening of
optimized membrane activities and of cytotoxicity [81].
5. Pharmacological and therapeutic
effects
Studies concerning the pharmacological and therapeutic
effects of whole or partial Helleborus spp. extract have
been conducted in various animal models and in vitro
systems.
5.1 Antirheumatic and anti-inammatory
activity
Kerek presented for the rst time the benecial action
of a drug named Boicil [84], which was made from
H. purpurascens root and stem extract (V. Boici, 1977,
Analgesic substance derived from the Helleborus plant
and method of making same, U.S. Patent 4,012,505)
and has been successfully used in Romania for decades
to treat rheumatic pains. H. purpurascens extracts
exhibited high biological activity with lasting analgesic,
myorelaxant, and blood vessel regulating actions.
H. orientalis was tested in mice, using a carrageenan-
induced hind paw edema model, to determine anti-
inammatory activity and a p-benzoquinone-induced
abdominal constriction test, to determine antinociceptive
activity. The authors reported that a dose of 500 mg/kg
ethanolic extract of roots of H. orientalis showed anti-
inammatory activity without inducing any gastric
damage. Root and herb extracts, both alcoholic and
aqueous, were reported as antinociceptive, when a
dose of 500 mg/kg was tested [85].
5.2 Immunomodulatory activity
Many studies made on animals have demonstrated the
immunostimulatory effect of Helleborus spp. extracts.
Immunostimulatory effect of H. purpurascens was
reported in sheep. Increased number of lymphocytes
(2×) and neutrophiles (3.5×) after 48 h of injecting young
sheep with 5% decoct of radix and rhizome showed an
improved immune response of the animals [86]. Activation
of rapid, non-specic defensive mechanisms and poor
haemolysis has been reported when subcutaneous,
intraperitoneal and intramuscular application of different
concentrations of the extract of rhizome and root of
H. odorus were performed. A pronounced leukocytosis
and an increased percentage of neutrophil granulocytes
have been recorded [87]. These results were conrmed
by other authors supporting the idea that Helleborus
extracts can trigger unspecic immune response in
animals [20].
A feature related to immunomodulation was
assigned to chemical compounds found in Helleborus
spp. MCS-18 (macrocyclic carbon suboxide), a
multi-anionic compound extracted and puried from
H. purpurascens roots, classied as New Chemical Entity
(NCE) and originated in plants [88], was characterized
as a highly potent inhibitor of Na, K-ATPase and of the
SR Ca-ATPase [89,90].
MCS-18 has a complex structure and in the last
years has been intensively studied. It induces in vitro
up-regulation of the immune modulatory cytokines IL-10
and TGF-β [91]. Also, MCS-18 efciently downregulates
T-cell-dependent antibody (Ab) formation in mice, where
a Toll-like-receptor (TLR) blockade might be involved.
MCS-18 leads to a strong attenuation of antibodies
against tetanus toxoid if administered together with
the Ab elicitor Freund’s Complete or TLR antagonists
in various combinations. These ndings suggest that
MCS-18 could be a potent, non-toxic antagonist or
a down-regulator of the TLR signaling pathway [92].
MCS-18 proved to be a potent antagonist of the capsaicin
activated vanilloid type pain receptors (TRPV1) and
explains its local analgesic action [93].
To MCS-18 was assigned an important role in
autoimmunity. MCS-18 strongly reduced the paralysis
associated with the experimental autoimmune
encephalomyelitis (EAE), which is a murine model
for human multiple sclerosis. This compound can be
used in the EAE model not only as a prophylactic, but
also as a therapeutic setting. It was also proved that
MCS-18 induces a long-lasting suppressive effect and
is able to inhibit the expression of typical molecules of
mature dendritic cells (DC). This compound impeded
the formation of the typical DC/T-cell clusters, which
are essential to induce potent immune responses [94].
Another paper that supported the above mentioned facts,
added that MCS-18 also reduced B-cell proliferation and
immunoglobulin production [95].
277

Natural compounds with important medical
potential found in
Helleborus
sp.
MCS-18 treatment almost completely reduced
leukocyte inltration in the brain and in the spinal
cord. Using EAE assay in vitro as well in vivo the
authors were able to show that MCS-18 exerts a
strong immunosuppressive activity with remarkable
potential for the therapy of diseases characterized by
a pathologically over-activated immune system. They
concluded that MCS-18 is an efcient modulator of
immune response in vitro and in vivo and that is able
to inhibit the disease symptoms at different stages [94].
The authors demonstrated that MCS-18 has a very
potent non-toxic immune-suppressive activity and their
results were strongly supported by clinical trial data
reporting that MCS-18 is a highly effective drug in the
treatment of arthritis.
Type I diabetes is still a serious problem for which
the medical community is looking for new ways to
prevent and overcome. MCS-18 could become a
promising answer for people suffering diabetes.
NOD-mouse model was used to observe the MCS-18
treatment, indicating signicantly reduced islet T-cell
inltrates and the rate of T-cell proliferation. Periinsular
inltrates in the MCS-18 treated animals showed a
signicantly enhanced number of Foxp3(+) CD25(+) T
cells, indicating the increased presence of regulatory T
cells. In the animal group which had been treated with
MCS-18, 70% of the animals showed a diabetes free
survival, in comparison with untreated animals where
only less than 10% were free of diabetes. These
studies showed that MCS-18 exerts an efcient
immunosuppressive activity with remarkable potential
for the therapy of diseases characterized by pathological
over-activation of the immune system [96].
5.3 Antioxidant activity
ROS (reactive oxygen species) are products of cellular
metabolism having important functions in cell signaling,
homeostasis and apoptosis, activation of host defense
genes and mobilization of ion transport systems. ROS
have an important role in immune system functioning.
Five progressively puried MCS-products from
H. purpurascens were tested as possible antioxidants
and/or modulators of ROS production and released
from human polymorphonuclear granulocytes cells.
One of the fractions MCS-Dx proved to be a potent ROS
scavenger and it could be used as adjuvant in antioxidant
therapy [97]. Even a simple aqueous or hydroalcoholic
extract, concentrated by ultraltration, proved to have
high antioxidant activity. The DPPH (2,2-diphenyl-
1-picrylhydrazyl) inhibition value of hydroalcoholic
extract showed the highest antioxidant activity (79%),
while the concentrated aqueous extract showed 73%
DPPH inhibition [98]. Čakar et al. measured antioxidant
activity in root and leaf extracts from H. odorus,
H. multidus and H. hercegovinus leaf extract exhibiting
a higher antioxidant activity (IC50 values between
0.12-0.89 mg/ml) compared to root extract (IC50 values
between 0.72-3.10 mg/ml) [99]. Another compound
found in Helleborus spp. that proved to be antioxidant
is 20-hydroxyecdysone, an ecdysteroid that acts
as a uoride-stimulated respiratory burst modulator
in the same manner as water soluble antioxidants,
chlorpromazine and emoxipin [100]. This compound,
alone or acting in synergy, could render the antioxidant
activity assigned to these plants.
5.4 Antimicrobial activity
Although many Helleborus spp. have been used to treat
infectious diseases in animals, the antimicrobial activity
of these species was not screened. There are just a few
studies concerning this aspect [101,102]. Roots from H.
bocconei ssp. siculus were tested for their antibacterial
activity against microorganisms responsible for
respiratory infections. Seven strains of microorganisms
responsible for these types of infections were tested:
Staphylococcus aureus ATCC 29213, Streptococcus
pneumoniae ATCC 49619, Escherichia coli ATCC
25922, Haemophilus inuenzae ATCC 49247, Moraxella
catarrhalis ATCC 25238, Pseudomonas aeruginosa
ATCC 27853, and Stenotrophomonas maltophilia ATCC
13637. The root methanolic extract and its bufadienolide
fraction showed the lowest Minimum Inhibitory
Concentration values (100 μg/ml) against Moraxella
catarrhalis (0.2, 0.1) and Streptococcus pneumonia
(0.4, 0.1) [101].
5.5 Cytotoxicity and anticancer properties
Many species of Helleborus are seen today as potential
sources for anticancer drugs. Studies involving extracts
or chemical compounds gave optimistic results related
to cancer inhibition and cytotoxicity. Lindholm et al.
[103] tested, through a large-scale screening protocol,
100 fractionated plant extracts, seven of them showing
interesting cytotoxic properties. The cytotoxic activity
was not fully characterized, but H. cyclophyllus extract
proved to have antitumoral potential. Another species,
H. caucasicus, also showed cytotoxic activity. Root and
rhizome alcoholic extracts were tested against human
lung cancer (A-549), human colorectal cancer (DLD-1)
and normal skin broblasts (WS1) and their cytotoxic
dose was reported as being rather low (0.002 μg/ml)
[104].
H. niger extracts were used for in vitro tests
involving hematological malignancies. Inhibition of cell
proliferation is caused by specic apoptosis induction
via mitochondrial pathway and caspase-3 processing.
278

M.C. Maior, C. Dobrotă
Apoptosis induction was observed in lymphoma (BJAB),
leukemia (REH, Nalm6, Sup-B15) and melanoma
(Mel-HO) cells. A better efciency in inducing apoptosis
was recorded when whole plant and root extracts were
used compared to leaf and blossom extracts. When
a concentration of 0.75 mg/ml H. niger extract was
added to a BJAB cell line, an inhibition up to 96.5% of
the proliferation rate was recorded. Synergistic effects
were also observed when a cytostatic drug - vincristine
- was added to the BJAB cell culture in combination with
H. niger whole plant extract.
Although apoptosis in Mel-OH cell line was correlated
with Bcl-2 protein overexpression, sensitivity to H. niger
extract was clearly recorded [105]. Another extract from
roots of H. bocconei ssp. intermedius proved to have
cytotoxic activity against rat C6 glioma cells. Three
isolated compounds, a hydrolyzed form of helleboroside
B, a furospirostanol saponin and polypodyne B showed
signicant cytotoxic activity, recording a 60-70%
inhibition of the cell growth [73].
In a similar comparative study, root extracts from
H. odorus, H. multidus and H. hercegovinus proved to
be more efcient than leaves extract of the same species
on inhibiting growth of BJAB cell lines. The strongest
antiproliferative activity was indicated for root extracts of
H. multidus and H. hercegovinus which inhibited BJAB
cell lines growth with 50.14% and 49.04%, respectively
[99]. Hellebosaponin C, a spirostanol glycoside,
and another two furostanol glycosides isolated from
rhizomes of H. orientalis exhibited a moderate cytotoxic
activity against human oral squamous cell carcinoma
(HSC-2) [66,74].
Two new bufadienolides (tigencaoside A, B) found in
rhizomes of H. thibetanus were also tested for cytotoxic
activity against human cancer cells 3LL, MCF-7, QGY-
7701 and BGC-823. The IC50 values of tigencaoside A
on the tested cell lines ranged between 105.23–253.12
μg/ml and for tigencaoside B exhibited values were
between 56.54–86.45 μg/ml [75].
Potential cytostatic and cytotoxic effects of
H.purpurascens hydrous extract (HphE) and
H. purpurascens hydroalchoholic extract (HphaE) were
recently evaluated by Vochita et al. [106]. Total and
fractionated polyphenolic compounds were extracted
from roots and rhizomes of H. purpurascens and tested
on HeLa cancerous cells. Tested biopreparations as
0.45 µm MF-HphE (microltrate of HphE), 30,000 Da
UF1P-HphE (permeate of rst ultraltrate), 10,000 Da
UF2C-HphE (concentrate of second ultraltrate),
10,000 Da UF2P-HphE (permeate of second ultraltrate)
exert a very strong cytostatic effect with values over
90%. Other biopreparations as HphE (total hydrous
extract), 30,000 Da UF1P-HphE, 3,000 Da UF3C-HphE
(concentrate of third ultraltrate), 3,000 Da UF3P-HphE
(permeate of third ultraltrate) induce an inhibition of the
tumor cell between 59.46% and 67.80%.
Thionins - the class of peptide that was found also
in H. purpurascens - have been proposed as potential
immunotoxins in tumor therapy. Cytotoxicity of thionins
has led to the development of a potential application:
targeting thionins by tumor-specic antibodies, which is
expected to support antitumor therapy [81]. Anticancer
properties of hellethionins were reported by Kerek.
Acting alone or synergistic, hellethionins proved
to inhibit proliferation in different cancer cell lines.
Hellethionin C, at very low concentration (2 µg/ml),
causes a clear inhibition of proliferation in MFC-7 cell
line (breast cancer cell culture). Hellethionin C and
hellethionin D, at a concentration of 100 µg/ml, strongly
inhibit (48.36% and 58.66%, respectively) the culture of
Colo 205 (colon cancer cell line). Hellethionin C at 50 µg/ml
in combination with MCS-18 at 100 µg/ml inhibits the
growth of Colo 205 with 66%. A mixture of hellethionins
B (B1: B2: B3 = 1:1:1), hellethionin C and hellethionin D
at a concentration of 100 µg/ml were also able to inhibit
with 50% the growth of a lung carcinoma cell line (Kerek
F., 2010, Petides having a high cysteine content, U.S.
Patent 7,750,114 B2).
Another recently registered patent is describing
methods for obtaining new cardiac steroids starting
from hellebrin and hellebrigenin and using conventional
techniques of synthetic organic chemistry (J. Dewelle, M.
El Yazidi, E. Van Quaquebeke, N. De Neve, T. Mijatovic,
L. Ingrassia, et al., 2010, Hellebrin and hellebrigenin
derivatives, WO2010/102673). Apparently, the 21 novel
hellebrin and hellebrigenin derivatives have a cytotoxic
activity with reduced general toxicity. These compounds
were tested against six human cancer cell lines in
the MTT tests, and a number of these compounds
showed very potent in vitro antitumor activity with IC50
values in the nanomolar range. That was why these
compounds were proposed as medicines for cancer
treatment. The authors also used two normal broblast
cell lines to investigate potential compound selectivity
towards cancer. Almost all described compounds
displayed in vitro marked selectivity toward cancer cells
(J. Dewelle, M. El Yazidi, E. Van Quaquebeke, N. De
Neve, T. Mijatovic, L. Ingrassia, et al., 2010, Hellebrin
and hellebrigenin derivatives, WO2010/102673). We
may consider that Helleborus spp., having so many
valuable chemical compounds, are potential pools for
a wide range of pharmacological agents and bioactive
molecules.
H. niger aqueous extracts were tested in order
to asses a DNA destabilizing risk. Sister Chromatid
Exchange assay (SCE) was used to detect DNA
279

Natural compounds with important medical
potential found in
Helleborus
sp.
damage. The authors found that H. niger aqueous
extract possesses immunomodulating properties
and also exerts slight effects of DNA destabilization
and might have a mutagenic effect on human PBMC
(peripheral blood mononuclear cells) [107].
6. Micropropagation and genetic
transformation
Harvesting wild medicinal plants is often nonproductive.
In the case of plants such as Helleborus, where the
targeted medicine is located in roots and the seeds have
a low germination rate, sustainable propagation could
be a better option. The latest considerable discoveries
concerning hellebores have used in vitro cultivation as
an easier way to study them and for nding new methods
to use their features. In vitro techniques consisting of
micropropagation, callus culture, cell suspension culture
or somaclonal variation, could improve the production of
secondary metabolites. Smulders and de Klerk reported
that 4 different lines of Helleborus spp., having the same
source, remained unmodied during many subcultures,
a feature which may be used in producing new
varieties and in synthesizing new chemical compounds
[108]. The antimicrobial activity of hellethionins could
be used in genetic engineering. Transgenic plants
containing thionin genes can enhance resistance
against pathogens. Hellethionins are peptides that are
promising candidates for engineered plant resistance
in the agricultural industry (81, Y. Ohashi, I. Mitsuhara,
M. Oshshima, M. Ugaki, H. Hirochika, R. Honkura,
et al., 1998, Method for producing disease resistant
plant with thionin gene from Avena sativa, U.S. Patent
6, 187, 995).
After the isolation and identication of these new
important chemicals, the next step in using this potential
would be molecular farming. Genetically engineered
plants with new traits could have extra resistance to
insect attack and improved weed control, or could
produce a large quantity of pharmaceutically active
compounds.
Computer- based analysis revealed that natural
products exhibit a remarkable structural diversity of
molecular frameworks and scaffolds that could be
systematically exploited for combinatorial synthesis.
Natural products offer a rich pool of unique molecular
frameworks and desirable drug-like properties,
rendering them ideal starting points for molecular design
considerations [109].
7. Conclusions and perspectives
Important progress on isolation and identication
of bioactive compounds from Helleborus species
has been made in the past few years. However,
further studies concerning the assessment of their
pharmacological and therapeutic effects and clinical
trials concerning the target-organ toxicity or side
effects, are still required.
There are many studies which strongly support
the view that extracts of the plants belonging to this
genus have benecial therapeutic actions. It is known
that the active principles are effective remedies in anti-
inamatory and antirheumatic conditions, and have
proven efciency as immunostimulators, antioxidants,
antimicrobial and antitumoral agents.
Even if there are some patents concerning active
compounds, Helleborus species are still insufficiently
explored as a source of valuable products and that
further studies need to be done. Precise clinical
trials using a large number of patients have to be
encouraged in order to evidence the efficacious and
possible side effects of the newly found compounds.
New research approaches on this topic may concern
the assessment of the effects of novel compounds
discovered, the mechanisms of their action, the
interplay with known regulatory components at the
protein level to provide an understanding of their
dynamic interactions and how these interactions
orchestrate the biosynthesis of secondary
metabolites in plants. Achieving these goals will
translate into a major advance in our understanding
of how plant bioactive compounds actually work,
since our understantding of interactions at the
cellular level is limited. This will not only generate
fundamental knowledge about the dynamics of plant
biosynthesis, but will ultimately provide us with the
tools to rationally reprogram secondary metabolism
for genetically engineering more useful plants.
Acknowledgements
This work was possible with the nancial support of the
Sectoral Operational Programme for Human Resources
Development 2007-2013, co-nanced by the European
Social Fund, under the project number POSDRU
89/1.5/S/60189 with the title „Postdoctoral Programs
for Sustainable Development in a Knowledge Based
Society”.
280

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