Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family [617487]

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

17 1. Ranunculaceae buttercup : General considerations
1.1. Distribution and taxonomy
Ranunculaceae Juss. (buttercup family) is one of the early lineages flowering
plants comprising about 60 genera and 2500 species, whereas 27 genera can be found
in Europe (HAO et al ., 2015 ).
Aconitum (c. 3 00 sp p), Anemone (c. 200 sp p), Clematis (c. 325 spp), Delphinium
(c. 3 60 sp p), Ranunculus (c. 600 sp p), and Thalictrum (c. 330 spp) are the most
represe ntative genera of the buttercup family ( HEYWOOD et al. , 2007 ).
Plants o f this family are distributed widely all over the world but are centered in
the temperate and cold areas of the northern and souther n hemisphere s, with few
tropical species (Figure 1) . Around 75 % of the genera are distributed in East Asia,
including many endemic plants, while 45 % of the genera are founded in America and
Europe . Species of the buttercup family can be founded from sea level to high altitude,
but m any of them grow in mesic or wet environments , while several are aquatic (e.g.
Ranunculus and Batrachium ) (HEYWOOD et al. , 2007 ).

Figure 1. World d istribution of Ranunculaceae species / Distribuția mondială a speciilor de
Ranunculacee (HEYWOO D et al., 2007)

The Ranunculaceae is one of the largest families among the Dicotyledons .
According to Angiosperm Phylogeny Group , the Ranunculaceae family together
with the Eupteleaceae, Lardizabalaceae, Menispermaceae, Berberidaceae, and
Papaveraceae ar e included in Ranunculales order of Dicotyledonopsida class ,
Angio spermatophytina subdivision, Spermatophyta division .

Cristina -Daniela Kelemen
18
1.2. Morphological traits
Most members of the buttercup family are annual or perennial herbs (e.g.
Anemone , Ranunculus and Thalict rum ), while some are woody climbers (e.g. Clematis ),
lianas or shrubs with adventitious roots, rhizomes, bulbs or tubers (CRISTEA, 2014 ).
The stems are unarmed. The leaves are opposite or spirally arranged, simple
(e.g. Caltha ) palmately (e.g. Ranunculus ), whole or divided. Most species have both
basal and cauline (stem) leaves, which are usually compo und or lobed but can be also
simple. The petioles are broadened into a sheathing base. Stipules are absent, except
Caltha , Thalictrum and Trollius (HEYWOOD et al., 2007).
In Ranunculaceae species , the floral parts such as petals, sepals, stamens , and
pistils are all of an indefinite numbe r and separate, while, the advanc ed plant families
have reduced the numbers of floral parts and the parts are often fused tog ether.
The inflorescence is terminal. The flowers are bisexual rarely unisexual , which
can be solitary (e.g. Anemone ), but more usually they are grouped in cymes , panicles , or
spikes . The floral wrapping can be simple or double on the type (4) 5 -6 (20) and the
floral elements arranged spirally or cyclically on a conical elongated receptacle
(CRISTEA, 2014). In many species, the sepals are colourful and appear petal -like. In
these species, the petals can be inconspicuous or absent . The flowers are usually
hermaphrodite and regular (e.g. Anemone , Ranunculus ) or irregular in same genera
(e.g. Aconitum , Delphinium ) as illustrated in Figure 2.

Figure 2. The regular and iregular type of flower founded in Ranunculaceae family/ Floarea de tip
regular și iregular regasită în familia Ranunculaceae .
(https://www.wildflowers -and -weeds.com/Plant_Families/Ranunculaceae.htm)

The fruit is most commonly represented by an achene (e.g. Ranunculus ,
Clematis ), follicle (e.g. Helleborus , Nigella ), or berr y (e.g. Actaea ).
The seeds have a small straight embryo and copious endosperm.

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

19 Most of the annual species are self pollinated, but there are also species which
are insect -pollinated (e.g. Anemone ), wind – pollinated (e.g. Thalictrum ) or bird
pollinated (e .g. Aquilegia ) (HEYWOOD et al. , 2007) .
Various reproductive strategies such as vegetative reproduction, self –
compatibility, and agamospermy are used within buttercup (HÖRANDL et al. , 2005) .
Considering all above mentioned morpholo gical characters, t he family shows a
wide variation especially in flow er structure (Figure 3) , fruit types and pollination
methods.

1.3. Phylogeny and genetic diversity
The Ranunculaceae represent a highly diverse and cosmopolitan tribe wich has
been considered "simple" from an evolutionary point of view , due to the flower
structure ( all the parts are independently attached ), which has retained the most
ancestral characteristics over time . Besides, the buttercup has been considered as one
of the most basal families within the eudicots and its crown age has been estimated as
ca. 75 Ma (SIMPSON, 2006; ANDERSON et al., 2005 ).
Polyploidy and hybridization may be regarded as important factors for
speciation and evolutionary success of buttercup. Autopoly ploidy and allopoly ploid
origin of species ha s been suggested for many taxa o f the family ( e.g., Ranunculus
cassubicifolius , R. auricomus , R. kuepferi ). Hybridization occurs within many groups,
including Ranunculus montanus and Ranunculus polyanthemos and water -buttercups
(Ranunculus subg. Batrachium ). The hybridization be twee n spe cies of different
sections has been documented only for s ome white -flowering alpine spe cies of Europe
(HÖRANDL et al. , 2005 ).
Systematics and phylog enetic relationships within the Ranunculaceae buttercup
has always been controversial due to the great dive rsity of morphological features and
lack of molecular phylogeny within the family (EMADZADE et al., 2010 ).
Various studies have been performed to clarify the systematic s of
Ranunculaceae family based on morphological characters (e.g. features of achenes ,
carpels, flowers, fruit s and the perianth ) (TAMURA, 1993, 1995 ), on molecular data
(JENSEN et al., 1995; WANG et al. , 2005; EMADZADE et al., 2010 ; ) and on a combined
molecular and morphologi cal dataset ( WANG et al. , 2009 ). However, there is a
discrepancy between these classific ations probably due to the great d iversity of

Cristina -Daniela Kelemen
20 morphological features especially the ample variation of floral characters e.g., bisexual
or unisexual flowers, petaloid or sepaloid sepals and the presence or absence of petals.
Molecular studies have given i nsights into the phylogenetic relationships within
the family by using several molecular methods such as chlorop last restriction site
variation, the ehloroplast gene sequences, ribosomal DNA seq uences, and nucl ear DNA
sequenees .
Recently, t he classificati on of the buttercup family have been considerably
improved based on data of nuclear (e.g ISSR markers ) and plastid markers ( e.g. nrITS,
matK, trnK, rbcL , psbJ -petA) , that provide a framework for understanding
relationships and character evolution within th e tribe (WANG et al., 2005, 2009 ,
HÖRANDL et al., 2005; PAUN et al., 2005; LEHNEBACH et al., 2007; HOOT et al., 2008;
HOFFMANN et al., 2010 ; XU et al., 2013 ; CAI et al. , 2010 ).
The main division within the family is trad itional ly between 2 types of
chrom osomes, T type which is typical for Thalictrum and R type founded in Ranunculus
and its relatives.
According to the widely used classification of TAMURA (1993) , based on the
chromosome and floral characteristics of Ranun culaceae species, five sub -families are
recognized within buttercup family namely Hydrastidoideae, Thalictroideae,
Isopyroideae, Ranu nculoideae, and Helleboroideae. Most recent , PENG et a l., (2006)
divided the Ranunculaceae family into six sub -families based on a comprehensive
study of the p hylogeny, chemo -taxonomy, ethnopharmacology, and bioactivity of the
Ranunculaceae species including Helleboroideae, Ranunculoideae , Cimicifugoideae,
Isopyroideae, Thalictroideae, and Coptidoideae . In other study Wang et al. (2009)
retrieved three major cla des within buttercup family defined by th eir basic
chromosome numbers: x=7, corresponding to Thalictroideae, x= 8, corresponding to
Ranunculoideae and; x= 9, corresponding to Coptidoideae .
Therefore, nowadays the family is mainly treated as comprising three or five
subfamilies based on the molecular data.
As stated in the cladogram (Figure 4) proposed by HAO et al., (2015) based on
molecular , cytology and morphologic data, previously reported by APG II and WANG et
al., (2006) , Ranunculaceae buttercup consists of five sub-families namely
Glaucidiaceae, Hydrastidoideae, Coptoideae, and the evolutionarily young
Thalictroideae and Ranunculoideae. Ten tribes (e.g. Aconitum , Anemone , Caltha,
Hepatica , and Ranunculus ) are included in sub -family Ranunculoideae , while, the
subfamily Thalictroideae includes Isopy rum , Dichocarpum and Aquilegia , which belong
to the previously proposed sub -family Isopyroideae ( Figure 3 ) (HAO et al., 2015 ). The
underlined genera ( Aconitum , Anemone , Caltha, Hepatica , Ranunculus and Trollius )
from Figure 4 are those that have been treated in the studies of this thesis.

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

21

Figure 3 Ranunculaceae cladogram according to HAO et al (2015) / Dendograma familiei
Ranunculaceae dupa HAO si colab . (2015)

1.4. Plant economic use
Many genera of the butterc up family have species that are used as ornamental
plants. Most of these genera are familiar to nurserymen and gardeners across the
world including Aconitum (monkshoo d), Anemone (columbines), Clematis (showy
herbaceous border plants), Helleborus (Christmas rose ), and Trollius (globe flowers) .
Likewise , other genera of the buttercup include ornamental species, many with
cultivated varieties such as Actaea , Aquilegia , Delphinium , Caltha , Hepatica, Nigella,
Ranunculus and Thalictrum (HEYWOOD et al ., 2007) .

Cristina -Daniela Kelemen
22 Although many species are poisonous, a number of them have been used for
centuries as spices (e.g Nigella sativa seeds) and some as vegetables (e.g. Aconitum sp.,
Caltha palustris , Clematis vitalba , and Ranunculus ficaria ) after cautious processing
(dry or co oking) to reduce their toxicity (VITALINI et al., 2010; HAO e al. 2015) .
Besides, Nigella sativa seeds are used as sniffed to restore a lost sense of smell
or used against insects as an insecticide . Insecticidal prop erties were also repor ted for
Aconitum species (e.g. Aconitum napellus) , while Anemone species a re d escribed as a
pesticide (DUKE, 2002 ).
In addition, many plant s belonging to the Ranunculaceae family (e.g. Aconitum
chinensis , Aconitum kusnezoffii , Cimicifuga foetida , Nigella sativa , and Pulsati lla
chinensis ) are important components of herbal preparations used in the eastern and
western therapeutics since ancient times , including Ayurveda a nd traditional Chinese
medicine. Although several species with traditional medicinal uses have been
researc hed for their bioactivities and active constituents, many species of the
buttercup remains unexplored from this point of view ( SHYAULA et al ., 2012 ).
1.5. Toxicity
Most of the genera belonging to the buttercup family are well -known as
poisonous plants. Th e compounds involved in the toxicity of the plans are various , but
mainly they are represented by alkaloids (from Aconitum and Delphinium ),
protoanemonin (from Anemone, Clematis , and Ranunculus ) or cardiac glycosides (from
Helleborus ).
The roots of Aconitu m species have been used as poison due to the presence of
fast-acting toxin namely aconitin e higher in roots and flowers than in leaves and stems
(SHYAULA et al. 2012) . The sym ptoms of aconitin e toxicity affect mainly the central
nervous system and the hea rt, together with gastrointestinal signs. There is n ot
described any specific therapy for Aconitum poisoning, usually , a cardiovascu lar
supportive treatment is applied. However, the tubers or aerial parts of Aconitum have
been used for centuries as a herba l drug or vegetable after carefully processing .
Anemone , Clematis and Ranunculus genera contain a high amount of protoanemonin,
which may cause irritation to skin, mucous mem branes and internal bleeding of the
digestive tract when fresh plants are handled or eaten (FROHNE and PFANDER, 2005 ).
However, many studies reported that during drying of plant material,
protoanemonin (an unstable unsaturated lactone) rapidly dim erizes with itself to form
anemonin (an innocuous compound), which is further hydrolyzed to a non -toxic
carboxylic acid ( BERGER and WACHTER, 1998; CHAWLA et al., 2012 ). Likewise , the
fresh plant of Ranunculus contains acrid sap which ca n cause blistering of the skin,
however , its toxicity is lost along drying or cooking (ORAK et al., 2009) .

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

23 1.6. Chemical composition
Ranunculaceae plants contain a broad variety of chemical constituents including
several representative metabolic group s such as alkaloids, flavonoid s, glycosides ,
phenols, lactones, polysaccharides , saponins, and terpenoids among ot hers. According
to HAO et al. , (2015), the chemical constituents of Ranuncu laceae species are not
distributed randomly, thus having taxonomic implications. Alkaloids togethe r with
flavonoids are founded in most of the Ranunc ulaceae family . Alkaloids as
benzylisoquinoline (Figure 5 (1), bisbenzylisoquinoline (Figure 5 (2)), apophinoid
(e.g., magnoflorine) (Figure 5 (3)), and protoberberine types are the most prominent
on buttercup family. Rest of the compound groups are distributed selectively, follow
the ph ylogenetic relationships between taxonomic groups (XIAO , 1980; HAO et al .,
2013) .
Diterpenoid alkaloids with the domina nt compou nd aconitine type (C19 type
III), (Figure 5 (4)) are widely distributed Aconitum genus (HAO et al. , 2013 ). The
chemical profiles of Adonis species consist mainly of magnoflorine and cardiac
glycosides (e.g. Figure 5 (5)) follow by flavonoids and lactones (KUBO et al., 2016; DAI
et al. , 2010)
Triterpene saponins were described as the most abundant compounds founded
in Anemone genus. Besides , Anemone contains a significant amount of ranunculin
(Figure 5 (6)), anemonin (Figure 5 (7)), and protoanemonin (SUN et al. , 2011).
Compounds such as triterpenoid lactones caltholide , epicaltholide , and
protoanemonin toge ther with oleanane -type sa ponin polypetalosides are predominant
in Caltha genus (JÜRGENS and DÖTTERL, 2004) .
In the leaves of Hepatica taxa substances such as anemonol, hepatrilobine
(glycoside), tannins, have been identified. Their r oots and rhizome contain various
saponins ( ALEXA N et al., 1991 ).
The chemical profiles of Ranunculus species are mainly characterized by
alkaloids (berberine -type ) and l actones including protoanemonin , anemonin,
ranunculin , and isoranunculin which have been found in high amount in Ranunculus
species (JÜRGENS and DÖTTERL, 2004)
Results of the chemical constituents studies of genus Trollius suggest the
presence of the following main compound classes flavonoids, organic acids,
carotenoids, terp enes, ceramides , steroid compounds, derivatives of fatty acids ,
benzenoids and carbohydrates. Flavonoids in particular C-glycosides and O-glycosides
are the most numerous group of compounds in Trollius species (WITKOWSKA –
BANASZCZAK EWA, 2015 )
Therefore the Ranunculeae and Anemoneae tribes are rich in saponins,
especial ly the pentacyclic triterpenoid derivatives, embodying the close relationship
between these tribes . Moreover , the r anunculin is only abundant in the tribes
Ranunculeae, Anemoneae, and Helleboreae (XIAO , 1980)

Cristina -Daniela Kelemen
24

Figure 4 Chemical structure of com pou nds detected in Ranuncula ceae species /
1.7. Essential o ils content
Essential oils are complex mixtures of volatile compounds present in plants ,
having diverse ecological functions including internal messengers, defensive
substances against herbivores or attrac tants for pollinating insects. Terpenoids mainly
represented by mono – and sesquiterpenoid hydrocarbons and their oxygenated
(hydroxyl and carbonyl) derivatives (Figure 6 ) are described as the most important
group of natural products isolated from essential oils Alcohols, aldehydes , shikimates ,
polyketides , esters (the acetates in particular) and some alkaloids are also commonly
encountered in essential oils (CROTEAU et al ., 2000) .

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

25

Figure 6. Chemical structure of common terpenoids widespread in essential oils/ Structura
chimc ă a terpenoidelor larg răspândite în uleiurile esențiale.

The volatiles are extracted from different parts of the plant using several
techniques, including water or steam distillation, solvent extraction, expression under
pressure, supercritical fluid and subcritical water extractions (EDRIS , 2007 ). Moreover,
it is also of great importance to highlight that a large number of essential oil are widely
used in the food (e.g. as food flavorings , as feed additives, as flavoring agents ),
fragrance, (compounding of cosmetics, deodorizers , and perfumes. ), and
pharmaceutical industry (massage, homeo pathy , aromatherapy and aromachology .)
due to their odor or flavor . Besides, the essential oils have various biological properties
such as anticancer effect, while others have antioxidant, anti -inflammatory,
antispasmodic , antiviral , antitussive , antibacterial or antifungal act ivities among other
properties (SCHMIDT, 2010 ).
Although all plants have the ability to produce volatiles oils, their presence in
many species remai ns unknown. However, many of the investigated plants contain
essential oils only in low amounts or traces. (SVOBODA et al. , 2000 ).

Ranunculaceae is not described as the major essential oil -bearing plant family
such as Apiaceae, Lamiaceae and Lauraceae (Young, 2014), the volatile oils have been
identified in several species of the family.
The first phytochemical investigation revealing the presence of essential oils in
this plant family was reported by ABOUTABL et al . (1989), who isolated and identified
the volatiles in seeds of Nigella sativa . Consequently , until now, most studies have
concentrated their attention on the chemistry of seed essential oils of Nigella species
due to their pharmacological activities (Kokoska et al., 2008) . A large number of

Cristina -Daniela Kelemen
26 stud ies show that essential oils extracted from Nigella specie s exhib ited several
beneficial effects including antioxidant, antimicrobial , anticancer and hepatoprotective
activities (Baser and Buchbauer, 2010; Edris, 2007 ; Fico et al., 2004; Kokoska et al. ,
2008) . Due to these properties the seedsoil of
Furthermore, other reports, from the scientific literature also describe the
occurrence of volatile oils obtained by hydrodistillation from different genera of
buttercup family such as Aconitu m (Zhang et al. , 2009) , Adonis (Mohadjerani et al .,
2013) , Anemone (Shi et al ., 2012) , Aquilegia (Radulovic et al., 2007) , Consolida
(Kokoska et al., 2012) , Delphinium (Gulec et al ., 2007) , Ficari a (Tavakoli et al. 2012)
Ranunculus (Terzioglu et al. , 2008 ; Erdogan et al ., 2014) and Trollius (Witkowska –
Banaszczak, 2013) .
Regarding genera studied in this paper, various parts of Aconitum tanguticu m
(Zhang et al., 2009 , Anemone rivularis (Shi et al. 2012) , Ranunculus arvensis (Hachelaf
et al. 2015),(Terzioglu et al. 2008) , Ranunculus constantinapolitanus (Terzioglu et al.,
2008) , Ranunculus marginatus var. trachycarpus (Tugce , et al., 2014) and Ranunculus
nipponicus var. submersus (Nakaya e t al. 2015) have previously been described a s
species containing volatiles.
The essential oil of A. tanguticum was mainly represented by monoterpenoids
and sesquiterpenoids , while the aliphatic compounds and benzene derivatives were
the dominant compounds of A. rivularis oil (Chun Jiang Zhang, Chun Mei Liu, Tian Yun
2009),(Shi et al. 2012) . On the other hand, the essential oils isolated from Ranuncul us
species have particularly been represented by a relatively higher content of phytol
(Terzioglu et al. 2008) .
Besides, the antimicrobial activity of A. tanguticum , A. rivularis , R. arvensis , and
R. constantinapolitanus essential oils was previously reported against several
pathogenic microorganisms
In our best knowledge despite the previous reports on th e presence of volatile
oils in the above -mentioned taxa of the Ranunculaceae family, other species have not
been studied in detail.
1.8. Ethnopharmacologic properties and bioactivity
According to the International Union for Conservation of Nature and the W orld
Wildlife Fund, there are between 50,000 and 80,000 flowering plant species used in
folk medicine worldwide. Among these about 1300 medicinal plants used in Europe, of
which 90 % are harvested from wild resources ( BALUNAS et al ., 2005 ). In Romania
abou t 283 species have therapeutic effects including also same Ranunculaceae species
(Adonis vernalis and Helleborus purpurascens ), however , only a small number of these
species are studied ( MOCANU 1999 ).
In folklore medicine, many species belonging to Ranuncu laceae family have
been used for centuries as a natural re medy for a diverse range of ailments such as
fever, cough, nasal congestion, bronchitis, asthma, dyspnea, hypertension, diabetes,

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

27 inflammation, milk production, eczema, dizziness, and gastrointestin al disturbances
etc. Mor eover , many of these plants have been characterized as an analgesic ,
antimicrobial , anti -inflammatory, antitumor , antioxidant and antiparasit ic agents due
to the presence of various medicinal phytometabolites such as alkaloids, flavonoid s,
phenols, saponins, steroids, and terpenoids (NYIRIMIGABO et al ., 2014) .
Nigella sativa is reported as t he most important medicinal herbs within
Ranunculaceae buttercup . N. sativa or black cumin is described by prophet Muhammad
1400 years ago as a “cure -all” having healing powers for every disease except death
(BAHAREH and HOSSEIN, 2016). Traditional uses of the seed of this herb originate
from the ancient Egyptians, Greeks, and Romans. N. sativa has been a dvocated as the
bodyʼs energizing compound and have been used as a remedy for a huge range of
diseases, such as, asthma, bronchitis, cough, diabetes , fever, inflammation , headaches ,
hypertension, milk production, toothaches, skin disorders, wounds, and external
irritations among others diseases ( HEISS et al. 2012; JUNEMANN et al. , 1998; ZIAEI et
al., 2012) . Additional ly, the seeds ' oil of N. sativa has been frequently prescribed as a
treatment for various diseases. Thymoquinone is reported as the main active
constituents of N. sativa oil and a broad -spectrum of bioactivities have been attributed
to it. Until now more than 150 studies have been conducted over the last five decades
to investigate the pharmacological and chemical properties of black cumin . Therefore
more than 100 constituents have b een identified and various therapeutic proprieties of
N. sativa have been demonstrated such as analgesic , anticancer, antioxidant ,
antimicrobial , anti -inflammatory , cardioprotective , hepatoprotector , hypotensive , as
well as antitussive diuretic and expectorant effects (ALI et al . 2003; ZIAEI et al., 2012,
DUKE, 2002 ; Tariq et al ., 2007; Abdel -Fattah et al., 2000; Daba and Abdel -Rahman,
1998).
Aconitum aerial parts are externally used to treat abrasions, wounds and
traumatic injuri es. For internal use, despite t he high toxicity of this genus, it was
historically employed as a remedy for arthritis, diarrhea, cough, fever, gastroenteritis,
menstrual disorders , neuropathic pain, rheumatism , ulcers, stroke, paralysis and other
(VITALINI et al., 2010 ). Furthermore, a broad spectrum of bioactivities such as
analgesic, antimicrobial, anticancer, antineuralgic, anti -inflammatory , diuretic and
cardioactive effects have been observed for various Aconitum species (HAO et al., 2013;
DUKE, 2002 ).
The plants of the genus Adonis genus have been traditionally used in various
indications, such as arrhythmia, cardiac insufficiency, ulcer disease and urinary
problems ( KAYANI et al ., 2015 ).
More than 50 Anemone species are used in various tr aditional medical systems
(HAO et al. , 2017) . Current studies showed that the t riterpenoid saponins founded in
Anemone species have an important role in the bioactivity f the genus (WANG et al. ,
2012 ). Many Anemone species have been used in folk medicine as a cure for arthritis ,

Cristina -Daniela Kelemen
28 constipation, fever, hepatitis , malaria, parasitic disease, ulcers and sores (SUN et al. ,
2011 ; KAYANI et al. , 2015 ). Besides , pharmacological activities, including analgesic,
antitumor, antimicrobial, anti -inflammatory , anticonvulsant , and anti -histamine effects
are well -described for several species of the genus (HAO et al. , 2017 , 2015 ).
The plant 's species belonging to of the genus Delphinium have numerous
pharmacological activities such as anti -cancer, antibacterial, anti -inflammatory,
immunosuppressive, analgesic, cardiot onic, anti -hypertensive, and vasodilative among
other effects (HAO et al ., 2015; KAYANI et al. , 2015 ).
Caltha is traditiona lly used in the treatment of arthritis, fever, muscular pain,
skin problem and toothache ( KAYANI et al., 2015 ). According to DUKE (2002), Caltha
palustris shows anti -arthritis antiedemic , antitumor , anti -inflammatory , antirheumatic ,
antispasmodic and immunomodulatory effects.
A large number of Clematis species are particularly used in folk medi cine in
Europe and Eastern Asia for treatin g rheumatic pain, fever, eye infections, gonorrheal
symptoms, bone illnesses, chronic skin disorders, gout , and varicosity and as diuretic,
antimal arial, antidote in snake bites and antidysentery (Chawla et al., 2013) .
Helleborus extracts contain many active molecules including bufadi enolide
heterosides (hellebrin), that exhibit various pharmacologic effects such as
antibacterial, antioxidant, antiproliferative , anti -inflammatory and antinociceptive
(ČAKAR et al. 2011; CHENG et al., 2014; PUGLISI et al., 2009)
Hepatica genus has a limited phytotherapeutic use, in official hemp from
Hepatica nobilis the aerial parts are used as an infusion for oral mucosal inflammation
and throat sore. Traditional medicine uses the aerial part of the plant in the treatment
of chronic bronchitis, hematuria , and liver diseases (Alexan and et al., 1991 ). Hepatica
nobilis had been described to possed analgesic, a ntibacterial circulotonic ,
contraceptive, diuretic , hepatotonic and laxative effects among others.
Extracts of Ranunculu s species are used as a parasiticide and as medicine for
various dise ases including arthritis , asthma , fever, malari a, jaundice , ulcers and skin
infections (HAO et al., 2015 ). Ranunculus has anti cancer, anti -inflammatory,
anti oxidant, a nalgesic, antimicrob ial, anti parasitic, and cardiovascular effects (ASLAM,
2012 ).
Flowers of the Trollius taxa (e.g. T. ledebourii and T. chinensis ) are widely used in
Chinese folk medicine to treat various ailments including respira tory tract infections ,
bronchitis, cold , fever, tonsillitis , otitis, lymphangi tis, conjunctivitis, appendici tis, and
dysentery ( SUN et al.,2011; WITKOWSKA -BANASZCZAK EWA , 2015 ). Trollius species
showed in vitro antibacterial, anti -inflammatory , antioxidant and antiviral effects
(WANG et al., 2014; LI et al., 2002).

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

29 I.1.7.1 . Anticancer activity
The anti -cancer activity is usually regarded to the ability of the compound to
inhibit tumor cell proliferation or induce cell death of tumor cells ( HAO et al. 2017 ).
Previous research studies described at least 17 genera of the Ranunculaceae
buttercup family, which have been enriched with anti cancer phytometabolites
including a lkaloids, terpenoids, saponins, and polysaccharides (HAO et al. 201 5).
Many of these phytometabolites founded in Ranunculaceae modu late multiple
signaling pathways for cancer initiation and progression . Therefore, these constituents
are able to induce the cell cycle arrest and apoptosis of cancer cells, while others
inhibit the proliferation, invasion, angiogenesis, and metastasis, o r reverse the multi –
drug resistanc e of cancer cells thereby regu lating all known hallmarks of cancer (HAO
et al. 2017 ). The cancer hallmarks modulated by Ranunculaceae phytometabolites are
summarize d in Figure 5.

Fig. (5). The cancer hallmarks modulated by Ranunculaceae phytometabolites /
Caile distincte ale cancerului modulate de fitometaboliti prezenți în Ranunculacee (HAO
et al., 2017) .

The promis ing effects against cancer have been already reported for several
genera of the family including Aconitum , Adonis , Anemone, Clematis, Pulsatilla,
Ranunculus and Trollius (HAO et al. 2017 ; ASLAM , 215; WITKOWSKA -BANASZCZAK et
al., 2015 ).

Cristina -Daniela Kelemen
30 Anemone , Clematis , Cimicifuga , and Pulsatilla species are a rich source of
multiple therapeutic s aponins, which usually exert anticancer activity especially via
induction of cell cycle arrest and apoptosis of the cancer cells (HAO et al. 201 5, 2013 )
Triterpene saponin (raddeanin A, saponins B and s aponin 1 ) isolated from some
Anemone species inhibits proliferation and induces apoptosis of multiple cancer cells ,
and would be a potential antitumor medicine (WANG et al., 20 12). The Anemone taxa
were effective against human leukemia , glioblastoma multiforme, and human cervical
cancer (XUE et al., 2013; LI et al., 2013 ).
Clematis phytometabolites have shown a cytotoxic effect against colon cancer ,
human glioblastoma , hepa toma cervical cancer, leukemia, gastric cancer and prostate
cancer (ZHAO et al. 2014; TIAN et al., 2013 ). Cimicifuga species had notable
cytotoxicity against human breast cancer and human gastric cancer cells . The Pulsatilla
saponin s (saponin A and D) and terp enoids suppress the prol iferation of human colon
cancer, pancreatic cancer cells , human chronic myelogenous leukemia and
hepa tocellular carcinoma ( RAO et al., 2013; LIU et al., 2014; HAO et al., 2017 ).
Diterpenoid alkaloids are reported as t he main effecti ve anticancer constituents
of Aconitum and Delphinium plants. C18 -, C19 -, C20 -, and bis -diterpenoid alkaloid are
described as the most promising, naturally, com pounds for treating multiple types of
cancer (e.g, b reast cancer , colon cancer , epidermoid carci noma , gastric carcinoma, and
prostate cancer among other) via various pathways include inhibiting cell growth,
inducing apoptosis, interfering with the cell cycle, and altering MDR. (REN et al., 2017 ).
Thymoquinone (TQ, 2 -methyl -5-isopropyl -1, 4 -benzoquino ne), a monoterpene
abundant in Nigella sativa , induces apoptosis in human colon cancer , hepatocellular
carci noma , human colorectal cancer , and prostate cancer via multiple pathways (KOKA
et al., 2010; EL -BABA et al., 2014; RAGHUNANDHAKUMAR et al., 2013) . Adonis
cardenolide therapeutic glycosides (e.g. strophanthidin glycoside, and pregnane
glycosides, cymarin, and cymarol) have shown also anti -cancer activity (HAO et al.,
2015 ). Additionally , the bufadienolides founded in Helleborus species exhibit potent
cytotoxic activities against cancer cells (Čakar et al. 2011) .
Cytotoxicity effect s against various cancer cells have been also described for
several Ranunculus species namely R. sieboldii , R. ternati and Ranunculus sceleratus
(ASLA M et al., 2012 ).
I.1.7 .2. Antioxidant activity
Free radicals are aggressive, unstable, and highly reactive atoms or compounds
because of their single electr on. They attack other molecules changing their proper ties
and making disorders inside possible. Free radicals result from different meta bolic
activities while, a large number occur because of smog, nitrogen oxides, ozone,
cigarette smoke, toxic heavy metal , pesticides, and cytostatic drugs (Schehl and
Schroth, 2004 ).

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

31 The oxidation induced by reactive oxy gen species(ROS) play a major role in the
development of oxidative stress that can results in protein damage and DNA mutation,
which can lead to to many illnesses including anemia, cancer, cardiovascular diseases,
degenerative diseases (e.g. Alzheimer’s di sease), diabetes, inflammation and ischemia
(CAI et al., 2004 ).
Antioxidants have the ability to protect the living system from these free
radicals and/ or delay the development of diseases caused by these free radicals.
Several investigations revealed tha t phenolics and flavonoids compounds widely
distributed in the plant kingdom are potent scavengers of reactive oxygen species and
thus prevent peroxidation of lipids contribute to the antioxidant activities (GILL et al.,
2010). Therefore, these plant -deriv ed compounds often exhibit a notably antioxidant
activity and may play a significant role in the prevention and therapeutics of the
diseases associated with oxidative stress.
According to previous reports many members of family Ranunculaceae (e.g.
Aconitum heterophyllum , Helleborus odorus , Nigella sativa , Ranunculus arvensis ,
Ranunculus marginatus , R. sprunerianus , Trollius chinensis ) were described to posed a
strong free radical scavenging activity ( MUNIR et al., 2016; AHMED et al., 2015; ČAKAR
et al., 201 1; BHATTI et al., 2015b; BOURGOU et al., 2012; SONG et al., 2013 ).
The phytochemical studies on Aconitum genus reported the antioxidant activity
of certain flavonol glycosides isolated from A. anthora , A. napellus ssp. tauricum , A.
chiisanense Nakai, A. napellus ssp. neomontanum (MARIANI et al. 2008; JEONG et al.,
1997; FICO et al., 2001a; FICO et al., 2001b ), Besides, a number of fractions isolated
from Anemone cathayensis had shown a strong radical scavenging activity in vitro
(WANG et al., 2012) .
Delphinium extract shows protective eff ects against Parkinson's disease and the
whole plant of Consolida is used as an analgesic in the treatment of rheumatic disease
through antioxidant activity ( AHMAD et al., 2006 ; XIAO et al., 2015 ). Likewise, Clematis
species namely C. armandii , C chinensis , C. flammula n, and C. trichotoma exhibited in
vitro antioxidant effect ( CHAWLA et al., 2012 ).
Another study by NEAG et al. (2017) indicates the antioxidant activity of
glycerol -ethanol extracts obtained from the herb of R. ficaria , R. sardous R. bulbosus ,
and R. sceleratus extracts.
The antioxidants identified in Ranunculaceae plants might play important roles
in fighting against genome instability and mutation of cancer cells. .

Cristina -Daniela Kelemen
32
2. Investigated Ranunculaceae genera and species
from Romania
In Romania, the family is represented by 23 genera and approximately 110
species of annual to perennial herbs, subshrubs, or woody climbers, distributed in all
regions of the country (CRISTEA, 2007).
The genus Ranunculus is one of the largest taxa of Romanian Ranuncula ceae
represented by 35 species (SAVULESCU, 2010).
Aconitum moldavicum Hacq . and Anemone transsilvanica Fuss Heuff . are
respective examples of rare and endemic plants also typical for the region (CIOCÎRLAN,
2000) .
In the traditional medicine, local species have been used for the treatment of
various ailments such as bronchitis, cough, diarrhea, fever, hepatitis, gout, malaria,
rheumatism, and skin diseases (TAMAS, 2005; ALEXAN et al., 1991) . Moreover, leav es
of some plants are locally consumed as food ( e.g. Caltha palustris L., Clematis vitalba L.,
Ficaria verna Huds), whil e some are used as spices and ornamentals, however, most of
them are highly poisonous (CIOCÎRLAN, 2000) .

2.1. Aconitum
Aconitum is the botanical name of the genus commonly kown as aconite,
monkshood etc. The genus is widely distributed in the mountain a nd subalpine
regions, g rowing in the moisture -retentive , but well -draining soils of mountain
meadows .
The species belonging to the genus are usually perennial or biennial herbs, often
with stout leafy stems, bulbs or creeping rhizomes. Leaves are mostly c auline, lobed,
rarely divided and dentate. The leaves have a spiral (alternate) arrangement. Aconitum
has zygomorphic flowers, si mple or branched racemes, of blue or yellow colour with
numerous stamens . These plants are distinguishable within buttercup by having one of
the five petaloid sepals , of a cylindrical helmet shape. The carpels (3 -5) are partially
fused at the base. The fruit is an aggregate of follicles with many seeds.
The high variability of Aconitum genus morphologic traits has always attracte d
the attention of bota nists. Many taxonomic studies have been performed to clarify the
systematic of the genus that appears particularly complex . It is generally accepted that
this genus consists of three well -defined subgenera : Aconitum , whose members
produce biennial tuberous roots; Lycoctonum , whose species have perennial rhizomes;
and Gymnaconitum , which is monotypic and has the only annual species of the genus .
The European species of the Aconitum genus were classified based on phenotypic
characterist ics into four groups namely Vulparia, V ariegatum, Napellus and Anthora.

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

33 The base chromosome number of the genera is 2n=16. Based on the data of
morphological characters , molecular and chemotaxonomic markers the Aconitum
genera is closely related with Consolida and Delphinium genera , however a great deal
of variation occurs within genera , perhaps as a result of hybridization.
Characteristic compounds of these plants alkaloids, amide alkaloids, flavonoids,
flavonol glycosides, diterpenoid and norditerpenoi d compounds which possess
medicinal values . The aconitine type (C19 type III) is the dominant diterpenoid
alkaloid HAO et al. , 2013 ).
Although essential oil s are not tipical constitue nts for this genera, CHUN et al .
(2009) reported the chemical compositio n of the essential oil hydrodistillat ed from
aerial parts of A. tanguticum . The essential oil obtained was mainly represented by
monoterpenoids and sesquiterpenoids with (-)-tranpinecarvyl acetate, heptane,
cineole , 3-pinanone, pinocamphone, cadino l and cu benol as main compounds .
Aconitum species are among the most toxic Ranunculaceae and several species
have been used as arrow poisons and as ornamental plants . However, their toxicity can
be reduced using different techniques and then benefit from its phar macological
activities . A particular use of the roots is in a soup with different kinds of meat and
vegetables (SINGHUBER et al. , 2009) .
Many species in this genus have been used for medicinal purpose at least 76
Aconitum species have been used in Eastern and Oriental traditional medicine (XIAO et
al., 2006 ; SINGHUBER et al. , 2009 ). Aconitum aerial parts are externally used to treat
abrasions, wounds and traumatic injuri es. For internal use, despite the high toxicity of
this genus, it was historically empl oyed as a remedy for arthritis, diarrhea, cough,
fever, gastroenteritis, menstrual disorders , neuropathic pain, rheumatism , ulcers,
stroke, paralysis and other (VITALINI et al., 2010 ). Furthermore, a broad spectrum of
bioactivities such as analgesic, antim icrobial, anticancer, antineuralgic, anti –
inflammatory , cardiotonics , diuretic and cardioactive effects have been observed for
various Aconitum species (HAO et al., 2013; DUKE, 2002 ).
Diterpenoid alkaloids isolated from Aconitum species have been describe d as
the main effective anticancer constituents. The cytotoxicity effect has been reported
until now for various species of the genera including A. carmichaeli , A. vaginatum , A.
taipeicum . Moreover, the phytochemical studies on Aconitum genus reported the
antioxidant activity of certain flavonol glycosides isolated from A. anthora , A. burnatii ,
A. chiisanense , A. napellus sp. Lusitanicum , A. napellus ssp. tauricum , , A. napellus ssp.
neomontanum , A. variegatum (MARIANI et al. 2008; JEONG et al., 1997; FICO et al.,
2001a; FICO et al., 2001b ; VITALINI et al ., 2010 ).
The increasing pharmacological significance of these species have determined
our further inestigation on four poorly examined Aconitum species native to the
Carpathian Basin namely Aconitum moldav icum , A. toxicum, A. variegatum and
Aconitum vulparia .

Cristina -Daniela Kelemen
34

A number of molecular studies based on internal transcribed spacers (ITS)
region of nuclear ribosomal DNA (rDNA) and chloroplast DNA sequencing were carried
out in order to examine the genetic var iability and the phylogenetic relationship of this
species. The results revealed that Aconitum moldavicum differed only by a single point
mutation from A. lycoctonum , however, there is not significant genetically
distinguishable between these both species and other investigations are needed in this
direction (UTELLI et al. , 2000; MITKA 2003).

2.1.1. Aconitum moldavicum Hacq .
A. moldavicum (syn . Aconitum carpaticum )
is subendemic to the Carpathian and is dispersed
in the eastern and central parts of Europe,
extending to Romania and West Ukraine. It is a
forest species occurring in mountains and
subalpine zone especially in the forest –edges,
bushes, and in alder woods.
Aconitum mol davicum is a perennial plant
at 60 –200 cm tall with a branched or simple
rhizome. The stem is erect, high -branched with 1 –
5 very large basal leave. The inflorescence is
around 10 to 20 cm. The zygomorphic flowers are
about 30 mm long of purple -violet color . The
helmet is composed of five petaloid sepals . The
fruit is represented by several pubescent follicles
with several triangular black seeds.
Several diterpenoids and norditerpene alkaloids
have been already isolated from the MeOH extract
prepared from th e fresh roots of A. moldavicum
(BORCSA et al ., 2014) .
Rhizome extracts or tinctures of Aconitum
moldavicum are locally used as treatments of
neuralgia, rheumatism, and cough.
Although Aconitum moldavicum have been
used in folk medicine its traditional c laims have
been not validated scientifically. Furthermore, the
plant is not described as containing essential oils.

Figure 10 . Aconitum moldavicum
(www.natura -2000.eu)

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

35 2.1.2 . Aconitum toxicum
Aconitum toxicum Rchb. is native to
Romania and Yugosla via. The plant is distributed
from the mountain and subalpine regions in
forests and bushes .
The plant is at most 200 cm in height, with
leaves divided to the base, the wide segments
often being divided beyond the middle, and deeply
incise -dentate. The inf lorescence is branched and
glandular, withovate bracts and blue flowers. The
helmet is about twice as high as wide 2.5 -4 cm.
The nectary spurs are not spirally curved.
The chemical composition of A. toxicum is
slightly studied and only a few alkaloids have been
isolated from its root . Furthermore, there are no
reports in its pharmacological use and essential oil
content.

Figure 11. Aconitum toxicum
(original photo)

2.1.3. Aconitum variegatum
Perennial plant spread to central Europe and
Russia growing in damp and nitrogenous mountain
meadows.
The plant has tuberous roots, alternate and
palmately leaves, blue -violet flowers, brownish seeds ,
and follicles as fruits. The helmet is higher than large
around 2.5-4 cm .
In particular, the chemical compositio n of A.
variegatum is characterized by alkaloids
(norditerpene and diterpene compounds) together
with various flavonoids.
The methanol extract of aerial parts of A.
variegatum exhibited strong antioxidant a ctivity
(VITALINI et al., 2010) .
Despite, the above -mentioned report , there is
no other studies regarding its biological activities.
Moreover , the characterization of their essential oils
is missing from the literature .

Figure 12 . A. variegatum
(Alexandru Badarau photo)

Cristina -Daniela Kelemen
36
2.1.4. Aconitum vulparia
Aconitum vulparia Rchb. ( syn. A.
lycoctonum L. subsp. vulparia (Rchb.) Nyman)
is distributed throughout Europe including
Romania .
The plant is 40 -120 cm in height, with
dark -green leaves segmented into 4 –6 (leaf –
segments 3 -fid to the middle). The
inflorescen ce is small and few -flowered, with
yellow flowers. The perianth -segments are
deciduous; the conical -cylindrical helmet
measures 12 – 25×3 –10 mm, and the nectar –
spurs are spirally curved.
Identified A. vulparia compounds
include norditerpene and diterpene a lkaloids.
A. vulparia is locally used in the
treatment of chronic skin disorders, gout,
neuralgia , and rheumatism .
The ethanol extract of this plant showed
in vitro anti -inflammatory and antioxidant
activity (MALIK et al., 2017 ).

Figure 12. Aconitum vu lparia
(original photo)

2.2. Anemone
Anemone ("wind flowers .") consists of more than 150 species of flowering
plants, distributed to the temperate zones of both Northern and Southern
hemispheres. They inhabit a wide range of habitats but occur most com monly in
woodlands and meadows of the North Temperate Zone .
Anemone species are perennial low-growing herbs with cylindric rhizomes . The
species are characterized by a rosette of basalleaves , an involucrate pedunele bearing a
single fiower with 4 –27 sepa ls or compound infiorescence (cymes of 2 –9 flowers, or
in umbels) , a perianth of colorful sepaIs . The flowers have nectaries , but petals are
missing in the majority of species . The pistils have one ovule. The fruits are ovoid to
obovoid shaped achenes . The achenes are beaked and some species have feathery
hairs attached to them . However, there is considerable morphological variation with in
the genus.
Base chromo some numbers are either 7 or 8. Based on morphological genetic,
The genus is closely related to Pulsatilla , Clematis , and Hepatica and phyto -chem ically .
Identified Anemone compounds include triterpenoids, saponins, steroids,
lactones, fats and oils, saccharides, and alkaloids, etc . Oleanolic acid triterpene saponin
is described as the most abundant compounds founded in Anemone genus. Besides,

Genetic diversity, bioactivity and essential oil content of several species belongi ng to Ranunculaceae family
from Romania

37 these species contains a significant amount of ranunculin, anemonin, and
protoanemonin ( SUN et al. , 2011). Coumarins, flavonoids, lactones, lignans, steroids,
phenolic compounds, and other compounds are also de tected in several Anemone
species
The e ssential oil was isolated from the roots of A. rivularis . The major
constituents were acetophenone, 3 -ethyl -2-methyl -hexane, 5,6 – dimethyl -decane and
4,5-diethyl -octane. Fatty acid derivatives, especially pentadecane and nonanal,
together with various sesquiterpe noids were the abundant compound classes in A.
sylvestris anther essential oils.
According to HAO et al . (2017b) , more than 50 species have various traditional
medicinal uses. Some traditional claims of Anemon e species have been validated
scientifically by pre -clinical and clinical studies .
Current studies showed that the triterpenoid saponins founded in Anemone
species have an important role in the bioactivity f the genus (Wang et al., 2012). Many
Anemone species have been used in folk medicine as a cure for arthritis, constipation,
fever, hepatitis, malaria, parasitic disease, ulcers and sores (SUN et al., 2011; KAYANI
et al. , 2015 ). Besides , pharmacological activities, including analgesic, antitumor,
antimic robial, anti -inflammatory, anticonvulsant, and anti -histamine effects are well –
described for several species of the genus (HAO et al., 2017 b, 2015) .
Triterpene saponin ( raddeanin A, saponins B and saponin 1) isolated from
Anemone species usually exert anti cancer activity via cell cycle arrest and apoptosis
induction and would be a potential antitumor medicine ( WANG et al., 2012 ). The
Anemone taxa were effective against human leukemia, glioblastoma multiforme, and
human cervical cancer ( XUE et al., 2013; LI et al., 2013 ).
Besides, a large numb er of triterpenoid sap onins isolated from Anemone plants
have been characterized as effective anti oxidants and anti -inflammatory agents (HAO
et al ., 2017b) .

De ce am ales A. transilvanica

Cristina -Daniela Kelemen
38

22.1.Anemone tr anssilvanica
A. transsilvanica is endemic species to
Carpathians of Romania. It is found in the in
shady areas, in forests and hedges.
The plant is 10 -20 cm in height, with
trilobate leaves, where each lobe is once
again trilobulated (distinct character o f
Hepatica nobilis ). The flower is
actinomorphic, with 6 -10 petals, blue to
violet, with numerous stamens. The fruit is a
achen .
A. transsilvanica is used as ornamental
plant and in traditional medicine its rhizome
have been used as remedy for rheumatism
and
The transsilvanic Hepatica species is
grown as an ornamental species in open,
tree -like areas in hill and mountain areas.