ON OVERVIEW OF BIOACTIVE COMPOUNDS, BIOLOGICAL AND PHARMACOLOGICAL EFFECTS OF MISTLETOE (VISCUM ALBUM L)

Eva Kleszken^{1, #}, Adrian Vasile Timar², Adriana Ramona Memete^{1, #}, Florina Miere (Groza)³, Simona Ioana Vicas²*

The genus Viscum includes many species that are mainly distributed in Europe, Africa, Asia, America, and Australia. Viscum extracts or their various preparations are widely used as complementary and alternative medicines in the treatment of various ailments. In the present review, articles related to the phytochemical composition of mistletoe were selected, depending on the host tree on which it grows, as well as articles in which its beneficial effects were highlighted. Viscum contains different active ingredients, including lectins, viscotoxins along with phenolic acids, flavonoids, alkaloids, terpenoids, and polysaccharides. Based on its composition, mistletoe extract is associated with multiple bioactivities, including anticancer, anti-inflammatory, and cardiovascular disease, attenuating the side effects of chemotherapy and enhancing immunity. The purpose of this review was to highlight the link between the host tree and the bioactive components of mistletoe such as lectin and viscotoxin, with a focus on phenolic compounds such as flavonoids and phenolic acids. The potential therapeutic effects of mistletoe are summarized by subspecies and host trees. Numerous mistletoe-based patents with various applications have been developed and presented in this review. Mistletoe is a medicinal plant with great biological potential that is worth exploring for various targeted treatments.

Keywords: Viscum album, Host trees, Flavonoids, Phenolic acid, Antioxidant, Anticancer

Introduction

Parasitic angiosperms are over 4,700 species of 277 genera, of which 87 genera and at least 1,670 species belong to the mistletoe which is considered hemiparasites that grow on the host branches of various species of shrubs and trees looking like a highly developed shrub  [1, 2]. Living organisms are involved in parasitic relationships, either as parasites or as hosts, these interactions having an essential role in the proper functioning of the biosphere and the process of biological evolution [3, 4].

Mistletoe (Viscum album L.) is an evergreen plant that depends on the host tree for some nutrients and water, while it produces carbohydrates in a process of photosynthesis, normally found growing on a variety of trees, both deciduous and coniferous trees [2]. Mistletoe (V. album), is a large hemiparasitic species found in northern, central, and southern Europe, Africa, America, Australia, and Asia, accompanied by a wide variety of habits, host preferences, morphologies, and development patterns depending on the geographical distribution [5-7].

Several chemical and pharmacological studies have identified various types of compounds in mistletoe, such as lectins, viscotoxins, lignans, amines, flavonoids, and polysaccharides [8, 9]. Among them, flavonoids and phenolic acids are natural antioxidants involved in the biological activity of the plant and used in the prevention of diseases such as cancer, caused by oxidative stress induced by free radicals in the body [10-13]. Cancer therapy is one of the most important uses of mistletoe, proving to have beneficial effects on various types of cancer such as breast [14,  15], pancreatic [16], laryngeal [17] bladder [18], or leukemia [19]. Over time, other bioactive activities of mistletoe have been reported, such as those related to neurological disorders, antiviral, antibacterial, anti-inflammatory, antiepileptic, or immunostimulatory activities [2, 11, 13, 20].

Due to its bioactive compounds and its beneficial effects on the human body, mistletoe has become more and more studied, developing various pharmaceutical formulations (Iscador, Isorel, Iscucin, Lektinol, Eurixor, Helixor, Abnoba-Viscum and recombinant lectin ML -1) [13, 21].

This review aimed to highlight the link between the host tree and the bioactive components of mistletoe such as lectin and viscotoxin, with a focus on phenolic compounds such as flavonoids and phenolic acids. The potential therapeutic effects of mistletoe are summarized by subspecies and host tree. Numerous mistletoe-based patents with various applications have been developed and presented in this review.

Materials and Methods

This review was performed using the PRISMA 2020 flow chart based on the suggestion of Page et al., 2021 [22]. The steps and selection criteria, followed by the number of studies used for our review, are shown in Figure 1. Databases such as PubMed, Scopus, Science Direct, Elsevier, Google Scholar, Google Patents have been accessed to search the literature. The keywords of the medical subject titles included in the search were: ″Viscum album″, ″V. coloratum″, ″V. articulatum″, ″mistletoe viscotoxin″, ″mistletoe lectin″, ″mistletoe effects″, ″mistletoe patents″, ″mistletoe compounds″, ″mistletoe history″, etc. All the information systematized in the tables was obtained from research articles. Studies published in languages ​​other than English and Romanian were excluded. A total of 100 studies were selected and included in this review.

The Historical Approach of Mistletoe

Given the co-evolutionary history between plants and the organisms that use them, over 150 years ago, the co-adaptation between mistletoe and birds caught the attention of Charles Darwin, stimulating considerable scientific research. Equally important for other researchers was the understanding of the interactions between herbivorous insects and plants, which can make the transition between plant taxa. The mistletoe is also considered the ancestral host of butterflies [23-25].

The number of host species infected with mistletoe is critical in that it influences the overall distribution of the parasite, its prevalence, and virulence. Therefore, macroecological analyzes of this feature related to the history of mistletoe life are missing for many regions, and with the evolution of mankind, geographical and environmental changes have influenced the parasitic species, through hybridization processes [1, 26, 27].

Mistletoe was later discovered for its miraculous effects, and traditional medicine spread it over time to many parts of the world, in particular, V. album L. and Loranthus (Taxillus Chinensis (DC.) Danser) [28, 29]. The latter belongs to the family Loranthaceae, distributed mainly in southern and southwestern China, is known as "Sang Ji Sheng", and its leaves and stems have a long history in traditional Chinese medicine, is used to treat rheumatism, arrhythmia, angina pectoris, hypertension, stroke [28]. V. album L. also has a long traditional history of about 100 years, used in German-speaking countries in cancer treatment therapy [29].

In addition to the fact that mistletoe was used for medicinal purposes, there are also several legends gathered from different regions of the world, related to this plant, legends that our ancestors respected and mentioned and which are still remembered today.

Because of its beliefs to bring good luck, to heal illnesses, and to attract angels, mistletoe is known since old times. It is said to bring health and prosperity. It had a very important part in the Celtic rituals. They believed that mistletoe was created by a bolt of lightning that hit a tree giving him magical powers. The Celts believed that a very precious mistletoe was grown by the oak, also known as the „Oak Tear”, and poaching this kind of mistletoe was always a moment of ceremonial cutting. Thus the mistletoe was cut only after the winter solstice with the Golden Sickle, by a white dressed priest. Mistletoe was also considered the symbol of femininity (Naturalis Historia, books XVI, XXIV, XXXIII).

The Greeks considered mistletoe from the underworld, believing that a branch of mistletoe was the key Aeneas that opened the underworld. The Romans put on Goddess Diana's head a crown of mistletoe, as a symbol of fertility. In the Scandinavian legends, mistletoe was something very special symbolizing peace, love, harmony, spiritual cleansing, and throwing away bad spirits.

In European mythology, mistletoe has a unique place. Around Christmas and New Year's Eve, people put mistletoe twigs on doors and windows to bring luck and health.  Other legends talk about a peculiar custom that if two enemies were met under a tree that grew mistletoe, they come to terms on behalf of the mistletoe. Later this custom was taken by the lovers, who kissing under the mistletoe felt like a love pledge, bringing happiness and fulfillment. In the town Tenbury Wells, from the UK, a mistletoe festival is held every year and a mistletoe Queen is chosen, bearing a crown of mistletoe [30].

Botanical Characterization

V. album is a hemiparasite shrub species that grows on branches and twigs of trees. It is a plant with slow development, usually being a nod to the blooming season.  Every year mistletoe develops more and more branches, so its growth tells us its age. Mistletoe can live up to 70 years [31, 32]  It looks like a very big bush, having a diameter of up to one meter (Figure 2a). The mistletoe is stuck on the host tree. It looks like a vertical, well-developed ax that enters the bark of the host tree, pervading its woody tissue. This network continues to grow, creating a root system (haustorium) that absorbs water, sugars, amino acids, and minerals from host trees, being able to biosynthesize their own primary and secondary metabolites [33]. Its stem is cylindrical, thick, dichotomic ramification, wide off 30 - 60 cm, thicker towards the nods, where it can be torn easily, having a yellow-greenish color. Leaves are green all year round, have no stalk, full edges, being seen like a ribbon, thick, skinny, bearing no wintergreen shrubs, having a yellow to intense green color, and are set in opposite pairs. On its lower part, one can notice 4 - 5 veins (Figure 2b). The size of the mistletoe leaves varies according to the host tree and the harvesting time.

The leaves harvested in April are larger, especially in the case of mistletoe leaves hosted by acacia. Leaves have a specific smell and a soft bitter sour taste. The optimal period to harvest is November to April, as the flowers are small (2-3 mm in diameter), yellow-greenish color, dioice, can be seen on top of young twigs. It blooms in March- April (Figure 2c). The fruits are spherical, having a glassy look, with a diameter of approximately 8 mm, green at the base. Between September - October, (Figure 2d) the fruits are white translucent, while in November- March they are toxic (Figure 2f). As a rule, the fruits are gathered thrisome during September-December (Figure 2e), containing 1-2 seeds covered by a jelly, sticky liquid (Figure 2g).

Up to the 18th-century botanists confused white mistletoe (V. album) with oak mistletoe (L. europaeus). The V. album belongs to the Viscaceae family, while L. europaeus belongs to the Loranthaceae family. The link between them is of taxonomic order and both belong to the Santalales order [7, 34].

The L. europaeus has younger twigs, brownish color, lance-like leaves, short stark and whole edge. It loses its leaves during wintertime, while white mistletoe keeps its leaves the entire year-round, being always green. The last leaves are small, all yellow-greenish, like a spike shape, blooming around May - June.  The fruits of V. album are small, yellowish, in the form of a small cluster shape. Its fruit is ripe in October, usually has a seed, that is spread by birds [35].

Taxonomy

The Viscaceae family has seven genres: Viscum, Phoradendron, Notothixos, Korthalsella, Ginalloa, Dendrophthora, and Arceuthobium. Regarding the scientific rating the mistletoe (V. album L) belongs to the Viscum genus, part of the Viscaceae family, but genetic research, new approach in APG (Angiosperm Phylogeny Group) III system, show that mistletoe belongs to the Santalaceae family.  The genus Viscum comprises about 90 species, of which two-thirds are native to Africa and one-third to Eurasia and Africa [36].

 The Phoradedron mistletoe is set in a part of Northern America (the North American Oak mistletoe, Phoradendron serotinum) and South America, Argentina. Loranthus europaeus belongs to the family Loranthaceae, which is widespread in America. Genera Notothixus mistletoe is found in the East of Australia and the Island. The Korthalsella kind is seen in East Asia, Malaysia, Australia, New Zealand. The genus Ginalloa includes only a few species that can be found in the South East of Asia. The mistletoe belonging to the genus Dendrophthora has a distribution similar to that of the genus Phoradendron, with an area in Central America. Genera Arceuthobium has approximately 24 species, being found in the USA and Mexico [37].

Host Trees and Geographical Area

The European mistletoe has several accepted subspecies that can be easily differentiated according to the host tree, as they are morphologically very similar. Mistletoe species differ in leaves’ shape and size, their fruits color, and host tree. V. album species parasitize over 400 host trees [38]. Table 1 summarizes literature data on mistletoe species/subspecies, their host trees on different continents, leaf size, and fruit color.

Table 1. Mistletoe species from different continents, their host trees, leaf size, and fruit color

Mistletoe can be seen in forests, on hills, and in the mountains, on various host trees, such as birch, ash, maple, poplar, lime, willow, or on conifers: fir, pine. The mistletoe also parasitizes fruit trees such as apples, pears, plums, cherries. The genus Viscum included species seen in the temperate areas of Europe, in Asia, in the tropical and subtropical parts of Africa, in Madagascar, in Australia, and the north and south of America

Mistletoe Bioactive Compounds

The mistletoe species (V. album L) has presented and still has a high interest in researching the bioactivity of its compounds. Many types of metabolites have been isolated from European mistletoe, some of which are not synthesized by mistletoe, but are obtained from the host tree, for example, some alkaloids [50].

Jäger et al., 2021 identified the specific biomarkers (arginine, pipecolic acid or lysine, dimethoxycoumarin, and sinapyl alcohol) of V. album ssp. album grew on three different host trees (M. Domestica, Q. Robur, and U. carpinifolia) [33].

The metabolite profile of V. coloratum harvested from three different host plants (Ulmus pumila L., Salix babylonica, and Populus ussuriensis Kom.) in two habitats (temperate continental climate and warm temperate humid monsoon climate, China) was investigated by Zhang et al., 2021 [51]. Three main metabolites, from the flavanone class, were identified, their synthesis and accumulation in mistletoe were dependent both on the host plant and environment [51].

Polyphenols - Chemical Structure and Classification

The flavonoids are a class of polyphenol compounds, secondary metabolites of vegetal origin, with antioxidant properties, which are found in the free state or seen as esters or glycosides in mistletoe leaves. More than 4.000 types of flavonoids have been identified in the plant kingdom [52].

Polyphenols occupy an important place in the life of plants by intervening in their metabolism. There are known about 8.000 plant-derived polyphenol compounds with antioxidant and antitumor properties [53-58]. Polyphenols are organic, water-soluble compounds that contain in their molecule one or more aromatic rings with one or more hydroxyl groups. Due to their chemical structural characteristics, polyphenols are compounds with high antioxidant power [52, 59-61]. The main polyphenols identified in mistletoe are from phenolic acids class, both hydroxybenzoic acid (gallic acid, protocatechuic acid) and hydroxycinnamic acid (caffeic, ferulic acid, synaptic acid) and flavonoids including flavanone (Naringenin, Eriodictiol), flavone (Apigenin), flavonol (3-O-Met Quercetin, Myricetin, Kamferol). The main polyphenols identified in the different subspecies of V. album and its host trees are presented in Figure 3.

The amount and composition of mistletoe flavonoids can vary significantly depending on the host tree, vegetation period, and harvest period [33, 41, 51, 62, 63].

The most important flavonoids identified so far in mistletoe leaves or stems are shown in Table 2.

Table 2. Polyphenols identified in different subspecies mistletoe leaves or stems depending on the host tree

V. album is a medicinal plant that contains a wide variety of phytochemicals. Quercetin, with high antioxidant activity and a strong antitumor effect, is one of the flavonols present in mistletoe being identified in V. album ssp. the album, V. album ssp. abietis, V. album ssp. austriacum and V. album ssp. coloratum [12, 63]. Among the flavanones, Naringenin and Eriodictyol are found both in V. album ssp. album and V. album ssp. austriacum [12, 51, 65]. Caffeic acid, a hydroxycinnamic acid, has been identified in various subspecies of V. album [12, 67].

Lectins and Viscotoxins

V. album is a medicinal plant that contains a wide variety of chemical compounds, from small molecules such as phenolic acids, flavonoids, to high molecular weight molecules proteins: lectins and viscotoxins [69].

Lectins are a class of proteins or glycoproteins, that can reversibly bind specific carbohydrates without altering their structure. Based on the specificity of carbohydrate-binding, lectins can be: monospecific - binds to one carbohydrate, (glucose, galactose) and polyspecific - binds to several sugars [24, 70]. In terms of chemical structure, mistletoe contains three lectins that differ in their molecular weight, about 60000Da and depending on the binding of carbohydrates [71, 72]. These lectins are coded as follows: ML 1, ML 2 and ML 3; (ML – Mistellectin) [73]. Lectin ML 1, is a glycoprotein that has two polypeptide chains linked together by a disulfide bond [74]. The chain A of lectin ML 1 is composed of three distinct individual domains, having a molecular weight of 29 kDa, 27 kDa, and 25 kDa, possessing RNA N-glycosidase activity [75]. The lectin B chain consists of two domains with the same configuration, with a molecular weight of 32 KDa and 25 KDa, respectively [24]. It has a specific carbohydrate-binding system, it binds specifically to D-galactose [76, 77]. The specificity of lectin B chain binding to carbohydrates plays a key role in explaining the selective cytotoxicity of lectins to tumor cells when interacting with various receptors. The specific interaction of lectins with receptors on the surface of cancer cells causes agglutination, apoptosis, and the stopping of the cell cycle and thus inhibition of angiogenesis and cell proliferation [78]. Thus, both lectin chains have cytotoxic action [24]. Lectin ML-2 binds specifically to D-galactose / N-acetyl-D-galactosamine [79]. Lectin ML-3 has a specificity for N-acetyl-D-galactosamine [72]. V. an album that grows on the following species: oak (Quercus ssp), poplar (Populus nigra) contains mostly lectin ML-1. V. album on conifers: fir (Abies alba), pine (Pinus sylvestris) contain mainly lectin ML-3 [71].

Viscotoxins are small polypeptides, rich in cysteine, and consist of about 46 amino acids, containing 3-4 disulfide bridges. They have a molecular weight of about 5KDa [80]. Depending on the amino acid sequence, the V. album contains 6 isomeric compounds: A1, A2, A3, B, B2, C1, 1-PS [72]. Viscotoxin has cytotoxic activity against different types of tumor cells and has immunomodulatory effects [15]. The composition and content of lectin and viscotoxin vary depending on the host tree.

In the V. album ssp album viscotoxin A2, A3 predominates, and 1-PS is missing while the V. album ssp austriacum is dominated by viscotoxin 1-PS, and viscotoxins A2, A3 have been detected in small amounts. V. album ssp abietis contains all viscotoxins, A3 predominates and viscotoxin A2 was not detected. [81]

Biological Properties and Pharmaceutical Action

Many biological effects of mistletoe, such as anticancer, apoptosis-inducing, antiviral,  antibacterial, and immunomodulatory activities have been reported [2, 11, 20, 82, 83].

A series of in vitro and in vivo studies confirm a broad spectrum of the therapeutic action of mistletoe (V. album). Table 3 summarizes the pharmacological activities of mistletoe (V. album ssp / Viscum coloratum / Viscum articulatum) depending on the host tree.

Table 3. The pharmacological activity of V. album ssp/ Viscum coloratum/ Viscum articulatum

*ns- not specified; MDA- malondialdehyde; MES- Maximum electroshock; INH-Isoniazid; 

Therapeutic, Cosmetic, and Functional Applications of Mistletoe Extract

Table 4 shows the patents published with mistletoe extracts (V. album) with therapeutic, cosmetic, and functional applications in the period 2011-2021.

Table 4. List of patents published from on V. album (2011-2021)

GGT gamma-glutamyltranspeptidase, GPT glutamic—pyruvic transaminase, NF-κB Nuclear factor-κB

Conclusion and Future Prospects

Mistletoe is a plant with a high potential for the treatment of various diseases. Its phytochemical composition and at the same time its biological effects depend on the host tree. The key components responsible for anticancer activity are lectin and viscotoxin. In addition, mistletoe also contains a sufficient amount of secondary metabolites from the class of flavonoids and phenolic acids, compounds with important beneficial effects. These compounds are widely distributed in plants and there is sufficient evidence to show that their consumption is closely linked to a decrease in the incidence of cancer, diabetes, and cardiovascular disease. Studies on the synergistic effects between mistletoe polyphenols and lectins should be further developed to identify new targeted therapeutic applications of mistletoe preparations.

Acknowledgments: The authors acknowledge the support provided by the University of Oradea through the grant competition "Excellent scientific research related to the priority fields with the goal of technology transfer: INO-TRANSFER-UO ", Project number 309/21.12.2021.

Conflict of interest: None

Financial support: This research was funded by the European Union, through the Horizon 2020 project “NextFood”, Grant agreement No. 771738.

Ethics statement: None

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