European Viscum album: a potent phytotherapeutic agent with multifarious phytochemicals, pharmacological properties and clinical evidence

Abstract

Viscum album L. or European mistletoe (Loranthaceae), a semi-parasitic shrub, has been used as a traditional medicine in Europe for centuries to treat various diseases like cancer, cardiovascular disorder, epilepsy, infertility, hypertension and arthritis. V. album contains diverse phytochemicals, which exert a large number of biological and pharmacological activities. The aim of this review is to compile the developments in the domain of V. album and research trends, with a focus on ethnopharmacology, phytochemistry and pharmacological properties, to illustrate the potential of this phytotherapeutic as an attractive commercial herbal medicine. Crude extracts and isolated chemical constituents from V. album have exhibited significant medicinal effects in experimental models and in patients with cancer, autoimmune and inflammatory conditions. Importantly, recent randomized clinical trials have suggested improved overall survival and quality of life in cancer patients treated with different mistletoe preparations. The current phytochemical studies have shown that lectins, hetero-dimeric glycoproteins, polysaccharides, viscotoxins, alkaloids, lipids, triterpenes, peptides, vesicles, flavonoids, cyclitols and amines are principal bioactive phytochemicals of V. album. Clinical studies and experimental models have revealed that V. album exhibits several pharmacological functions such as immunomodulatory, anti-hypertensive, anti-oxidant, cytotoxicity, anti-tumor, anti-inflammation, anti-diabetic, anti-microbial and sedative activities. It is conceivable that the heterogenous profile of biochemical compounds provides the basis for the broad diversity of pharmacological activities of mistletoe, as each single component contributes diverse modes of action in addition to imparting a synergistic beneficial action in conjunction with other molecules.

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Brahma N. Singh

Dr Brahma Singh obtained MS (2004) and PhD in Biochemistry (2010) from National Botanical Research Institute (NBRI)/Dr Ram Manohar Lohia Avadh University, India. Currently, he is a scientist at Pharmacognosy & Ethnopharmacology Division of NBRI. He did his postdoctoral studies at USA. His research is focused on the design of bacterial quorum sensing nanoinhibitors from plants and green nanomaterials for the development of next-generation anti-microbials and the design of anti-cancer bionanomaterials. He has also worked on bio-prospection of plants and bio-nanomaterials for anti-microbial and anti-cancer applications and phytoantioxidants for the development of naturaceuticals and functional foods.

Chaitrali Saha

Dr Chaitrali Saha received Master of Science degree in Bangalore, India in 2008 and PhD from the Université de Technologie de Compiègne, Compiègne, France in 2015. Her PhD research was focused on understanding the molecular mechanisms of action of Viscum album. Currently, she is pursuing postdoctoral research on the critical role for Notch signaling in controlling regulatory T cell function at National Centre for Biological Sciences, Bangalore, India.

Danijel Galun

Dr Danijel Galun is an Assistant Professor at University Clinic for digestive Surgery, Clinical Centre of Serbia, Belgrade, Serbia. He obtained medicine degree from the Medical School, University of Belgrade. His research interests are liver regeneration, liver cirrhosis, surgery of liver and pancreas, hepatocellular carcinoma and mistletoe extract therapy in gastrointestinal cancers. He is an Editorial Board member of Clinical and Molecular Hepatology and a member of EASL, EAES, EHS, Serbian chapter of A-EHPBA, Surgical Chapter of Serbian Medical Society. He was an active member of several clinical trials on Viscum album.

Dalip K. Upreti

Dr Dalip Upreti obtained his PhD in Botany (1983) from Lucknow University, Lucknow, India. After his postdoctoral fellowship at NBRI, India he joined as a scientist at plant diversity and lichenology, NBRI. He worked on various aspects including synthesis of anti-quorum sensing nanoparticles using lichen metabolites, identification of medicinally important Indian lichens, and their bio-prospection. He is elected as a fellow of National Academy of Science of India in 2014. He had been to Antarctica in 1991–1992 and is also the member of steering committee of Council of Scientific & Industrial Research, India for antarctic researches.

Jagadeesh Bayry

Dr Jagadeesh Bayry is a Principal Investigator at Institut National de la Santé et de la Recherche Médicale (French National institute of Health and Medical Research, INSERM). He received Veterinary Medicine degree in Bangalore, India in 1996, and completed his postgraduate studies in Virology and Immunology from the Indian Veterinary Research Institute. He obtained a PhD from the Université Pierre et Marie Curie, Paris in 2003. He later carried out postdoctoral research at the Edward Jenner Institute for Vaccine Research, University of Oxford, UK. In 2006, he was recruited as a scientist by INSERM. His research is aimed at understanding the cellular and molecular mechanisms of immune homeostasis and deciphering the host–pathogen interaction. He has authored more than 200 papers in leading journals.

Srini V. Kaveri

Dr Srini V. Kaveri is a Director of Research at Centre National de la Recherche Scientifique (CNRS), France. He received his Veterinary Medicine degree in Bangalore in 1978, and his PhD at the Pasteur Institute, Paris in 1987. He carried out his post-doctoral research at University of California, USA. In 1990, he was recruited as a scientist by CNRS. He has been involved with the basic and applied aspects of research in the area of immunopathology and immunotherapy. He has put forward various novel concepts towards understanding the basis for diseases of the immune system and his current interests are aimed at improving the therapeutic strategies for these diseases. He has authored more than 300 papers in leading journals.

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1. Introduction

Viscum album L., (Loranthaceae) commonly known as mistletoe or European mistletoe, is recognised by various names: European white-berry mistletoe, bird lime, birdlime, all-heal, and masslin. In German, V. album is called by Mistel, Vogelmistel, Leimmistel, Affolter, and Bocksfutter; gui, gui commun, and gui de druides in French; vischio, visco, vescovaggine, guatrice, pania, and scoaggine in Italian; muerdago in Spanish, and common mistletoe in Asia and Africa.^1–4 It is native to Europe and western and southern Asia.^5,6 V. album has been commonly used in local medicine in Europe and Asia for thousands of years.² In Europe, folk medicinal uses of V. album are recorded for curing various ailments such as cancer, hypertension, anxiety, insomnia, headache, internal bleeding and atherosclerosis. The compounds isolated from V. album so far mainly include hetero-dimeric glycoproteins, polysaccharides, lectins, amines, triterpenes, viscotoxins, alkaloids, lipids, peptides, cyclitols, vesicles and flavonoids.^7–10 European V. album with its bioactive phytochemicals is possessed with wide-reaching biological activities including immunomodulatory, anti-oxidant, cytotoxicity, anti-tumor, anti-hypertensive, sedative, anti-diabetic and hepato-protective. Meanwhile, V. album has also displayed significant inhibitory bio-activity against human cancer cell lines.^11,12 In addition, extract and phytochemicals have ability to inhibit inflammation and to prevent the development of cancer.^4,6 A number of phyto-pharmacological providers like WELEDA, ABNOBA HEILMITTEL, HELIXOR HEILMITTEL, NOVIPHARM and MADAUS market a range of different mistletoe preparations. Brand names of various preparations of mistletoe extract are Iscador®, ABNOBAViscum, Cephalektin, Eurixor®, Helixor®, Isorel and Lektinol™.

Owing to its extensive use as a potent phytotherapeutic agent in European countries, V. album has been in the spotlight of researchers for a long time. The present review provides an update on the developments in the domain of V. album and research trends, with a focus on ethnopharmacology, phytochemistry, pharmacological properties and clinical use.

2. Botany and distribution

V. album L. is a dioecious, insect-pollinated and hemi-parasitic evergreen shrub that grows on a number of host trees. It is commonly found on the crowns of broad-leaved deciduous trees including apple, ash, hawthorn, lime, cedar of lebanon, larch and other trees. On the oak and pear, it grows very rarely. V. album receives additional nutrients via haustorial attachment to a host, but is also able to photosynthesize. It has 30–100 cm long yellowish and smooth stems with dichotomous branching. The leaves are tongue-shaped, in apposite pairs, broader towards the end, 2–8 cm long, 1–2.5 cm wide, leather textured and of a dull yellow-green colour. Small flowers are inconspicuous and yellowish-green in colour, cluster in the forks of the branches and 2–3 cm diameter. Neither male nor female flowers have a corolla. The fruit is a white or yellow berry, smooth, ripening in December, containing one (very seldom) seed covered in the very sticky pulp.¹³ Based on the fruit colour, leaf shape and size, and most obviously the host trees, numerous subspecies of V. album have been classified. These include V. album subsp. abietis (Wiesb.) having white fruit and leaves up to 8 cm; V. album subsp. album having white fruit and leaves 3–5 cm; V. album subsp. austriacum (Wiesb.) having yellow fruit and leaves 2–4 cm; V. album subsp. meridianum (Danser) having yellow fruit and leaves 3–5 cm; V. album subsp. creticum having white fruit and short leaves; and Viscum album subsp. coloratum Kom, which is now considered as a separate species Viscum coloratum (Kom) Nakai by the Flora of China.

Geographically, Mistletoe is distributed from North Africa to Southern England and Southern Scandinavian regions, across Central Europe to Southwest and East Asia to Japan. In Europe, three subspecies have been identified depending on the species of host trees. V. album subsp. album grows on hardwoods, V. album subsp. abietis uses fir as host tree and V. album subsp. laxum is dependent on pines and spruce. V. album with coloured fruits are also recorded in further east.¹³ In United Kingdom, V. album is distributed from East Devon to Yorkshire, and is exceptionally common across London and regions of Central and Southern England.

3. Traditional uses and ethnopharmacology

The European V. album is a pharmaceutical plant and a symbol in mythology. It is the first plant, which was termed as “mistletoe”. According to G. P. Secundus (23–79 AC) this plant was considered to be an antidote for poisons and the plant became a miracle because of its ability to cure each illness.

Traditionally, mistletoe infusion has been used for high blood pressure, dizziness and hives. Ancient Greek records suggest that during 460–377 BC, spleen-related diseases were treated using Oak tree mistletoe. During 23–79 AC, Plinius explained the beneficial role of mistletoe in the treatment of infertility, ulcers and epilepsy. Platonist around 150 AC, described the utilization of mistletoe to treat tumors. In a French work on domestic remedies, in the year 1682, it was considered as a golden herb for treating epilepsy. During the year 1731, mistletoe was used for various purposes including labour pain and deworming in children. Later, it had been used for curing convulsions delirium, hysteria, neuralgia, nervous debility, urinary disorders, heart disease and many other complaints arising from a weakened and disordered state of the nervous system. Mistletoe extracts contain several toxic components, several of which are lectins, or proteins capable of binding to specific sugars. In 1921, the Austrian anthroposophical spiritual leader Rudolf Steiner recommended that mistletoe could be used to treat cancer, based on the observation that mistletoe, like cancer, is a parasitic and lethal to its host. Swiss and German clinics were founded to implement this idea and still actively use mistletoe preparation fermented with a strain of Lactobacillus for 3 days.

Despite having a strong historical background of mistletoe, in the 19^th century scientific community rejected mistletoe remedy and the interest was re-awakened in the 20^th century when Gaultier demonstrated oral/subcutaneous administration of fresh mistletoe extract to cure blood pressure-related issues both in animals and humans. Traditionally, the European mistletoe has been widely used for many years with remarkable therapeutic effects for the treatment of hypertension, anxiety, insomnia, internal bleeding, atherosclerosis and in complementary cancer therapies. In the year 1920, the founder of anthroposophy, Rudolf Steiner, introduced V. album as an anti-cancer remedy.¹⁴ Although local medicine at the end of the 19^th century still regarded mistletoe as a crucial part of the medicine box, academic medicine in the growing scientific age lost considerable attention in mistletoe as a remedy.

4. Bioactive constituents of European V. album

European mistletoe is characterized by a number of phytochemicals including lectins, polysaccharides, alkaloids, terpenoids, proteins, amines, peptides, polyphenols, flavonoids, phytosterols and amino acids (Table 1). Interestingly, some therapeutic phytochemicals such as certain alkaloids are not produced by the mistletoe rather absorbed from the host tree.

Table 1 Chemical constituents of V. album

S. no.	Chemical constituents	Content	Source	References
1	Viscotoxins	0.05–0.1%		18
	Isoforms: A1, A2, A3, B, B2, C1 & 1-PS		Leaves	17
			Stem
2	Lectins	0.34–1.0 mg g^−1 dried material	Older stems	22 and 24–26
	Isoforms: ML-I, ML-II, & ML-III
3	Carbohydrates	44–58%/dry wt	Leaves	28
			Stem
			Berries
	Methylated homogalacturonan		Stem	30
	Pectin		Berries	18
			Leaves
	1→α4-Galacturonic acid methyl ester		Berries	29
	Arabinogalactan		Berries	28
4	Polyphenols and phenylpropanoids	50–85 mg/100 g dry wt	Leaves	33
			Stem
			Berries
	Flavonoids			35 and 39
	Phenolic acids			33
	Lignans			40
	Kalopanaxin D			43
5	Vitamin C	750 mg/100 g fresh wt	Leaves	18
			Berries
6	Proteins	9.3%	Leaves	136
			Stem
			Berries
7	Lipophilic compounds	—
	Terpenoids		Leaves	47
			Stem
			Berries
	Phytosterols		Berries	46
	Saturated fatty acids		Leaves	48–50
			Stem
8	Inorganic elements
	Potassium, calcium, manganese, sodium, nickel, phosphate, selenium, silica, magnesium, and zinc	—	Leaves	18
			Stem
9	Others
	Cyclic peptides, alkaloids, amines (histamine and cetylcholine), jasmonic acid, cysteine, glutathione, and xanthophyll	—	Leaves	54
			Stem
			Berries

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4.1. Viscotoxins

Viscotoxins, a mixture of low-molecular weight cysteine-rich and basic proteins belong to plant thionins (α and β) and are synthesized in the leaves and stems.^15,16 They are amphipathic in nature consisting of 46 amino acids with a molecular mass of 5 kDa. The polypeptide chains are attached through three or four disulphide bonds at highly conserved positions (Cys3/Cys40, Cys3/Cys32, and Cys16/Cys26), giving them a compact structure and high stability against denaturing conditions such as heat and proteases. To date, seven different isoforms have been characterized (A1, A2, A3, B, B2, C1 and 1 PS) and are differed mainly in their sequence of amino acids.¹⁷ The content of viscotoxins varies from 0.05 to 0.1%, while composition depends on the host tree. For example, the presence of viscotoxins A2 and A3 were observed in V. album ssp. album (VAA), however the predominance of PS-V was detected in V. album ssp. austriacum. All viscotoxins, with the exception of A2, were detected but A3 was predominant in V. album ssp. abietis.¹⁸

Investigation on the 3D-structures of viscotoxins also provided information on a specific phosphate-binding site.¹⁹ It has also been assumed that the phosphate-binding site and amphipathic structures of the viscotoxins promote cytotoxicity in eukaryotic cells by interfering with cell membrane and altering its integrity. In addition to their high structural homology, biological effect of viscotoxins can vary according to their isoforms.²⁰ The viscotoxins reveal a high structural and pharmacological association with snake (cobra) cardiotoxins.²¹

4.2. Mistletoe lectins (MLs)

The main compounds isolated from V. album are MLs (a mixture of high-molecular-weight polypeptides) and the total content is in the range of 340–1000 μg g⁻¹ dried plant material or their content is not less than 2% of total polypeptides and proteins.^22,23 The lectin content is highest in the winter. Sprouts and shoots contain the highest concentrations. Three different MLs (ML-I, ML-II, and ML-III) with differential sugar-binding specificities have been isolated from European mistletoe by affinity chromatography on partially hydrolyzed Sepharose and human immunoglobulin-Sepharose.^18,24,25 These include galactose-specific ML-I (115 kDa, dimer), galactose- and N-acetyl-D-galactosamine-specific ML-II (60 kDa) and N-acetyl-D-galactosamine-specific ML-III (60 kDa). All three MLs have high reactivity with human erythrocytes without specificity for the A, B, and O blood groups.

Peumans and colleagues reported that deciduous trees contain mostly ML-I and European mistletoe growing on fir and pine trees found to be rich in ML-III.²² The subdomains of ML-I and ML-III were identified to be responsible for sugar binding. These authors also first described chitin-binding (cb) MLs with a molecular weight of 10.8 kDa. The primary structures of three cbMLs including cbML-1, cbML-2, and cbML-3 are very closely related to hevein. MLs are categorized as type-2 ribosome-inactivating proteins, which consist of two peptide chains namely chain A comprising three distinct individual domains and chain B having two domains with similar configurations.^18,26,27 The chains are linked by a disulfide bridge. The chain A inhibits protein synthesis by degrading the 28S rRNA in ribosomes of eukaryotic cells and also accelerates apoptosis. While, the chain B is capable of binding to glycoconjugates of cell surface and thereby permitting the cellular entry of toxic subunit.²² Based on glycosylation patterns of MLs, more than 20 different isoforms have been separated by isoelectric focusing.

4.3. Carbohydrates

Further constituents of European V. album include oligo- and polysaccharides. Structurally different types of mistletoe polysaccharides have been identified in the berries and leaves. A highly methylated homogalacturonan, pectin (42 kDa), 1→α4 galacturonic acid methyl ester and arabinogalactan (110 kDa) were characterized in leaves and stem, while berries were specially rich in other polysaccharides such as rhamnogalacturonans with arabinogalactan side chains (1340 kDa), arabinogalactans and small amounts of xyloglucans.^28–30 Monosaccharides and polyols were also identified, but after hydrolysis of mistletoe extract. The content of polysaccharides is varied depending on the host plants. For example, inositol (58%) was recorded at early stage of lime tree, however in the latter stage, galactose (44%) was dominant.

4.4. Polyphenols and phenylpropanoids

A range of flavonoids, phenylpropanoids, and phenolic acids were isolated from European V. album and the host tree has an influence on their contents. For example, high contents of salicylic acid and rosmarinic acid were detected in Sorbus aucuparia and Malus domestica, respectively. It has also been noticed that mistletoe grown on Fraxinus excelsior had the highest quantity of total phenolic acids and total flavonoids. The diversified qualitative and quantitative amount of phenolic acids including caffeic, phydroxybenzoic, salicylic, protocatechuic, ferulic, and sinapic acids were observed in a free state and as glycosides.^31,32 Recently, two new phenolic acids have been isolated from European white-berry V. album, including gallic acid and 3-(3′-carbomethoxypropyl)-7→3′′-protocatechoyl galloate.³³

Two classes of flavonoids such as chalcones and flavanones were isolated from alcoholic extract of V. album in their glycosidic form and with methoxyl groups in the molecules.¹⁸ They were 5,7-dimethoxyflavanone-4′-O-[2′′-O-(5′′′-O-trans-cinnamoyl)-apiosyl]-glucoside, 2′-hydroxy-4′,6′-dimethoxychalcone-4-O-[2′′-O-(5′′′-O-trans-cinnamoyl)-apiosyl]-glucoside, 5,7-dimethoxy-flavanone-4′-O-glucoside, 2′-hydroxy-4′,6′-dimethoxychalcone-4-O-glucoside, 2′-hydroxy-3,4′,6′-trimethoxychalcone-4-O-glucoside, 5,7-dimethoxyflavanone-4′-O-[apiosyl-(1→2)]-glucoside and (2S)-3′,5,7-trimethoxyflavanone-4′-O-glucoside, and 2′-hydroxy-4′,6′-dimethoxychalcone-4-O-[apiosyl(1→2)] glucoside.^34,35 A promising anti-oxidant flavonoid quercetin has been detected only after acid hydrolysis of V. album extract.³⁶ Other flavonoids including quercetin, kaempferol and their mono-, di- and trimethylethers were also characterized in epicuticular waxes of different subspecies of the European mistletoe.³⁷ Chaudhary and colleagues reported that 80% methanolic extract of mistletoe contains many polyphenolic constituents viz. 5,7-dimethoxy-4′-hydroxy flavanone, 3-(4-acetoxy-3,5-dimethoxy)-phenyl-2E-propenyl-β-glucoside, 5,7-dimethoxyflavanone-4′-O-β-glucoside, 4′-O-[bapiosyl(1→2)]-β-glucosyl]-5-hydroxy-7-O-sinapylflavanone, 3-(4-hydroxy-3,5-dimethoxy)-phenyl-2E-propenyl-β-glucoside and 4′,5-dimethoxy-7-hydroxy flavanone.³⁸

Recently, four new flavonoid glycosides were isolated and identified from the leaves and twigs of V. album such as 3,7,3′-tri-O-methylquercetin-4′-O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside, 7,3′-di-O-methylquercetin-4′-O-β-D-glucopyranosyl-3-O-[6′′′-(3-hydroxy-3-methylglutaroyl)]-α-D-glucopyranoside, 7,3′-di-O-methylquercetin-4′-O-β-D-glucopyranosyl-3-O-[(6′′′′→5′′′′)-O-1′′′′′-(sinap-4-yl)-β-D-glucopyranosyl-6-(3-hydroxy-3-methylglutaroyl)]-α-D-glucopyranoside and (2S)-5-hydroxy-7,3′-dimethoxyflavanone-4′-O-β-D-apiofuranosyl-(1→5)-O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside.³⁹

Phenylpropanoids are the important bioactive molecules of the European mistletoe, with extremely diverse structures and wide-spectrum medicinal effects. The leaves and stem were found to contain several phenylpropanoids including coniferyl alcohol 4-O-β-D-glucoside (coniferin), syringenin 4-O-β-D-glucoside (syringin), syringenin 4-O-β-D-apiosyl(1→2)-β-D-glucoside, and lignans such as syringaresinol 4′,4′′-di-O-glucoside (eleutheroside E) and syringaresinol-O-glucoside.^40,41 Lignan skeleton also contained trihydroxy-tetramethoxy-epoxy glucosides such as ligalbumosides A to E and alangilignoside C.⁴² The quantity of phenylpropanoids also varied according to the mistletoe subspecies. Maximum levels of syringin and coniferin were noticed in the extract of VAA by HPLC, having isocratic mobile phase (methanol : water : 0.1 N sodium acetate; 20 : 73.5 : 6.5). Apart from these phenylpropanoids, kalopanaxin D (4-[2-O-(apiosyl)-β-D-glucosyloxy]-3-methoxycinnamyl alcohol) was detected. V. album ssp. austriacum contains trace amount of coniferin, and both syringin and coniferin were characterized in V. album ssp. abietis. However, both these subspecies of mistletoe were unable to synthesize kalopanaxin D.⁴³

4.5. Lipid soluble compounds

Terpenoids, liposoluble compounds are the main components of European V. album. The lipid soluble extract of V. album showed the presence of oleanolic acid, β-amyrin acetate, β-amyrinacetate, lupeol, lupeol acetate, betulinic acid and ursolic acid.^44,45 A mixture of phytosterols including β-sitosterol and stigmasterol, and their esters were also identified.^46,47 Moreover, other lipophilic compounds specially saturated palmitic, arachidic, lignoceric, behenic and cerotic acids and the unsaturated oleic, linoleic and linolenic acids have been identified in the Turkey V. album extracts.^48,49 Long-chain fatty acids and hydrocarbons including loliolide, vomifoliol trans-α-bergamotene and trans-β-farnesene have been detected in the extracts obtained from supercritical fluid extraction method.⁵⁰ Due to poor solubility of triterpene in water, it is difficult to extract from the mistletoe. However, 2-hydroxypropyl-β-cyclodextrin and sodium phosphate (pH 7.3) as solubilizers have been developed and represent excellent tools for the extraction of triterpenes.^47,51

4.6. Trace minerals

In addition to organic components, mistletoe contains trace mineral elements including potassium, calcium, manganese, sodium, nickel, phosphate, selenium, silica, magnesium and zinc. Importantly, calcium has been detected mainly in its non-soluble oxalate form.^52,53 Mistletoe grown on oak and fir have high levels of manganese.⁵³

4.7. Other chemical constituents

Cyclic peptides, amino acids, proteins (9.3%), alkaloids, amines (histamine and cetylcholine), jasmonic acid, cysteine, glutathione, vitamin C and xanthophyll are the other constituents of V. album.^54,55 Three new diarylheptanoids including (3S,5R)-3-hydroxy-5-methoxy-1,7-bis(4-hydroxyphenyl)-6E-heptene (1), (3S,5S)-3-hydroxy-5-methoxy-1,7-bis(4-hydroxyphenyl)-6E-heptene (2) and (3S)-3-hydroxy-1,7-bis(4-hydroxyphenyl)-6E-hepten-5-one have been isolated and determined from the leaves and twigs of V. album.³⁹ Arabinogalactan proteins (APs) were isolated from berries and aerial part of European mistletoe. The ratios of arabinose and galactose in APs are 1 : 0.7 and 1 : 1.18 in barriers and aerial part, respectively.

5. Multifarious pharmacological properties

V. album has been used as a folk herbal remedy in Europe and was a mythical shrub that had a strong influence on people in ancient times. Owing to the presence of a range of therapeutic and bioactive chemical constituents, European mistletoe exhibits multifarious pharmacological activities by altering molecular events in the cells (Fig. 1 and Table 2).

Fig. 1 Molecular targets of V. album for different biological properties.

Table 2 Pharmacological properties of V. album and its bioactive compounds

Bioactivity	Extract/constituent	Model system	Mechanism	Dose	References
Antioxidant activity	Extracts of V. album grown on lime tree or white locust tree	HeLa cells	Inhibits mitochondrial DNA damage induced by H2O2	10 μg mL^−1 for 48 h	110
	Organic extracts	In vitro	Show anti-glycation and antioxidant properties		38
	Lectin	LLC-PK(1) cells	Exhibits free radical scavenging, expression of cyclooxygenase-2, inducible NO synthase, SIN-1-induced nuclear factor kappa B and the phosphorylation of inhibitor kappa B alpha	IC50 42.6 μg mL^−1	111
	Methanolic extract	Rats	Shows DPPH radical scavenging and anti-lipid peroxidation activities	500 mg kg^−1	112
Anti-inflammatory	Preparation (VA Qu Spez)	A549 cells	Inhibits prostaglandin E2, by selectively inhibiting COX-2	100 μg mL^−1	6
	Preparation (VA Qu Spez)	A549 cells	Reduces COX-2 mRNA half-life	50 μg mL^−1	4
	Flavonoids	Rats	Inhibit carrageenan-induced hind paw edema without any toxic effects	30 mg kg^−1	60
	Preparation (Isorel)	Mice	Triggers abundant tumour necrosis with inflammatory response, oedema and destruction of the malignant tissue	100 mg kg^−1	61
Immunomodulatory	Preparation (QU FrF)	B16 mouse melanoma	Abrogates IL-12 expression	20 μg per mouse per day	190
	VA Qu Spez	Dendritic cells	Stimulates proliferation of CD^4+ T cells	5–15 μg mL^−1	69
	N-Acetyl-D-galactosamine-specific lectin	T Cells	Inhibits 3000 immune functions-regulating genes	600 ng mL^−1	70
	KME	Epinephelus bruneus	Enhances phagocytic activity	1% and 2%	71
	Aviscumine	In vivo and clinical phase I studies	Activates immune system	1.5 mg per kg per day	72
	KML	Tumoral implantation	Modulates lymphocytes, natural killer cells, and macrophages		74
	VCA	Murine splenocytes	Decreases interferon (IFN)-gamma secretion	4–64 ng mL^−1	80
	VCA	hPBMC cells T-lymphocytes	Releases IL-1α, IL-1β, IL-6, IL-8, and IFN-γ	4–16 pg mL^−1, 4–32 pg mL^−1	62
	Lectin	T-Lymphocytes	Enhances expression of IL-1 alpha, IL-1 beta, IL-6, IL-10, TNF-α, interferon-gamma, and granulocyte-monocyte colony stimulating factor genes	1–8 ng mL^−1	62
	ML-I	Peripheral blood mononuclear cells	Induces cytokines gene expression and protein production	1 ng mL^−1	82
	ML-I	Peripheral blood mononuclear cells	Induces IL-6 and TNF-alpha production	10 ng mL^−1	82
	VAA extract	Epithelial cells	Stimulates the levels of CD4(+)CD25(+) and CD8(+)CD25(+) T cells and CD3(−)CD16(+)CD56(+) natural killer cells	10% ethanolic extract	83
	VTA1 (85 nm), VTA2 (18 nm) and VTA3	K562 and NK effector cells	Increase natural killer cell-mediated cytotoxicity	6–25 nM	85
	Viscotoxins	Rats	Enhance phagocytosis and burst activity against E. coli infection	25 and 250 μg mL^−1	86
	Aqueous extract	Human trail	Induces the secretion of Th1-(IFN-γ) or Th2-(IL-4)		87
	rVAA	Rat splenocytes	Enhances the secretion of an active form of IL-12	100 pg mL^−1	89
	Iscador Pini	Peripheral blood mononuclear cells	Activates T-helper cells (CD4+)	0.1–1.0 mg mL^−1	88
Cytotoxicity	Mistletoe lectin	SK-Hep-1 (p53-positive) and Hep 3B (p53-negative) cells	Induces apoptosis by stimulating mitochondrial membrane potential (MMP) breakdown and stimulating caspase-3	10–50 ng mL^−1	91
	VA Qu FrF	CEM, HL-60 and MM-6 cells	Reveals cell cytotoxicity	100–200 μg mL^−1	92
	Viscotoxin-free V. album extract		Enhances granulocyte activity		93
	Preparations	Daoy, D342, D425 and UW-288-2 cells	Induces cytotoxicity by reducing mitochondrial activity	50 mg mL^−1	94
	MLI, MLII, and MLIII	Molt 4 cells	Exhibits cytotoxic activity		95
	Viscotoxins and alkaloids	Tumor MSV cells	Show cytotoxicity	10 μg mL^−1	97
	MLI		Exhibits cytotoxicity		96
	KML-C	Human and murine tumor cells	Shows strong cytotoxicity by inducing apoptotic cell death	0.4–307 mg mL^−1	98
Anti-angiogenic	ME	B16L6 melanoma cells	Suppresses tumor growth and metastasis by elevating fragmentation and nuclear morphological changes	100 ng mL^−1	191
	VA QU FrF	Human umbilical vein endothelial and immortalized human venous endothelial cells	Induces apoptosis	12.5–50 μg mL^−1	12
	FME	Glioblastoma cells	Regulates cytokine TGF-β and matrix-metallo-proteinases central genes expression	100 μl mL^−1	100
	VAA-I	PLB-985 and chronic granulomatous disease cells	Induces apoptosis via caspase activation	1 mg mL^−1	101
	VAA-I	LPS-treated human neutrophils and murine neutrophils	Activates apoptosis	1–100 ng mL^−1	102
	VAA-I	Human neutrophils	Induces apoptosis via acceleration the loss of antiapoptotic Mcl-1 expression and the degradation of cytoskeletal paxillin and vimentin proteins	1–10 mg mL^−1	103
	Iscador Qu	Endothelial cell cultures	Causes early cell cycle inhibition followed by apoptosis		104
	Epi-oleanolic acid	Human and marine cancer cells	Activates apoptotic cell death, characterized by morphological changes and DNA fragmentation	4, 20, and 100 μg mL^−1	105
	VCA	Hepatocarcinoma Hep3B cells	Induces apoptosis by increasing ROS production and a loss of DeltaPsim	20 ng mL^−1	106
	β-Galactoside, N-acetyl-D-galactosamine-specific lectin II, polysaccharides, viscotoxin	U937 cells	Induce apoptosis through activation of the phosphotransferase activity of c-Jun N-terminal kinase 1 (JNK1)/stress-activated protein kinase (SAPK)	100 ng mL^−1	107
	Viscotoxins	Lymphocytes	Induce cell death by producing mitochondrial Apo2.7 molecules and by generating ROS-intermediates	100 ng mL^−1	174
Anti-tumoral	Aqueous extract	Cancer cells	Depletion of hypoxanthine concentration and xanthine oxidase activation		113
	Lipophilic extract and its predominant triterpene oleanolic acid	Tumour cells	Decreases MCP-1 induced monocyte transmigration	25 μg mL^−1	114
	Ethanolic extract containing viscotoxin	Swiss female mice	Enhances the anti-tumor effect of doxorubicin	50 mg kg^−1	115
	VAE	C6 glioma cells	Induces apoptosis by activating caspase-mediated pathway	100 μg mL^−1	116
	VAA-1	Lung carcinoma A549	Enhances anti-proliferating potential of cycloheximide by inducing G1-phase accumulation		117
	Synergistic effect of mistletoe extract and its components	U937 cells	Induce apoptosis via activation of caspase cascades	100 ng mL^−1	118
	ML-II	U937 cells	Enhances apoptotic response through augmentation of Fas/Fas L expression and caspase activation	100 ng mL^−1	119
	ML-III	Tumour cell lines and human lymphocytes	Reduce the expression of nuclear p53 and Bcl-2		120
	Oleanolic acid	HaCaT cells	Induces apoptosis by altering cellular morphology as well as DNA integrity	12.5–200 μM	121
	Either solubilized triterpene acids or lectins and combinations thereof	Acute lymphoblastic leukaemia cell line NALM-6	Induce dose-dependent apoptosis via caspase-dependent pathways	8 ng mL^−1	122
	VE from apple and pine	Human macrophages	Increases anti-tumoral activity of activated human macrophages by inducing the production of NO		123
	Lectin containing Iscador M Spezial and Iscador Qu Spezial	Mammary cancer MAXF 401NL cells	70% growth inhibition	15 μg mL^−1	124 and 125
	VAE	Bladder carcinoma T24, TCCSUP, J82 and UM-UC3 cell lines	Induces necrosis and apoptotic cell death	10–1000 μg mL^−1	126
	Viscotoxin B2	Rat osteoblast-like sarcoma 17/2.8 cells	Exhibits antitumor activity	IC50 1.6 mg L^−1	127
	Viscin	Molt4 U937 leukaemia cells	Inhibits growth and induce apoptotic cell death	IC50 118 ± 24 & 138 ± 24 μg mL^−1	48
	VAC	Hepatoma cells	Induces apoptosis by decreasing Bcl-2 level and telomerase activity and by inducing of Bax	10 ng mL^−1	128
	Lectins	Rat liver	Modulate protein kinase activities	100 μg mL^−1	129
	Lectin	Yac-1 tumor cells	Increases natural killer-mediated cytotoxicity	50 ng per mouse	130
	KM-110	B16-BL6, 26-M3.1, L5178Y-ML25 cells	Inhibits lung metastasis	100 μg per mouse	131
	Iscador	Melanoma cells in mice	Inhibits lung metastasis by reducing nodule formation (92%) and by enhancing a life span (71%)	IC50 0.0166 mg per dose	132
	ME	Mice	Inhibits pulmonary metastatic colonization	3, 30 or 150 ng kg^−1	133
	KM-110	L5178Y-ML25 lymphoma cells	Inhibit liver and spleen metastasis	100 μg mL^−1	131
Anti-diabetic	VAC	Mice	Enhances the insulin secretion from the pancreatic β-cell without any effects of cytotoxicity	2 mg mL^−1	55
	VAC	Mice	Upregulates pattern of insulin genes such as PDX-1 and β2/neuroD	2 mg mL^−1	55
	ML-I		Mimics the sugar compound		137
	VE		Shows potent alpha-glucosidase inhibitory activity	IC50 10.1 mg mL^−1	138
	Aqueous extract		Stimulate secretion of insulin (1.1 to 12.2-fold) from clonal pancreatic B-cells	1–10 mg mL^−1	139
Anti-hypertensive	Ethanol extract	Wistar rats	Reduces the blood pressure	3.33 × 10^−5 mg kg^−1	140
Anti-microbial	Methanolic extract	Pathogenic microorganisms	Shows antimicrobial activity		141
	Aqueous extract	Vero cells	Prevent HPIV-2 replication and the virus production	1 μg mL^−1	142
Anti-mutagenic	VAC	Salmonella typhimurium strains TA98 and TA100	Prevents the mutagenicity of the indirect-acting mutagen 2-aminoanthracene	100–400 μg mL^−1	145
Anticonvulsant	VE	4.5 year old girl	Manages refractory childhood absence epilepsy		146
Wound healing activity	Lipophilic extract	Rats	Stimulates migration of NIH/3T3 fibroblasts	10 μg mL^−1	147
Anti-ageing	VAC	Caenorhabditis elegans and Drosophila melanogaster	Promotes the mean survival time	50 μg mL^−1	148
Anti-obesity	VAC	Mice	Protects against hepatic steatosis	3 g per kg per day	149
Endurance promoting	KME	Cell lines	Activates PGC-1α and SIRT1	400 & 1000 mg mL^−1	150
Others	Aqueous extract of leaves	Mice and rats	Exhibits sedative, antiepileptic and antipsychotic activities	50 & 150 mg mL^−1, p.o.	2

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5.1. Anti-inflammatory

Our group has found that VA Qu Spez impedes cytokine-induced prostaglandin E2 (PGE₂), by selectively inhibiting cyclooxygenase-2 (COX-2), which is transcriptionally activated in response to various pro-inflammatory cytokines.⁶ Further dissection of the molecular events revealed that V. album significantly reduces the COX-2 mRNA half-life without influencing its protein stability, implicating that V. album induces destabilization of COX-2 mRNA.⁴ Excessive amount of PGE₂ is also associated with pro-tumoral conditions and enhances dendritic cell-mediated regulatory T cell expansion.^56–59 Thus, inhibition of PGE₂ by V. album was also important for anti-tumoral functions. To assess the anti-nociceptive and anti-inflammatory activities of isolated flavonoids of V. album, p-benzoquinone-induced writhing test and carrageenan-induced hind paw edema model were used, respectively. The ethyl acetate fraction (250 mg kg⁻¹) as well as 2′-hydroxy-4′,6′-dimethoxy-chalcone-4-O-beta-D-glucopyranoside and 5,7-dimethoxy-flavanone-4′-O-[β-D-apiofuranosyl-(1→2)]-β-D-glucopyranoside at the concentration of 30 mg kg⁻¹ dose were shown to possess remarkable anti-nociceptive and anti-inflammatory activities, without inducing any apparent acute toxicity as well as gastric damage.⁶⁰

A single intraperitoneal (IP) injection of the mistletoe preparation Isorel (100 mg kg⁻¹) decreased the size of the tumour and triggered abundant tumour necrosis with inflammatory response, oedema and destruction of the malignant tissue.⁶¹ Moreover, the Isorel-treated melanoma cells were found to be more sensitive to the cytotoxic activity of the lymphocytes in the presence of Isorel-treated mice plasma than the control tumour cells.

5.2. Immunomodulatory

V. album and their chemical constituents have well-known immunomodulatory properties. V. album significantly enhanced interferon gamma (IFN-γ) responses.⁶² Our group has reported that VA Qu FrF mistletoe preparation significantly inhibits tumor growth in vivo in an IL-12-dependent mechanism.⁶³ As dendritic cells are key players in regulating the immune responses,^64–68 we examined the effect of V. album on these innate cells. V. album Qu Spez enhanced the expression of several antigen-presenting and co-stimulatory molecules on human dendritic cells and additionally induced secretion of pro-inflammatory cytokines such as IL-6 and IL-8, and stimulated the proliferation of CD4⁺ T cells.⁶⁹ A transcriptome analysis of the gene expression profile in human T cells following incubation with N-acetyl-D-galactosamine-specific lectin of V. album var. coloratum agglutinin (VCA, Korean mistletoe lectin) revealed activation and inhibition of 3000 genes, which are involved in a wide range of immune functions. These genes were related to cytokines, cell adhesion, cell motility, cell growth and maintenance, cell death and the response to stress and external stimulus.⁷⁰

A diet enriched by 1% and 2% of Korean mistletoe extract positively enhanced innate immune responses such as respiratory burst and phagocytic activity in kelp grouper Epinephelus bruneus against Philasterides dicentrarchi.⁷¹ A recombinant form of Escherichia coli, producing ML (aviscumine) was developed. Immunomodulatory and cytotoxic activities have been observed in in vivo and clinical phase I studies.⁷² The natural killer (NK) cells have been anticipated as one of the candidates for direct tumour cell destruction.⁷³ Under in vitro and in vivo systems, Korean mistletoe lectin was found to enhance the immune system through modulation of lymphocytes, NK cells and macrophages.⁷⁴ Subcutaneous (SC) administration of mistletoe causes increase in relative number of lymphocytes with activated phenotype, NK cells and specific subsets of lymphocytes including B cells, CD4⁺ T cells and cytotoxic T cells.⁷⁵ These results were also confirmed in other studies and found that V. album treatment can result in normalization of initial immune indices.^25,76–78 Further, mistletoe extracts obtained from apple (mali) or pine (pini) induced in vitro oligoclonal activation of CD4⁺ T cells from mistletoe-treated cancer patients.⁷⁷ A placebo-controlled study in healthy individuals found that Iscador Quercus causes eosinophilia due to stimulation of IL-5 and GM-CSF by ML.⁷⁸ Non-toxic doses of ML-1 or its carbohydrate-binding subunit prompted significant increase in components of the cellular host defense system including NK cell cytotoxicity or release of various cytokines including IL-1, TNF-α and IL-6.⁷⁹

The effect of VCA on murine splenocytes was investigated to examine whether VCA acts as an immunomodulator. VCA in a dose-dependent manner (4–64 ng mL⁻¹) decreased IFN-γ secretion in concanavalin A (ConA)-stimulated murine splenocytes without changing IL-4 levels.⁸⁰ Treatment of VCA also resulted in an anti-proliferative effect at 2–8 ng mL⁻¹ and 1–8 ng mL⁻¹ in human peripheral blood mononuclear cells (hPBMC) and T lymphocytes, respectively. However, at lower doses (4–16 pg mL⁻¹ and 4–32 pg mL⁻¹ respectively), a proliferative effect was noticed in hPBMC and T lymphocytes.⁶² The RT-PCR results confirmed the release of pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6, IL-8 and IFN-γ, when cells were treated with low doses of VCA (4–32 pg mL⁻¹). Another report also confirmed enhanced expression of aforementioned cytokine genes upon stimulation of hPBMC with V. album agglutinin-I.⁸¹ These data might suggest new perspective of VCA to regulate the balance between cell proliferation, cytokine production and apoptotic cell death. Induction of these cytokine genes and protein production in the cultures of hPBMC was also observed upon treatment with ML-I.⁸²

VAA extract increased phagocytic activity and candidacidal activity of neutrophils, and decreased adhesion function of epithelial cells. Furthermore, extract stimulated the levels of CD4⁺CD25⁺ and CD8⁺CD25⁺ T cells and CD3⁻CD16⁺CD56⁺ natural killer cells.⁸³ A study reported that the cell killing capacities of mistletoe extracts are host tree-specific and not correlated with ML or viscotoxin content.⁸⁴ The newly isolated mistletoe viscotoxins such as VTA1 (85 nm), VTA2 (18 nm) and VTA3 were found to increase natural killer cell-mediated cytotoxicity.⁸⁵ Impact of the viscotoxins on human granulocytes was studied by flow cytometry and it was found that viscotoxins at 25 and 250 μg mL⁻¹ concentrations enhanced phagocytosis and burst activity against E. coli infection in respiratory track.⁸⁶ The SC treatment of aqueous mistletoe extract in 8 volunteers was reported to induce the secretion of Th1 (IFN-γ) or Th2 (IL-4) cytokines and also the release of TNF-α and IL-6.⁸⁷

Numerous studies have reported that Iscador Pini (a fermented extract of mistletoe) induces a strong stimulatory response of hPBMC in normal and allergic individuals. A study was conducted to examine the cell subtypes involved in this in vitro reactivity. Flow cytometry results clearly showed that Iscador activates T cells (CD3⁺), especially CD4⁺ T helper cells, as well as monocytes at concentrations of 0.1 to 1.0 mg mL⁻¹. No evidence for a key involvement of B cells (CD19⁺), NK cells (CD56⁺), and T suppressor cells/cytotoxic T lymphocytes (CD8⁺) was detected.⁸⁸ A recombinant V. album agglutinin (rVAA) enhanced the secretion of an active form of IL-12 and potentiated the cytokine-induced NK cell activation in cultured rat splenocytes. These authors also stated that the effects of rVAA could be associated with its enhancing effects on MHC-unrestricted cytotoxicity in vivo.⁸⁹ However, the contradictory report on phagocytic activity of lectin is also reported and investigators found that various concentrations of lectin ranging from 0.025 to 20 ng mL⁻¹ had only marginal effect on phagocyte activity.

5.3. Cytotoxicity

ML can induce apoptosis via apoptosis-associated factor-1 (Apaf-1) pathway by stimulating mitochondrial membrane potential (MMP) breakdown and stimulating caspase-3.^90,91 The c-Jun N-terminal kinase (JNK) stimulation by ML-I leads to translocation of the pro-apoptotic proteins Bax and Bad. ML-I down regulates B-cell lymphoma 2 (Bcl-2) and up regulates TNF-α and hence provoke apoptosis. We have demonstrated that VA Qu FrF, induces significant cell toxicity in vitro in the human T cell lines CEM and in monocyte cell lines HL-60 and MM-6.⁹² The viscotoxin-free V. album extract significantly enhanced granulocyte activity and this effect was correlated with the content of the ML.⁹³

Treatment of V. album preparations from eight dissimilar host trees (Iscucin Abietis, Pini, Populi, Mali, Salicis, Crataegi, Quercus and Tiliae) showed a significant cytotoxic effect on the medulloblastoma cell lines including Daoy, D342, D425, and UW-288-2, yet the cell susceptibility was unrelated against the different extracts. The reduction in mitochondrial activity and enhancement in apoptotic cell death correlated with the lectin content of the used preparation in a dose-dependent manner.⁹⁴ ML-I, ML-II, and ML-III were found to be toxic for Molt 4 cells at pg concentrations, ML-III being the most cytotoxic. Interestingly, the digalactosides Gal beta 1,2-Gal beta-allyl and Gal beta 1,3-Gal beta-allyl were able to bind to the B-chain of these lectins and inhibit their toxic activity. N-Acetyl-D-galactosamine and rho-nitrophenyl N-acetylgalactosamine prevented the toxic effects of ML-II and III.⁹⁵

Major cytotoxic components were fractionated from Korean mistletoe and the changes in their cytotoxic effects due to heat treatment were studied. ML-I showed maximum toxicity, but was disappeared upon heating for 30 min. The study suggested that the ML is not responsible for inducing apoptosis rather it might be due to other components.⁹⁶ Viscotoxins and alkaloids were found to retain their effects even after heating for 60 and 180 min, respectively. Moreover, the alkaloid fraction was more effective to induce cytotoxic effects on tumor MSV cells than non-tumor A31 cells.⁹⁷ The isolated KML-C showed strong cytotoxicity against various human and murine tumor cells by inducing apoptosis mediated by Ca²⁺/Mg²⁺-dependent endonucleases. However, the cytotoxic activity of KML-C was higher than that of a lectin from European mistletoe V. album spp. loranthaceae.⁹⁸

5.4. Anti-angiogenic

Treating B16L6 melanoma cells with V. album suppressed tumor growth and resulted in DNA fragmentation and nuclear morphological changes, suggesting that V. album inhibits tumor growth and metastasis by elevating apoptosis and blocking angiogenesis.⁹⁹ Our group has shown that VA Qu FrF induces apoptosis of endothelial cells in human umbilical vein endothelial cells and in immortalized human venous endothelial cell line.¹² Fermented mistletoe extract (FME) treatment of glioblastoma cells down-regulated TGF-β and matrix-metallo-proteinase genes expression, which involve in glioblastoma progression and malignancy. In addition, FME reduced the migratory and invasive potential of glioblastoma cells.¹⁰⁰ VAA-I is a plant lectin, which possesses anti-tumoral properties. VAA-I was reported to induce apoptosis in PLB-985 cells and cells from chronic granulomatous disease via caspase-mediated pathway.¹⁰¹

The role of VAA-I on activated neutrophils and pro-inflammatory properties have not much explored so far. Lavastre et al. demonstrated that VAA-I at 1000 ng mL⁻¹ activates apoptotic cell death in lipopolysaccharide (LPS)-treated human neutrophils in vitro as well as in murine neutrophils isolated from LPS-induced neutrophil influx.¹⁰² They concluded that VAA-I can inhibit LPS-induced pro-inflammatory response in vivo.¹⁰² VAA-I induces apoptosis in human neutrophils by accelerating the loss of anti-apoptotic Mcl-1 expression and the degradation of cytoskeletal paxillin and vimentin proteins via caspases.¹⁰³ Iscador Qu, an aqueous fermented extract from the European mistletoe grown on oaks caused early cell cycle inhibition followed by apoptosis in a dose-dependent manner in endothelial cell cultures. Apoptosis was induced by activating the mitochondrial activity.¹⁰⁴

Epi-oleanolic acid, a triterpene was isolated from the dichloromethane extract of Korean V. album (KVA) by repeated silica gel chromatography and recrystallization. Treatment of triterpene showed a typical pattern of apoptotic cell death, including morphological changes and DNA fragmentation in human and murine cancer cells.¹⁰⁵ A study on the anti-cancer mechanisms of action of VCA from Korean mistletoe suggested that VCA induces apoptosis in hepatocarcinoma Hep3B cells by inducing ROS production and a loss of DeltaPsim, in which JNK phosphorylation plays a key role in these events.¹⁰⁶ The β-galactoside- and N-acetyl-D-galactosamine-specific lectin II, polysaccharides, and viscotoxin of mistletoe were found to induce apoptosis in U937 cells through the activation of phosphotransferase activity in JNK1/stress-activated protein kinase and was characterized by DNA ladder pattern fragmentation.¹⁰⁷ However, protein kinase A or C protected the apoptosis induced by MLII of KVA in the human leukemic HL-60 cells.¹⁰⁸ The viscotoxins induced cell death by producing mitochondrial Apo2.7 molecules and by generating ROS-intermediates in lymphocytes.¹⁰⁹

5.5. Anti-oxidant

It is well known that the anti-oxidant activity of V. album extracts are varied depending on the host tree and the harvesting time. It was observed that extract from the lime tree or white locust tree completely inhibits mitochondrial DNA damage induced by H₂O₂ in HeLa cells, while extract from hedge maple tree inhibits mitochondrial DNA damage only by 50%.¹¹⁰

Organic extracts of V. album, which contains polyphenolic compounds were reported to exert anti-glycation and anti-oxidant properties.³⁸ Oxidative stress protective activity of Korean mistletoe lectin was examined under in vitro system using LLC-PK1 renal epithelial cells. Korean mistletoe lectin exhibited strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging potential with an IC₅₀ value of 42.6 μg mL⁻¹.¹¹¹ In addition, it exerted free radical quenching potential against nitric oxide (NO), superoxide anion, and hydroxyl radical in a concentration-dependent manner. Further, inhibition of COX-2, inducible NO synthase, SIN-1-induced nuclear factor kappa B, and the phosphorylation of inhibitor kappa B alpha was also seen in lectin-treated LLC-PK1 cells.¹¹¹

Methanol extracts of mistletoe grown on different host trees were studied for their potential anti-oxidant activity. The extract from mistletoe grown on lime tree in summer showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ant-lipid peroxidation activities.¹¹²

5.6. Anti-tumoral

A pre-clinical study suggested that aqueous mistletoe extract exhibits potent anti-tumoral activity by depleting hypoxanthine and activating xanthine oxidase in the cancer cells, which lead to lowered salvage pathway activity required for the cancer cells to proliferate in the cancerous colon tissue.¹¹³ Lipophilic extract of V. album and its predominant triterpene oleanolic acid significantly decreased monocyte chemotactic protein-1-induced monocyte transmigration.¹¹⁴ Ethanol extract of V. album containing viscotoxin enhanced the anti-tumor effect of doxorubicin.¹¹⁵ Pretreatment of C6 glioma cells with 100 μg mL⁻¹ of VAE before heat shock significantly decreased expression levels of Hsp27 (73%), 14-3-3β (124%), 14-3-3γ (23%), and 14-3-3ζ (84%) proteins. Increased apoptosis was also observed through caspase-3 activation (60%).¹¹⁶ VAA-I enhanced anti-proliferating potential of cycloheximide in the human lung carcinoma cell line A549 by inducing G1-phase accumulation.¹¹⁷ The KML-II was also found to induce apoptosis in U937 cells via activation of caspase cascades.¹¹⁸

A large number of studies on synergistic effect of mistletoe extract and its components are available. IFN-γ enhanced the apoptotic response to ML-II through augmentation of Fas/Fas L expression and caspase activation in human myeloid U937 cells.¹¹⁹ The MLs activated apoptotic pathway in various tumour cell lines and human lymphocytes. The ML-III was also found to reduce the expression of nuclear p53 and Bcl-2.¹²⁰

Oleanolic acid, a component of the leaves and roots of V. album induced apoptosis by altering cellular morphology as well as DNA integrity in HaCaT cells in a dose-dependent manner, with comparatively low cytotoxicity.¹²¹ Either solubilized triterpene acids or lectins and combinations thereof were found to induce dose-dependent apoptosis in the acute lymphoblastic leukaemia cell line NALM-6 via caspase-8 and -9 dependent pathways in vitro and in vivo.¹²² V. album from apple and pine increased anti-tumoral activity of activated human macrophages by inducing the production of NO.¹²³ Anti-tumor activity of Iscador M Spezial, Iscador Qu Spezial and Iscador P preparations of V. album at high concentrations were investigated in a panel of 12 cell lines.^124,125 The lectin-containing Iscador M Spezial and Iscador Qu Spezial showed a noticeable anti-tumor activity in the mammary MAXF cancer 401NL cells at 15 μg mL⁻¹ concentration with more than 70% growth inhibition compared to untreated control cells.^124,125

Anti-proliferative effect of V. album extracts was characterized in the bladder carcinoma T24, TCCSUP, J82 and UM-UC3 cell lines. Necrosis and apoptotic cell death were the fundamental mechanisms of anti-tumoral effect of V. album extracts.¹²⁶ The primary structure and anti-tumor activity of a novel peptide from stem and leaves of mistletoe (V. coloratum (Kom.) Nakai) was examined on the rat osteoblast-like sarcoma 17/2.8 cells.¹²⁷ The primary structure of the peptide named as viscotoxin B2 was KSCCKNTTGRNIYNTCRFAGGSRERCAKLSGCKIISASTCPSDYPK and its IC₅₀ value was 1.6 mg L⁻¹. Viscin, betulinic acid, oleanolic acid and ursolic acid, which are lipophilic compounds of V. album were found to inhibit growth and induce apoptotic cell death in Molt4, K562 and U937 leukaemia cells. However, the growth inhibitory effect of viscin was more prominent on Molt4 and U937 cells with IC₅₀ values 118 ± 24 and 138 ± 24 μg mL⁻¹ respectively.⁴⁸

VCA has been shown to induce apoptosis by decreasing Bcl-2 level and telomerase activity and by inducing of Bax through p53- and p21-independent pathway in hepatoma cells. Later on, the induction of apoptosis via activation of caspase-3 and the inhibition of telomerase activity through transcriptional suppression of hTERT in the VCA-treated A253 cells was reported.¹²⁸ Treatment with VCA also induced apoptosis in both SK-Hep-1 (+p53) and Hep 3B (−p53) cells through p53- and p21-independent pathways. Apoptosis induction was related to down-regulation of Bcl-2 and up-regulation of Bax functioning upstream of caspase-3. Moreover, VCA caused down-regulation of telomerase activity in both the cells.⁹¹ Signaling through lectins could involve modulation of protein kinase activities. However, the alpha/beta-galactoside-binding lectins, isolated from mistletoe leaves, did not inhibit the epidermal growth factor receptor tyrosine kinase activity of rat liver.¹²⁹

Administration of lectin (KML-C) from Korean mistletoe (20–50 ng per mouse) for 2 days by intravenous route before tumor implantation significantly reduced lung metastases of B16-BL6 and colon 26-M3.1 cells. Importantly, KML-C treatment one-day post-tumor implantation not only significantly suppressed lung metastasis of B16-BL6 and 26-M3.1 cells, but also reduced liver and spleen metastasis of L5178Y-ML25 lymphoma cells. Mechanistically, it was found that KML-C treatment (50 ng per mouse) for 2 days significantly increased NK cell-mediated cytotoxicity against tumor cells and tumoricidal activity of peritoneal macrophages.¹³⁰ Similar results were also observed with Korean mistletoe extract KM-110, wherein administration of KM-110 (100 μg per mouse) for 2 days by intravenous route before tumor implantation significantly blocked lung metastases of B16-BL6 and 26-M3.1 cells, and liver and spleen metastasis of L5178Y-ML25 cells. This effect on tumor metastasis was also mediated by NK cell activation.¹³¹ Additionally, multiple administration of KM-110 into tumour-bearing mice resulted in significant inhibition of primary tumour growth.¹³¹ Administration of the Iscador (1 mg Iscador per dose, IP) inhibited lung metastasis of B16F10 melanoma cells in mice by reducing nodule formation (92%) and enhanced the life-span (71%) of animals.¹³² The IC₅₀ was found to be 0.0166 mg per dose. However, galactoside-specific mistletoe lectin failed to inhibit N-methyl-N-nitrosourea-induced tumor development in the urinary bladder of rats and to mediate a local cellular immune response following long-term administration.¹³³ Therefore, anti-tumoral functions are not mediated by all lectin preparations of mistletoe.

Intravenous treatment with a standardized mistletoe extract at 3, 30 or 150 ng kg⁻¹ doses once daily for 3 weeks exerted inhibitory effects (58 to 95%) on the lung metastasis of B16. A significant reduction in the percentage of bronchoalveolar lavage pigmented cells was also noticed.¹³⁴ The study conducted by Srdic-Rajic and co-workers investigated synergistic anti-tumor effects of V. album and doxorubicin chemotherapeutic agent on chemo-resistant chronic myelogenic leukemia K562 cells. Authors found that V. album enhances the anti-leukemic efficiency of doxorubicin against chemo-resistant K562 cells by checking the G2/M arrest and by stimulating apoptosis.¹³⁵

5.7. Anti-diabetic

V. album has been well-documented as a traditional treatment for diabetes. The Korean mistletoe V. album var. coloratum enhanced the insulin secretion from pancreatic β-cells without any cytotoxicity effects. Moreover, upregulated pattern of insulin genes such as PDX-1 and β2/neuroD was also observed in V. album var. coloratum-treated mice. Thus, VAC could be considered as a useful source for the development of anti-diabetic drug to reduce the blood glucose level of type I diabetic patients.¹³⁶ Structural analysis of ML-1 complexed with galactose and lactose revealed unique sugar binding abilities.¹³⁷ Among the medicinal plants, V. album showed potent alpha-glucosidase inhibitory activity.¹³⁸ The aqueous extract of mistletoe (1–10 mg mL⁻¹) was reported to stimulate secretion of insulin (1.1- to 12.2-fold) from clonal pancreatic β-cells. The ability of extract to enhance insulin secretion was not mediated by lectins.¹³⁹ The results indicated the presence of insulin-releasing natural product(s), which might contribute to the reported anti-diabetic property of the mistletoe.

5.8. Anti-hypertensive

Acute effect of different extracts of mistletoe stem on arterial blood pressure was studied in Wistar rats.¹⁴⁰ The ethanol extract showed the superlative effect even at the lowest applied concentration (3.33 × 10⁻⁵ mg kg⁻¹) and significantly reduced the blood pressure after applied concentration (1.00 × 10⁻³ mg kg⁻¹). However, other extracts such as ether and ethyl acetate showed the activity only at higher concentrations.

5.9. Anti-microbial

Methanol extract of V. album showed anti-microbial activity against 9 out of 32 pathogenic microorganisms.¹⁴¹ Different extracts from the leaves of V. album L. ssp. album were prepared and analyzed for their effect on human parainfluenza virus type 2 (HPIV-2) growth in Vero cells.¹⁴² The aqueous extract (1 μg mL⁻¹) was found to prevent HPIV-2 replication and that virus production was inhibited >99% without any toxic effect on host cells. This activity could neither be credited to the direct HPIV-2 inactivation nor to the inhibition of adsorption to Vero cells. Five patients with chronic hepatitis C showed 6–20% reduction in the viral load and normalization of liver inflammation (two patients) without side effects following treatment with Iscador for one year.¹⁴³ Two other patients were also in complete remission of their elevated aspartate transaminase and alanine transaminase. However, IFN-γ increase in the serum of HIV-positive and healthy subjects was not noticed following subcutaneous injection of a non-fermented V. album extracts.¹⁴⁴

5.10. Anti-mutagenic

V. album var. coloratum was evaluated for its anti-mutagenic activity against the mutagens such as 2-aminoanthracene (2AA) and furylfuramide-2 for Salmonella typhimurium strain TA98, and sodium azide (NaN₃) and 2AA for S. typhimurium strain TA100 using Ames test. V. album var. coloratum was more effective in preventing the mutagenicity of the indirect-acting mutagen 2-AA, when tested with both the strains.¹⁴⁵

5.11. Miscellaneous properties

European mistletoe is known for its anti-cancer and immune enhancing activities but few data exist on anti-convulsant activity. Treatment with V. album managed refractory childhood absence epilepsy in a 4.5 year old girl.¹⁴⁶ V. album lipophilic extract (10 μg mL⁻¹) and its oleanolic acid (1 μg mL⁻¹) have shown excellent wound healing activity. It was associated with the stimulation of migration of NIH/3T3 fibroblasts.¹⁴⁷ Administration of V. album var. coloratum (50 μg mL⁻¹) increased the mean survival time by 9.61 and 19.86% in Caenorhabditis elegans and Drosophila melanogaster, respectively.¹⁴⁸ Treatment with V. album var. coloratum extract (3 g per kg per day) had an anti-obesity effect and protected against hepatic steatosis in mice with high-fat diet-induced obesity. The effects appear to be mediated through an increased mitochondrial activity.¹⁴⁹ KME was reported to induce mitochondrial activity possibly by activating PGC-1α and SIRT1, and improved the endurance of mice. Authors have suggested that KME could be used as a novel mitochondria-activating agent.¹⁵⁰ A pre-clinical study suggested that aqueous extract of V. album leaves exhibits sedative, anti-epileptic and anti-psychotic activities in mice and rats.²

To find out the promising pancreatic lipase (triacylglycerol acylhydrolase) suppressors from natural products, 61 medicinal plants from Korea were tested for their anti-lipase activity for the prevention of obesity. The V. album extracts showed anti-lipase activity with IC₅₀ values of 33.3 μg mL⁻¹ and 35.15 μg mL⁻¹ for anti-phosphodiesterase.¹⁵¹ Aqueous extract of V. album decreased the serum cholesterol, and HDL-cholesterol, triglyceride concentrations in the mice fed with high-cholesterol diet without inducing any gastric damage, suggesting potent hypocholesterolaemic activity.¹⁵² The aqueous extract of V. album leaves exhibited a significant coronary vasodilator activity in the Langendorff's isolated perfused heart model. Authors have also suggested that extract contains some bioactivity constituents that may act as inducers of the NO/soluble guanylate cyclase pathway.¹⁵³ Formation of lactose-resistant aggregates of human platelets and differential signaling responses to cell contact formation by ML were also been reported.¹⁵⁴

6. Clinical trials

Although mistletoe preparations are currently being used in different clinical settings, the most important clinical use has been in the field of cancer as a complementary therapy to reduce the adverse reactions of conventional chemotherapies. In fact, mistletoe extract therapy is among the most thoroughly studied complementary treatments in Europe. Several systematic reviews and meta-analysis have found a benefit from mistletoe treatment in cancer patients and in minimizing the side effects of anti-cancer chemotherapy.^155–159 However, these reviews have also found that nearly all studies suffered from methodological shortcomings to some degree and many of the studies were not conclusive. Earlier review had found that even statistical pooling is not possible because of the heterogeneity of the primary studies,¹⁶⁰ therefore only a narrative systematic review was conducted.

Furthermore, a Cochrane review was done with the objective to determine the effectiveness, tolerability and safety of mistletoe extracts either as a monotherapy or administered as an adjunct to conventional cancer treatment.¹⁶¹ Cochrane reviewers have found that from 80 mistletoe studies examined for the purpose of assessing mistletoe therapy in oncology, 58 had no prospective trial design with randomized treatment allocation and were excluded from the analysis. Although 6 trials among 13 that investigated survival upon mistletoe therapy showed certain evidence of therapeutic benefit, none of them met with high methodological quality. Among 16 trials that explored the efficacy of mistletoe extracts for either improved quality of life (QOL), psychological parameters, performance index, symptom scales or the reduction of adverse effects of chemotherapy, only 2 of them were of a superior methodological quality.¹⁶¹ Thus, the overall conclusion was that independent clinical research of superior quality is required to accurately assess the safety and therapeutic effects of mistletoe extracts.

A study published by Gerhard et al. in 2004 illustrated the difficulties in enrolment and randomization of cancer patients for the therapy with mistletoe.¹⁶² Among 1922 patients who were operated for breast tumor, 154 patients who met the inclusion criteria agreed to participate in the study. However, 80 patients were subsequently excluded from the study following evaluation of the final results on tumor staging and conventional treatment plan. This study suggested that only 29 (39%) of the remaining 74 patients would have agreed to participate in a randomized trial on mistletoe therapy for breast cancer. However, several randomized clinical trials assessing safety and effectiveness of mistletoe preparations have been published in recent years providing clear evidence for improved survival and QOL of cancer patients treated with mistletoe preparations.

6.1. Pancreatic cancer

Two hundred and twenty patients with inoperable or metastatic pancreatic cancer were included in a prospective randomized clinical trial. Hundred and ten patients received Iscador® and remaining 110 patients received no anti-neoplastic therapy. Patients treated with Iscador® survived better (4.8 months) than the control group (2.7 months) (HR = 0.55; p = 0.0031). Importantly, no therapy-related adverse events reported in the Iscador group. V. album therapy thus exhibited a significant and clinically-relevant prolongation of overall survival.¹⁶³ In the same single-center, group-sequential, randomized phase III trial (ISRCTN70760582),¹⁶⁴ data on QOL and body weight were obtained from 96 patients treated with mistletoe and 72 control patients. Patients treated with mistletoe performed better on all the 6 functional scales and on 7 of the 9 symptom scales European Organisation for Research and Treatment of Cancer QOL questionnaire (EORTC QLQ-C30), including pain (95% confidence interval [CI] −29 to −17), fatigue (95% CI −36.1 to −25.0), appetite loss (95% CI −51 to −36.7) and insomnia (95% CI −45.8 to −28.6). This was reflected by the body weight trend of the patients during the study period. The results indicated that mistletoe treatment significantly improves the QOL in comparison to best supportive care alone.¹⁶⁴ The study suggested that V. album is non-toxic and effective second-line therapy for patients with locally advanced or metastatic pancreatic cancer.

6.2. Breast cancer

A prospective randomized open label pilot study on 95 breast cancer patients showed an improvement of QOL when treated with a combination of chemotherapeutic agents cyclophosphamide, adriamycin and 5-fluoro-uracil (CAF), and Iscador® M special (IMS). The control group received only CAF.¹⁶⁵ A descriptive analysis of all 15 scores of the EORTC-QLQ-C30 displayed better QOL in the IMS group compared to the control group. Significant differences were observed among 12 scores (p < 0.02) and clinically-pertinent and significant difference of minimum 5 points were noticed in nine scores. IMS group showed a trend of lower frequency of CAF-induced neutropenia. This pilot study thus showed the importance of IMS to improve the QOL of the patients treated with CAF. A five-year follow-up study suggested that adding V. album during chemotherapy of early stage breast cancer patients does not influence the frequency of relapse or metastasis within 5 years.¹⁶⁶

6.3. Bone cancer

A recent randomized study investigated post second metastatic relapse (12 month) disease-free survival rate in osteosarcoma patients following treatment with Viscum sc or oral Etoposide (a topoisomerase inhibitor anticancer drug). Twenty patients with a median age of 34 years (ranging 11–65 years) were enrolled and were treated randomly with Viscum sc or oral Etoposide. Patients were monitored for a median follow-up time of 38.5 months (3–73). The median postrelapse disease-free survival in the oral Etoposide was 4 months (1–47) and it was 39 months (2–73) in the Viscum group. Also, because of lower toxicity, Viscum-treated patients reported a higher QOL.¹⁶⁷ However, authors have also suggested that a larger study is obligatory for the firm determination of the efficacy and immunomodulatory mechanisms of Viscum therapy in osteosarcoma.

6.4. Lung cancer

A randomized phase II study was conducted in chemotherapy-naïve advanced non-small-cell lung cancer (NSCLC) patients to evaluate the influence of Iscador therapy on carboplatin-containing treatments-related side-effects and QOL. Seventy-two patients were registered for this study with 39 patients for control and 33 for Iscador. Majority of the patients (65%) were in stage IV and had squamous histology (62%). Iscador therapy did not modify the overall survival of the patients and median overall survival was 11 months in both the groups. Although not significant, Iscador group showed a tendency of higher time to tumour progression (TTP). Median TTP was 4.8 months for the controls and 6 months in the Iscador. Grade 3–4 hematological toxicities were similar between both the groups. However, patients in the control group had significantly higher chemotherapy dose reductions (44% vs. 13%, p = 0.005), grade 3–4 non-hematological toxicities (41% vs. 16%, p = 0.043) and hospitalizations (54% vs. 24%, p = 0.016), suggesting that Iscador reduces the chemotherapy-related toxicity. Additional clinical trials are required to confirm and validate these results.¹⁶⁸

A multi-center randomized open prospective clinical trial was conducted to assess the impact of standardized mistletoe extract (sME) therapy on QOL in various types of cancers.¹⁶⁹ The study enrolled 233 patients with NSCL (n = 94), breast (n = 68) and ovarian (n = 71). The 224 patients who fulfilled all the criteria were grouped into two. One hundred and fifteen patients were treated with sME HELIXOR A and 109 control group patients were treated with the approved immunomodulating phytopharmacon Lentinan. All the patients with treated with sME or Lentinan complimentary therapy during chemotherapy regimen. QOL was determined by the Functional Living Index-Cance, Traditional Chinese Medicine Index and the Karnofsky Performance Index. Authors found that patients complementarily treated with sME had significantly improved QOL (p < 0.05) as compared to control group. Adverse effects were also less frequent and self-limiting in sME-treated patients.¹⁶⁹ This trial suggested that complementary sME therapy can improve the QOL in cancer patients by reducing the side-effects of chemotherapy.

6.5. Advanced solid tumors

The phase I study of gemcitabine (GEM) and V. album in patients with advanced solid cancers (ASC) was conducted for the evaluation of safety, toxicity, and maximum tolerated dose (MTD); absolute neutrophil count (ANC) recovery; formation of mistletoe lectin antibodies (ML Abs); plasma cytokine concentrations; clinical response and pharmacokinetics of GEM.¹⁷⁰ Forty four patients with advanced pancreatic, NSCLC, recurrent metastatic colorectal or breast cancer was included. In the first stage, increasing dose of V. album and fixed dose GEM was used. In the second stage, increasing dose of GEM and fixed dose of V. album was used. This study found that all the patients showed immune response to mistletoe injections as determined by ML3 IgG Abs. Compliance with mistletoe therapy was high and the median survival was 200 days with partial response in 6% patients and stable disease in 42%. Dose-limiting toxicities attributed to V. album were G4 neutropenia, G4 thrombocytopenia, G4 acute renal failure, and G3 cellulitis. MTD was GEM 1300 mg m⁻² and mistletoe 250 mg combined and V. album did not affect pharmacokinetics of GEM. This study indicated that combined GEM and V. album is well tolerated by patients with advanced sold tumors. Clinical response in the group received combination therapy was similar to GEM alone treated patients.

6.6. Cancer-related fatigue

Although not examined in a randomized clinical trial, the use of mistletoe preparations has shown an improvement of cancer-related fatigue (CRF).¹⁷¹ The fatigue levels among 324 patients with non-metastasized colorectal cancer (Union for International Cancer Control stage I–III) during the first-line chemo- or radio-chemotherapy protocols were assessed. Iscador(®) Qu was given to 181 patients compared to control group of 143 patients without this supportive care treatment. At the end of the median treatment period, CRF was diagnosed in 16 patients (8.8%) treated with Iscador(®) Qu and was 60.1% in chemo- or radio-chemotherapy group without Iscador(®) Qu. Multivariable-adjusted OR = 10.651 (95% CI 5.09–22.28; p < 0.001) at the first visit was dropped to OR = 0.054 (95 CI 0.02–0.13; p < 0.001) at the end of therapy.

7. Toxicological studies

For the development of remedies in general, knowledge on the toxicology is crucial to confirm drug safety. Cancer patients who received SC injections of mistletoe extracts were examined for adverse drug reactions (ADRs). Out of 1923 patients, 14.7% patients reported local reactions less than 5 cm and raised body temperature less than 38 °C. Among 162 patients who reported ADRs, these reactions were mild (50.8%) to moderate (45.1%) in majority of the patients. Only 4.2% patients reported severe ADR. There were no recognizable risk factors for ADRs. The ADR rate augmented as the dose of mistletoe increased. However, patients receiving concurrent conventional therapies reported less ADRs during mistletoe therapy. The study indicated that mistletoe therapy is safe.¹⁷² In another report, SC injections of ML-1 (1 mg kg⁻¹, weekly twice) for a month period resulted in significant enhancement of several acute phase reactants including C-reactive protein, haptoglobin and complement component C3.¹⁷³ Viscotoxins were also shown to stimulate the generation of reactive oxygen species in human lymphocytes, as well as cell death.¹⁷⁴ Altogether, mistletoe preparations at high doses were reported to cause hypotension, pupil contraction, vomiting, intestinal cramps, diarrhea and seizures. In addition to pain and irritation at the site of injection, SC route of administration of mistletoe can also trigger mild to severe headaches, chills, angina, fever and allergic reactions.

Experimental studies have shown that effects of mistletoe viscotoxins and phoratoxin on the circulation were responsible for the reflex bradycardia, negative inotropic effects and vasoconstriction observed in the cardiac muscles of cats.²¹ In addition, viscotoxins reduced isometric twitch and caused contracture and progressive depolarization in rabbit heart preparations.^21,175 It was also proposed that phenylpropanoids might mediate cardiovascular effects by suppressing cAMP phosphodiesterase.¹⁷⁶ Therefore, use of mistletoe in patients with cardiovascular diseases requires caution. A systemic review on the safety of mistletoe in animals and humans revealed that this therapy is not associated with immunosuppression. The side effects were mostly dose-dependent flu-like symptoms, local reactions at the site of injection of mistletoe and miscellaneous mild effects. Some reports of allergic reactions and reversible hepatotoxicity with high doses of recombinant ML were also recorded.¹⁷⁷

Toxicity of oral mistletoe exposure is controversially discussed. In 1952, Winterfeld stated that oral application of powdered V. album extracts or drops were well tolerated and did never induced toxic reactions.¹⁷⁸ Further, it was also observed that consumption of berries up to three or one to two leaves of American mistletoe Phoradendron serotinum (Loranthaceae) seems unlikely to cause severe toxicity.¹⁷⁹ However, consumption of a herbal product containing mistletoe as one of the ingredients caused hepatitis in a women.¹⁸⁰ However, the role of mistletoe was not proved in this case. Weeks and Proper also reported a case of chronic active hepatitis after ingestion of a herbal remedy containing mistletoe, skullcap, valerian and other plants. Again, this study could not prove mistletoe as an underlying cause.¹⁸¹

Toxicity of Iscador and a purified protein fraction of V. album by parenteral route were examined in vivo in mice.¹⁸² Authors could observe long-term toxicity only with the purified but not well-characterized proteins. Mortality accompanying with liver atrophy and other organs involved in metabolism, and thymus disintegration was recorded 3–4 days post V. album injection. However, 5–10% of the LD₅₀ concentrations of these proteins caused enlarged spleen and thymus. However, precise concentration of the ingredients in the injected preparation was not known in this study. Subsequent study by Rentea and colleagues tried to determine the LD₅₀ of a Iscador by using precise concentration of the product.¹⁸³ They found LD₅₀ dose following IP injection varies among different strains and species of the animals. Thus, LD₅₀ was 700 mg kg⁻¹ for CD-1 outbred albino mice; 348 mg kg⁻¹ for C57/BL6 mice and 378 mg kg⁻¹ for Sprague-Dwaley rats. These animals at lethal doses showed hemorrhagic peritonitis and died with tonic and clonic seizures. On the other hand, LD₅₀ of VA-E (Iscador Mali) in mice was lower (168 mg kg⁻¹).¹⁸⁴ However, LD₅₀ of VA-E (Iscador Quercus) in mice was at higher range: 500 mg kg⁻¹ by intravenous route and 1200 mg kg⁻¹ by SC route.¹⁸⁵ Studies in the animals did not give any indications on the adverse effects of mistletoe on the reproduction and genotoxic effects.¹⁷⁷

Administration of high dose VA-E (Lektinol) at 100 mg kg⁻¹ caused mortality of all the rats within 5 min.¹⁸⁶ These animals experienced dyspnea, ataxia, sedation, exophthalmos and spasms. However at 25 mg kg⁻¹, animals showed dyspnea and sedation with no death. The sub-chronic toxic doses of 0.2, 1.5 and 5 mg kg⁻¹ for 4 weeks did not reveal any organ toxicity.¹⁸⁶

Toxic effects of purified components of mistletoe were also been explored. In mice, the LD₅₀ of ML-1 was found to be 80 μg kg⁻¹.¹⁸⁷ Another report suggested that LD₅₀ of ML-1 and ML-3 were 28 and 49 mg kg⁻¹, respectively.¹⁸⁵ But the lectin activity and route of application were not clear in these reports. Subsequent study however reported lower LD₅₀ values when different lectins were injected by IP route: ML-1: 28 μg kg⁻¹, ML-2: 1.5 μg kg⁻¹ and ML-3: 55 μg kg⁻¹.²⁴ ML-1 in rat was lethal within 24 hours at 100 μg kg⁻¹ by IP route. However, 10 μg kg⁻¹ caused mortality in 3–4 days.¹⁸⁸ These animals experienced pancreatic hemorrhages, ascites and congested intestine, and these symptoms were similar to those observed with ricin. Thus, toxicological values for ML preparations showed large variations probably due to differences in the methods that calculate lectin activity. High production of TNFα and hemagglutinating activity of the lectins were proposed as underlying mechanisms of ML toxicity.¹⁸⁹

8. Conclusions and perspectives

V. album, a plant that has been described from mythological times as a potent remedy for several pathologies continues to evoke interest and scientific curiosity among researchers. Even after a century after its introduction as a treatment for cancer, the clinical use as a component of supportive care, and knowledge on the mechanisms of action of mistletoe continue to expand. Over hundred clinical studies have provided evidence in support of the beneficial effects of mistletoe in cancer patients and mistletoe thus remains as one of the complementary remedies most often used. The results from several randomized clinical trials suggested that mistletoe preparations are safe and improved overall survival and QOL of cancer patients.

Our essay is focused on the active components of mistletoe extracts and pluripotent biological activities. A wide spectrum of pharmacologically active metabolites that belong to a variety of chemical entities of proteins, polysaccharides, liposoluble compounds and secondary metabolites have been identified in V. album extracts. It is conceivable that the heterogenous profile of biochemical compounds provides the basis to the broad diversity of pharmacological activities of mistletoe as each single component contributes diverse modes of actions in addition to imparting to a synergistic beneficial action in conjunction with other molecules. Although a large number of the anti-tumoral properties of mistletoe preparations have been attributed to the lectins, it is possible that enlarging the scope of research to other components especially polyphenols would open new perspectives.

Although a number of elegant pre-clinical studies and numerous powerful clinical trials have provided ample lines of evidence in favor of potent anti-cancer activity of mistletoe if used as concomitant therapeutics in parallel to standard therapy, the field is plagued by a certain degree of skepticism. This may be due to homeopathic origin of the therapy, or inconclusive beneficial effects in Cochrane review or highly scattered technically-sound scientific reports of exploring the molecular and cellular mechanisms underlying the beneficial effects of mistletoe in cancer patients. Thus, further studies examining the results of V. album extracts in the adjunct therapy of cancer should aim at evidence-based clinical data, superior quality, transparent study-design and clear end-points to deliver higher perceptiveness into a supportive therapy that is frequently disapproved as ineffective. Such careful analysis of the effects of V. album should help in clarifying certain skepticism clouding over its use, and provide more effective pointers to the clinicians in adopting appropriate treatment regimes.

Abbreviations

2AA	2-Aminoanthracene
ADRs	Adverse drug reactions
Aps	Arabinogalactan proteins
Bcl-2	B-Cell lymphoma 2
Cb	Chitin-binding
COX-2	Cyclooxygenase
DPPH	2,2-Diphenyl-1-picrylhydrazyl
FME	Fermented mistletoe extract
HPIV-2	Human parainfluenza virus type 2
HUVEC	Human umbilical vein endothelial cells
IFN-γ	Interferon gamma
IL	Interleukin
JNK	c-jun N-terminal kinase
KME	Korean mistletoe extract
KML	Korean mistletoe lectin
KML-C	Lectins from Korean V. album spp. coloratum
KVA	Korean V. album
LPS	Lipopolysaccharide
ML	Mistletoe lectin
NK	Natural killer
NO	Nitric oxide
QOL	Quality of life
rVAA	Recombinant V. album agglutinin, TNF-α, tumor necrosis factor-alpha
VAA	V. album ssp. album
VAA-I	V. album agglutinin-I
VAC	V. album var. coloratum
VAE	Viscum album extract
VCA	V. album var. coloratum agglutinin
VF-2	Viscum fraxini-2

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