COMPARATIVE PHYTOCHEMICAL OF MISTLETOE LEAVES AND STEM AND THEIR TOXIC DOSES IN GUINEA PIGS AND RATS

COMPARATIVE PHYTOCHEMICAL OF MISTLETOE LEAVES AND STEM AND THEIR TOXIC DOSES IN GUINEA PIGS AND RATS

 CHAPTER ONE

1.0       INTRODUCTION

Mistletoe is an evergreen parasitic plant, growing on the branches of trees, Viscum album is also a small woody shrub, frequently globular in shape, where it forms pendent bushes about 2-5 feet in diameter. Mistletoe is a parasitic flowering plant found in the sandalwood order, that attaches itself to the stem of another plant (primarily gymnosperms and angiosperms), mistletoe,  any of many species of semi parasitic green plants of the families Loranthaceaeand Viscaceae, especially those of the genera Viscum, Phoradendron, and Arceuthobium, all members of the Viscaceae (Barlow, 1968).

The legendary mistletoe was known for centuries before the Christian era. It forms a drooping yellowish evergreen bush, 0.6 to 0.9 m (about 2 to 3 feet) long, on the branch of a host tree. It has thickly crowded, forking branches with oval to lance-shaped, leathery leaves about 5 cm (2 inches) long, arranged in pairs, each opposite the other on the branch. The flowers, in compact spikes, are bisexual, unisexual, or regular.(Barlow, 1968). They are yellower than the leaves and appear in the late winter and soon give rise to one-seeded, white berries, which when ripe are filled with a sticky, semi transparent pulp. Most tropical mistletoes are pollinated by birds, most temperate species by flies and wind. Fruit-eating birds distribute the seeds in their droppings or by wiping their beaks, to which the seeds often adhere, against the bark of a tree. After germination a modified root (haustorium) penetrates the bark of the host tree and forms a connection through which water and nutrients pass from host to parasite Mistletoes contain chlorophyll and can make some of their own food(Calvin and Wilson, 2006). Most mistletoe parasitizes a variety of hosts, and some species even parasitize other mistletoes, which, in turn, are parasitic on a host. The Eurasian Viscum album is most abundant on apple trees, poplars, willows, lindens, and hawthorns. Species of Phoradendronin America also parasitize many deciduous trees, including oaks. In some parts of Europe the midsummer gathering of mistletoe is still associated with the burning of bonfires, a remnant of sacrificial ceremonies performed by ancient priests, or druids. Mistletoe was once believed to have magic powers as well as medicinal properties. Later, the custom developed in England (and, still later, the United States) of kissingunder the mistletoe, an action that once was believed to lead inevitably to marriage. (Calvin and Wilson, 2006).

Mistletoe extracts contain pharmacologically active proteins (lectins). The composition of an extract may vary according to season, host tree, parts of the plant used and extraction method. Mistletoe extracts induce macrophage cytotoxicity, stimulate phagocytosis of immune cells, increase cytokine secretion and enhance cytotoxicity effects on various cell lines in vitro. 2-3 The plant also contains a host of other ingredients such as acids, alkaloids, amines, flavonoids, terpenoids and viscotoxins.(phytochemical) ( Franz, 1986).

Extracts of mistletoe (Viscum album) are highly popular in cancer care, particularly in Europe. They are usually injected subcutaneously. Proponents of this therapy claim that it improves quality of life, strengthens the immune system, has a positive impact on tumors remission and survival of cancer patients. However, the evidence to support these claims is weak. Mistletoe is generally well tolerated and safe. This project work aim at testing the toxicity of this plant and its phytochemicals on animal specie

1.1       Taxonomy of mistletoe

Kingdom- Plantae: It has cell walls made of cellulose and is photosynthetic.

 Phylum- Magnoliophyta: It is a Flowering plant.

 Class- Magnoliopsida: It is a Dicotyledon.

 Order- Santalales:  Hemi-parasitic.

 Family- Viscaceae: Christmas mistletoe family.

 Genus- Phoradendron Nutt. : mistletoe.

 Species- Phoradendron Leucarpum: American (Oak) Mistletoe (Barlow and Wien, 1974)

1.2       Morphology of Mistletoe plant

Being a parasitical plant, mistletoegrows as a hanging bush on the branches of trees. With its cylindrical stem, ramified in pairs and slightly thickened at the nodes, mistletoe can reach in time (it grows slowly) up to 60 cm in height. It has oval leafs, rounded at their tip and of a yellowish-green color. These maintain their fresh color all through the winter time. Also, in December, mistletoe fruits mature, having a rounded shape and white color. The flowers appear late in the autumn, and soon following, the fruits appear. The lifespan of this plant is approximately 70 years. Mistletoe can be found between tree branches (even those taller than 4-5 meters): poplar, willow, birch, pear and apple trees, hawthorn, plum, cherry, acacia, maple, chestnut, lime, or more rarely in ash or alder trees. The most “valuable” kind of mistletoe is that which grows in fruit bearing (especially apple) trees. Also, the ones which grow on pear trees, fir, birch, rose and ash trees are considered to be of superior quality (Barlow and Wien, 1974)

Mistletoes are slow-growing but persistent; their natural death is determined by the death of the hosts. They are pests of many ornamental, timber, and crop trees and are the cause of abnormal growths called “witches’ brooms” that deform the branches and decrease the reproductive ability of the host. The only effective control measure is complete removal of the parasite from the host (Barlow, 1968)  

1.3       Mistletoe Biology

1.3.1    Infection

The basic biology of mistletoes is remarkable. Host infection has been described in detail for some groups and not others, but is considered to be similar for all mistletoes. Upon germination, seeds form a hypocotyl that elongates until it forms a holdfast that attaches firmly to the host branch. As for other flowering plants, seed germination is  influenced by temperature, moisture, and light (Aukema and Martinez, 2004).

The seeds of mistletoes in the Viscaceae have a chlorophyllous endosperm and embryo and so are capable of producing simple sugars as an energy source after germination. A penetration peg develops on the lower surface of the holdfast that mechanically penetrates the epidermis or bark, eventually contacting the host’s phloem and/or xylem. Penetration of host tissue is evidently purely by mechanical means, as no chemical breakdown of host tissue has been identified thus far. Once the mistletoe has entered host tissue, it develops its haustorium and then aerial shoots. Many tropical mistletoes begin forming shoots soon after they establish their connection to their host, while the dwarf mistletoes may take 2 to 6 years to form aerial shoots ( Aukema, 2003).

1.3.2    Mistletoe Ecology

Ecological research on mistletoes has changed markedly over the last 50 years, in terms of both breadth and depth, reflecting changing priorities and a gradual shift in overall attitudes toward these parasitic plants. A key stimulus for discovering more about mistletoes was the perceived need to control them in commercial forests, orchards, and plantations worldwide. Initial investigations concentrated on host–parasite interactions, quantifying the effects of mistletoes on host growth, and describing the processes of mistletoe dispersal and establishment (Aukema and Martinez, 2004).

Subsequent research focused on several components of mistletoe–host interactions, host range, germination and establishment, the anatomical and physiological basis of parasitism, and detailed explorations of the role of frugivorous birds as seed vectors.

Seed dispersal studies were restricted primarily to the small number of mistletoe fruit specialists consistent with the view that few other species could detect or process the relatively cryptic and sticky fruits. Whereas many researchers viewed mistletoes as botanical anomalies or models for studying plant–animal interactions, the broader perception of these parasitic plants as destructive forest pathogens persisted. While this targeted research was being conducted, anecdotal and incidental information on mistletoe–animal interactions was accumulating, gathered by biologists working on other components of forests and woodlands throughout the world (Aukema and Martinez, 2004).

This highly dispersed information was synthesized by Watson, revealing an unprecedented breadth of interactions. In addition to documenting the wide range of opportunistic consumers of mistletoe fruit (in contrast to the prevailing view), this review also highlighted how many folivores and nectarivores feed on mistletoes..

These interactions were suggested to underpin a generalized positive effect of mistletoe occurrence on diversity, and mistletoes have been proposed to function as a keystone resource in many forest ecosystems. Building on these previous advances, current ecological research on mistletoes is dominated by three major themes: mistletoes influence on wildlife habitat; mistletoes as a food source; and mistletoe–ecosystem interactions (Aukema and Martinez, 2004).

1.3.3    Pollination

Mistletoes are pollinated by biotic agents (primarily birds and insects) as well as wind. Many tropical and subtropical mistletoes in Loranthaceae have large, colorful flowers borne in groups that produce large amounts of sugar-rich nectar that attract avian pollinators. Elaborate pollination and seed dispersal mechanisms involving birds have evolved in some of these loranth species. For example, birds pry open the fused corollas to reach their nectar reward, upon which the pollen “explodes” onto the bird’s head. (Amico et al 2007).

These mistletoes are often dichogamous (protandrous), and after the birds have visited flowers in the male phase, they eventually visit flowers in the female phase, thereby effecting pollination. The co-evolutionary relationship between mistletoes and their bird pollinators is so closely linked that disruption of this association could have long-term negative consequences for both interacting organisms and possibly the entire ecosystem. However, many bird-pollinated types of mistletoe are serviced by a broad range of species, and no bird can be considered amistletoe pollen specialist. In Mexico, Central America, and South America, hummingbirds are key pollinators of mistletoes with large, showy red or yellow flowers. A variety of insects are the key pollinators of mistletoes in the Viscaceae and Loranthaceae. While mammals are known to visit flowers, they have not yet been positively implicated as mistletoe pollinators. Bats are the most likely mammal pollinators of mistletoes, but studies have not yet confirmed their role in this process. (Amico et al2007).

1.3.4    Dispersal

The co evolution of mistletoes with their avian vectors has resulted in attractive and nutritious fruits that provide valuable food for many bird species throughout the world. The mature fruits of mistletoes are brightly colored (usually white, yellow, red, blue, or purple), and their seeds are coated with a natural “glue,” termed viscin. Birds either swallow mistletoe fruits whole, peel off the outer exocarp and ingest the seed and viscin, or eat only the viscin coating around the seed. Once the bird has eaten the seed, it is either regurgitated or defecated, but the seed is still covered with some of its viscin coat, which allows it to adhere to potential hosts. In many instances, seeds adhering to a bird’s beak, legs, or feathers are rubbed off onto a branch of a potential host. Approximately 90 bird species from 10 families are considered mistletoe fruit specialists, exhibiting a range of behavioral and morphological adaptations to their narrow diet. Most of these groups are represented by four or fewer species, except the flowerpeckers (Dicaeidae) of Asia (44 species) and the euphonias (Carduelinae) of Latin America (33 species) (Amico et al 2007). While most discussion of mistletoe dispersal is typically restricted to these dietary specialists, a wide range of other avian species disperse their seeds, accounting for all dispersal in Europe and most regions of North America. Within the United States, vectors of Phoradendronspp. (Viscaceae) are fairly well known, but only a few studies have examined in any detail the relationships between birds and Phoradendron. For many mistletoes, particularly those in Central and South America, the key vectors have not been investigated to any large extent (Amico et al 2007)

The control of economically damaging mistletoes in managed areas is often confounded by their reintroduction by birds. In South and North America, animals other than birds have also been implicated in the dispersal of mistletoe seeds. In South America, a marsupial disperses seeds, and in North America, squirrels and other mammals have been shown to rarely disperse dwarf mistletoe seeds adhering (Amico et al, 2007)

1.3.5    Haustorium

All mistletoes produce a morphologically diverse structure that allows them to interface with their hosts: the haustorium. Calvin and Wilson described four basic haustorial system types that are found in aerial parasitic mistletoes

1.     Epicortical roots that grow along a host branch surface and at intervals form haustoria

2.     Elasping unions where the mistletoe haustorium enlarges, partly encircling a branch

3.     Wood roses where host tissue enlarges forming a placenta to which the mistletoe’s haustorium attaches

4.     Bark strands that spread within the host bark and connect to host xylem and phloem

 Plants with wood roses, clasping unions, and bark strands are often described as having “solitary unions” with their hosts.

In contrast, plants with epicortical roots have multiple, visible haustorial connections to their hosts. Because of the diversity and possible phylogenetic implications of the morphology of the mistletoe haustorium, investigators have continued study of these diverse and intricate connections between mistletoes and their hosts.

1.4       Mistletoe Biochemistry

Mistletoe contains the following active chemical constituent

 Glycoproteins: mistletoe lectins I (galactoside-specific lectin), II, and III, Proteins: viscotoxin, Polysacchrides: galacturonan, arabinogalactan Alkaloids

·         Glycoproteins:Mistletoe’s lectins are cytotoxic glycoproteins of approximately 10,000 molecular weight; they cause cells to agglutinate14 and inhibit protein synthesis on the ribosomal level.The lectins, also known as viscumin or agglutinin, are dual chain molecules. Chain A inhibits protein synthesis and chain B activates macrophages and releases lymphokines from lymphocytes. Both the A and B chains of mistletoe lectin I also inhibit allergen-inducedhistamine release from leukocytes and collagen-induced serotonin release from platelets (. Franz, 1986).

Lectins are structurally similar to two highly biologically active toxic proteins, ricin and abrin. The amounts and biological activity of V. album lectins are dependent on the host tree,manufacturing process, and time of harvest (Bussing And  Schietzel, 1999).

·         Proteins:Viscotoxin is a 46-amino acid peptide that damages cell membranes. Viscotoxin is found only in V. album. A similar constituent of Phoradendron is phoratoxin, a polypeptide about twice the weight of viscotoxin; it makes up 0.01% to 0.23% of Phoradendron leaves and stems (. Andersson and Johannsson, 1973 ).

·         Polysacchrides: Various polysaccharides are thought to be involved in mistletoe’s antineoplastic effects. The leaves and stems contain esterified galacturonan, while the berries contain primarily arabinogalactan (Franz, 1986).

·         Alkaloids:mistletoes are also thought to contain alkaloids. Alkaloids are nitrogenous compounds that may contribute to mistletoe’s cytotoxicity (Franz, 1986).

1.5       Background of the Study

The amount of pharmacological substances and chemicals being used in the human community today, have increased to almost an innumerable amount. These may be presented today in the form or as constituents of food substances, medicines, and beverages, other industrial and household products. However, these chemicals or pharmacological substances may result in chronic toxicity in the living system when used over a long period of time or acute toxicity may also occur when large quantities capable of eliciting immediate toxic effect are used. These effects may be mild or severe, depending on the nature of the substance. Acute toxicity is defined as the unwanted effect (s) that occurs either immediately or at a short time interval after a single or multiple administration of such substance within 24 hours. The unwanted (or adverse) effect is any effect that produces functional impairments in organs and/or biochemical lesions, which could alter the functioning of the organism in general or individual organs.  Studies of acute toxicity however tends to establish the dose-dependent unwanted (or adverse) effect (s), which may take place and this includes all information that is important in the assessment of acute toxicity including mortality. The assessment of the lethal dose (LD₅₀) (the dose that kills 50% of test animals population) has now been used as a major parameter in measuring acute toxicity and also as an initial procedure for general screening of chemical and pharmacological agents for toxicity. Apart from mortality, other biological effects and the time of onset, duration and degree of recovery on survived animals, are also important in acute toxicity evaluation. Acute toxicity study solely gives information about LD₅₀, therapeutic index and the degree of safety of a pharmacological agent. The toxicity assessment of pharmacological agents is a very important procedure that is usually carried-out before they are allowed to enter the market for sale. Conversely, different methods have been developed and adopted for acute toxicity testing. This project work assesses the different of pharmacological agents in mistletoe plant and also its toxicity on animal species using the Lorke’s method.

1.6       Aim and Objective of the Study

The aim of this research studies are focused on;

i)        The screening of bioactive compounds (phytochemical) from leaves and stems of V. album that are growing on different host trees,

ii)       The second is to evaluated the toxic activity (toxicity)of V. album’ leaves and stems extract on animal (Guinea pig or rat).

1.7       Significance of the Study

Medicinal plants are a rich source of bioactive phytochemicals or bionutrients. Studies carried out during the past 2– 3 decades have shown that these phytochemicals have an important role in preventing chronic diseases like cancer, diabetes and coronary heart disease. The major classes of phytochemicals with disease-preventing functions are dietary fibre, antioxidants, anticancer, detoxifying agents, immunity-potentiating agents and neuropharmacological agents. Each class of these functional agents consists of a wide range of chemicals with differing potency. Some of these phytochemicals have more than one function. This research work helps in screening mistletoe which is one of the discovered medicinal plant for the presence of the different phytochemical in it and its toxic activity on animal cell which will aid in its administration and dosage level as a medicine.

CHAPTER TWO

2.0       LITERATURE REVIEW

            Life History and Phenology

Mistletoes are characterized by their growth habit and, excepting several root parasitic species (Kuijt 1969, Fineran and Hocking 1983), they typically form dense clumps in the crowns of their hosts. These clumps are generally composed of semisucculent mistletoe stems and leaves, but for dwarf mistletoes (Viscaceae, Arceuthobium spp.), the resultant clump (termed a witch’s broom) is actually the host tree’s response to infection composed of thickened and twistedP branches (Hawksworth and Wiens 1996). Although mistletoe plants are frequently regarded as detrimental to tree health, this is not necessarily the case. Mistletoe plants have low annual survivorship (Musselman and Press 1995), with estimates of 19% and 31% for two Australian species and are considered “low-grade K-selected forest

parasites” (Andrews and Rouse 1982). Many species are sensitive to fire and frost (Rowe 1983, Hawksworth and Wiens 1996); these factors are cited as limiting the distribution of mistletoe in some areas (Hawksworth 1969, Hawksworth and Wiens 1996). Mistletoe seeds require high light levels for germination, establishment, and subsequent maturation and they are frequently shaded out as the host canopy develops). Thus, despite high rates of dispersal and successful germination, establishment is rare, and all mistletoe species studied have narrow microsite tolerances (Hawksworth and Wiens 1996). Mistletoes obtain all of their water and minerals from the host through a vascular connection termed a haustorium. This swollen holdfast serves both to attach the mistletoe plant to the host and to divert water and minerals to the parasite. The term hemiparasitic is used because most mistletoes photosynthesize, although they may obtain up to 60% of their carbohydrates from the host (Hawksworth and Wiens 1996). The consequence of this growth-form is that mistletoes are less affected by the edaphic, hydrological, and nutritional factors that limit the distribution, growth, and phenology of most plants; the host plant buffers the parasite against large-scale fluctuations in resource availability (Ehleringer and  Marshall 1990).

Washington Irving described the tradition of stealing a kiss under this intriguing plant:

“the mistletoe, with its white berries,” he said, is “hung up, to the imminent peril of all the pretty housemaids.” At Christmas, the young men had the privilege of kissing ladies under mistletoe, “plucking each time a berry from the bush.” Once all the berries had been plucked, no more kissing was allowed. (Musselman and Press 1995)

First described by the Greek naturalist Theophrastus in the Third Century BC, mistletoe became embedded in European rituals, folklore, and folk medicine. It was a sacred plant of the Celtic peoples who dominated Europe in the first millennium BC. The Gauls and the Celts called it “all- healer” or “cure-all” (Musselman and Press 1995)

Both European and American mistletoe contain toxic proteins which are similar in their chemical composition and produce similar effects, including hypotension, bradycardia, and vasoconstriction, in test animals3. Despite the popular belief that the two types of mistletoe have opposite effects, the stems and leaves of these plants contain similar phytochemicals4. Anthroposophist Rudolf Steiner introduced the use of mistletoe extracts for the treatment of cancer in 19165. Nowadays, Europeans include V. album in oncology therapies under the trade names IscadorÒ and HelixorÒ. The German Commission E has approved mistletoe as a treatment for degenerative and inflamed joints and as a palliative therapy for malignant tumors. (Musselman andss Press 1995)

Mistletoe has been used medicinally for centuries and has been employed to treat cancer, epilepsy, infertility, menopausal symptoms, nervous tension, asthma, hypertension, headache, and dermatitis. Recent interest in mistletoe began in the 1920s after it was first proposed for the treatment of cancer by Rudolf Steiner, the founder of anthroposophy and anthroposophical medicine. Since the 1980s, mistletoe therapy has been researched systematically. A number of German phyto-pharmacological providers like WELEDA, ABNOBA HEILMITTEL, HELIXOR HEILMITTEL, NOVIPHARM and MADAUS market a range of different misteltoe preparations. Some products, e.g. those from WELEDA are anthroposophical, i.e. fermented and diluted preparations, others are herbal extracts. Indications within the anthroposophical approach depend on the host tree of the misteltoe plant.