The Genus Galanthus: A Source of Bioactive Compounds

The Amaryllidaceae family is one of the 20 most important alkaloid-containing plant families (Zhong, 2005). It comprises about 1100 perennial bulbous species classified in 85 genera, distributed throughout the tropics and warm temperate regions of the world (Willis, 1988). The specific alkaloids produced by the amaryllidaceous plants have attracted considerable attention due to their interesting pharmacological activities. One of them, galanthamine, is a long acting, selective, reversible and competitive inhibitor of the acetylcholinesterase enzyme (Thomsen et al., 1998), which is marketed as a hydrobromide salt under the name of Razadyne® (formerly Reminyl®) and Nivalin® for the treatment of Alzheimer’s disease, poliomyelitis and other neurological diseases (Heinrich and Teoh, 2004). After its discovery in Galanthus woronowii by Proskurina and co-authors in 1955 (Proskurina et al., 1955), the pharmacological properties of galanthamine soon attracted the attention of the pharmaceutical industry. It was first produced by Sopharma (Bulgaria) under the name of Nivalin® from G. nivalis in the early 1960s, but due to the small plant size and variability of galanthamine content, this species was soon replaced by other plant sources (Berkov et al., 2009b).


Introduction
The Amaryllidaceae family is one of the 20 most important alkaloid-containing plant families (Zhong, 2005). It comprises about 1100 perennial bulbous species classified in 85 genera, distributed throughout the tropics and warm temperate regions of the world (Willis, 1988). The specific alkaloids produced by the amaryllidaceous plants have attracted considerable attention due to their interesting pharmacological activities. One of them, galanthamine, is a long acting, selective, reversible and competitive inhibitor of the acetylcholinesterase enzyme (Thomsen et al., 1998), which is marketed as a hydrobromide salt under the name of Razadyne® (formerly Reminyl®) and Nivalin® for the treatment of Alzheimer's disease, poliomyelitis and other neurological diseases (Heinrich and Teoh, 2004). After its discovery in Galanthus woronowii by Proskurina andco-authors in 1955 (Proskurina et al., 1955), the pharmacological properties of galanthamine soon attracted the attention of the pharmaceutical industry. It was first produced by Sopharma (Bulgaria) under the name of Nivalin® from G. nivalis in the early 1960s, but due to the small plant size and variability of galanthamine content, this species was soon replaced by other plant sources (Berkov et al., 2009b).
The genus Galanthus (Snowdrop; Greek gála "milk", ánthos "flower") comprises about 19 species (World Checklist of Selected Plant Families), and to our knowledge 11 have been investigated for their alkaloid content. Although the genus has only been partially studied, phytochemical work has revealed an exceptional diversity of alkaloid structures, many of them reported for the first time and with still unknown bioactivity. The present article provides a brief overview of the phytochemical studies within the genus Galanthus.
The habitats of Galanthus species are varied, ranging from undisturbed broad-leaved or coniferous woodlands of, for example oak (Quercus spp.), beech (Fagus orientalis), maple (Acer spp.), pines (Pinus spp.), Cilician fir (Abies cilicia), and cedar of Lebanon (Cedrus libani), woodland edges, river banks, scrub, grassland, amongst large rocks, and pockets of soil on rocks and cliff faces. G. peshmenii can sometimes be found only 10 m from the sea-shore on Kastellorhizo, a typical hot and dry Aegean island. In contrast, G. platyphyllus is a plant of the subalpine to alpine zone, and occurs mainly at altitudes of 2,000 -2,700 m in alpine grasslands and meadows above the tree-line and at the edges of high-altitude woodlands (Davis, 1999). Typically, the Galanthus species are winter-to-spring flowering plants, but some species, like G. cilicicus, G. peshmenii and G. reginae-olgae, flower in autumn.
G. nivalis and G. elwesii are two of the best known and most frequently cultivated bulbous plants. Their popularity is due to their beauty, longevity and because they flower when little else is in season. A vast number of cultivars and clones are available (Davis, 1999). Huge numbers of wild-collected bulbs are exported annually from Turkey. In the early 1980s onwards this trade increased, with many millions of G. elwesii bulbs being exported via the Netherlands. The large numbers of Galanthus bulbs coming into commerce caused great concern because it was uncertain whether the collection of bulbs in such high numbers was sustainable. For this reason, Galanthus was placed on Appendix II of CITES in 1990. The wild harvesting of G. elwesii bulbs is now carefully controlled and monitored, and export quotas are set each year. Some snowdrop species are threatened in their wild habitats, and in most countries it is now illegal to collect bulbs from the wild. Under CITES regulations, international trade in any quantity of Galanthus, whether bulbs or plants, live or dead, is illegal without a CITES permit. This applies to hybrids and named cultivars as well as species. CITES does, however, allow a limited trade in wild-collected bulbs of just three species (G. nivalis, G, elwesii and G. woronowii) from Turkey.

Biosynthesis and structural types of Amaryllidaceae alkaloids
A particular characteristic of the Amaryllidaceae plant family is a consistent presence of an exclusive group of isoquinoline alkaloids, which have been isolated from plants of all the genera of this family. As a result of extensive phytochemical studies, over 500 alkaloids have been isolated from the amaryllidaceous plants (Zhong, 2005). The Amaryllidaceae type alkaloids have been structurally classified into nine main subgroups, namely lycorine, crinine, haemanthamine, narciclasine, galanthamine, tazettine, homolycorine, montanine www.intechopen.com Phytochemicals -A Global Perspective of Their Role in Nutrition and Health 238 and norbelladine (Bastida et al., 2006). In the genus Galanthus, however, two new structural subgroups, graciline and plicamine type alkaloids, have been found (Ünver, 2007). The following new subgroups have also been reported: specific augustamine-type structures in Crinum kirkii (Machocho et al., 2004), a carboline alkaloid in Hippeastrum vittatum (Youssef, 2001), mesembrane (Sceletium)-type compounds in Narcissus pallidulus and N. triandrus (Bastida et al., 2006), and phtalideisoquinoline-, benzyltetrahydroisoquinoline-and aporphine-type alkaloids in G. trojanus (Kaya et al., 2004b(Kaya et al., , 2011. Mesembrane-type compounds are typical of the genus Sceletium of the Aizoaceae, while phtalideisoquinoline-, benzyltetrahydroisoquinoline-and aporphine-type alkaloids are found in the Papaveraceae, both families being dicotyledonous. Tyramine-type protoalkaloids, which are biosynthesized in Poaceae, Cactaceae, some algae and fungi, have also been found in Leucojum and Galanthus species (Berkov et al., 2009a(Berkov et al., , 2011. Amaryllidaceae alkaloids are formed biogenetically by intramolecular oxidative coupling of norbelladines derived from the amino acids L-phenylalanine and L-tyrosine (Bastida et al., 2006). The key intermediate metabolite is O-methylnorbelladine. Ortho-para´ phenol oxidative coupling of O-methylnorbelladine results in the formation of a lycorine-type skeleton, from which homolycorine-type compounds proceed. The galanthamine-type skeleton originates from para-ortho´ phenol oxidative coupling. Para-para´ phenol oxidative coupling leads to the formation of crinine, haemanthamine, tazettine, narciclasine and montanine structures (Bastida et al., 2006). In the present article, for the structures reported by different authors we have adopted the numbering system according to Bastida et al., (2006, Fig. 1). The biogenetic pathway of gracilines possibly originates from the 6-hydroxy derivatives of haemanthamine-type species (Noyan et al., 1998), while plicamine-type alkaloids most probably proceed from tazettine-type compounds, considering their structural similarities (Ünver et al., 1999a).

Distribution of alkaloids in the genus Galanthus
The phytochemical studies of the genus Galanthus started in the early fifties of the last century. Two of the first alkaloids reported for the genus were galanthine (Proskurina and Ordzhonikidze, 1953) and galanthamine (Proskurina et al., 1955), which were isolated from G. voronowii. To the best of our knowledge, eleven species from the genus Galanthus have been phytochemically studied to date and ninety alkaloids have been found and classified in 11 structural types (Table 1, Fig.2).
Until recently, the distribution of alkaloids within the genus has been studied by classical phytochemical approaches. The collected biomass is extracted with alcohol, the neutral compounds removed at low pH and the alkaloids fractionated after basification of the extract. Individual alkaloids have been separated by column chromatography, preparative TLC, prep. HPLC, etc., and identified by spectroscopy, mainly 1D and 2D NMR. The GC-MS technique has proved to be very effective for rapid separation and identification of complex mixtures of Amaryllidaceae alkaloids obtained from low mass samples (Kreh et al., 1995). Thus, the assessment of alkaloid distribution at species, populational and individual levels and the detection of new compounds have become much easier and faster (Berkov et al., 2007a(Berkov et al., , 2009c(Berkov et al., , 2011).
An overview of the literature indicates that the genus Galanthus is a very rich source of novel compounds. Thirty-seven alkaloids (namely 12, 22, 26, 29, 34-39, 46-49, 53, 56-58, 62, 67, 69-75, 77-86) or ca. 40% of all identified compounds from the genus have been isolated for the first time from Galanthus. What is more, the biochemical evolution of the genus has led to the occurrence of two specific subgroups, namely graciline-and plicamine-type alkaloids.
The most studied species are G. nivalis and G. elwesii. Due to taxonomical changes over the years, the information on the alkaloids of G. nivalis is confusing. Thus, until 1966, only one Galanthus species had been recognized in Bulgaria, namely G. nivalis L. (Jordanov, 1964). This taxon was subsequently separated into G. nivalis L. and G. elwesii Hook. (Kozuharov, 1992). At present, it is unclear which plant species the alkaloids isolated in the early sixties from Bulgarian G. nivalis can be attributed to (Valkova, 1961;Bubeva-Ivanova and Pavlova, 1965). Kaya et al. (2004b) have reported five alkaloids for G. nivalis L. subsp. silicicus (Baker) Guttl.-Tann., a taxon regarded as a synonym of G. silicicus Baker by other authors (Davis and Barnett, 1997;Davis, 1999). A recent revelation has substantiated that G. nivalis subsp. cilicicus is identical to the newly introduced species, G. trojanus A. P. Davis and N. Özhatay, a plant species endemic to Northwestern Turkey (Davis and Özhatay, 2001). Latvala et al., (1995) isolated 18 alkaloids (6 new) from G. elwesii in addition to the already reported flexinine, elwesine, tazettine and haemanthamine (Boit and Ehmke, 1955;Boit and Döpke, 1961). The occurrence of elwesine (26) in the genus is particularly interesting. This compound displays a -configuration of its 5,10b-ethano bridge, which is typical of the South African representatives of the family (Viladomat et al., 1997). Although widely accepted that G. nivalis was the industrial source of galanthamine (in Bulgaria) during the 1960s (Heinrich and Teoh, 2004), later studies on 32 Bulgarian populations of G. nivalis and G. elwesii indicate that the distribution of this important compound is limited to a few populations of G. elwesii, while just one population of G. nivalis has been found to contain galanthamine and only as a minor alkaloid (Sidjimova et al., 2003;Berkov et al., 2011). These studies, however, have also shown a great intra-species diversity of alkaloid synthesis in G. nivalis and G. elwesii. The populations displayed between 6 and 31 alkaloids in their alkaloid patterns and about 70 compounds have been detected in total. Many of them were left unidentified due to the lack of reference spectra, possibly indicating new structures. This biochemical diversity has led to the isolation of eight more new alkaloids from these wellstudied species, after the collection of plant material from populations proven by GC-MS to be a rich source of unknown compounds (Berkov et al., 2007a(Berkov et al., , 2009c. Interestingly, many of the G. elwesii populations have accumulated the tyramine-type protoalkaloids as major compounds (up to 99 % of all alkaloids). In addition to the tyramine chemotype, homolycorine, lycorine haemanthamine and galanthamine chemotypes have also been found in the studied populations of G. elwesii. A galanthamine chemotype population was also found for G. nivalis, but in contrast with G. elwesii, this G. nivalis population accumulated the 4,4a-dihydrogenated derivatives of galanthamine (12), lycoramine (16) and its isomer (17) (Berkov et al., 2011). As well as a high level of alkaloid diversity and the existence of different chemotypes among the species populations, G. elwesii and G. nivalis have also shown some important differences in their alkaloid patterns, at least in the studied Bulgarian populations. A study of sympatric populations, and 32 populations from both species showed that the alkaloid pattern of G. nivalis is dominated by compounds coming from a para-para´ oxidative coupling of O-methylnorbelladine (haemanthamine-and tazettine-type alkaloids, Fig. 1). The conjugated and free lycorine-type alkaloids proceeding from an ortho-para´ oxidative coupling were relatively less abundant. Homolycorine-type alkaloids were not detected in this plant species. In contrast to G. nivalis, the alkaloid pattern of G. elwesii was dominated mainly by compounds coming from ortho-para´ oxidative coupling: free lycorine-and homolycorine-type alkaloids. The synthesis of para-para´ oxidative products in G. elwesii is relatively weak (only haemanthamine-and no tazettine-type compounds, Berkov et al., 2008Berkov et al., , 2011. In total, 46 and 38 alkaloids have been identified in G. elwesii and G. nivalis, respectively. In a study on sympatric G. nivalis and G. elwesii populations, it was found that the organs of the plants presented different alkaloid patterns (Berkov et al., 2008). Thus, the predominant alkaloids of G. nivalis roots were found to belong to the lycorine and tazettine structural types, bulbs were dominated by tazettine, leaves by lycorine and flowers by haemanthamine-type alkaloids. The predominant alkaloids in G. elwesii roots, bulbs and leaves were those of the homolycorine type, whereas the flowers accumulated mainly tyramine-type compounds. To the best of our knowledge, no studies of the dynamics of the alkaloid patterns during ontogenesis have been reported for either of these two species or any other Galanthus species. Such studies, however, may contribute to the understanding of the chemoecological role of the alkaloids in the genus Galanthus and the Amaryllidaceae as a whole. A remarkably high number of alkaloids conjugated with 3-hydroxybutyryl moieties occur in G. nivalis. Co-existence of free and conjugated alkaloids in the plant implies that the latter may have a chemoecological role. Such conjugated alkaloids have rarely been reported for Amaryllidaceae plants.

Biological and pharmacological activities of the alkaloid found in Galanthus
Alkaloids are important for the well-being of the producing organism. One of their main functions is to provide a chemical defence against herbivores, predators or microorganisms (Wink, 2008). The biological roles of the numerous alkaloids found in the genus Galanthus remain largely unknown and only a few have been studied for their pharmacological activities.

Galanthamine-type
The most studied Galanthus alkaloid, galanthamine (12), is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase (AChE) and an allosteric modulator of the neuronal nicotinic receptor for acetylcholine. AChE is responsible for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central cholinergic synapses. Galanthamine has the ability to cross the blood-brain barrier and to act within the central nervous system (Bastida et al., 2006;Heinrich and Teoh, 2006). Owing to its AChE inhibitory activity, galanthamine is used and marketed under the name of Razadine ® , formerly Reminyl ® , in the USA, for the treatment of certain stages of Alzheimer's Disease (AD). According to data presented by the Alzheimer's Association in 2007, the prevalence of Alzheimer's disease will quadruple by 2050. Galanthamine hydrobromide has superior pharmacological profiles and higher tolerance as compared to the original AChE inhibitors, physostigmine or tacrine (Grutzendler and Morris, 2001).
Epigalanthamine (13), with a hydroxylgroup at -position, and narwedine (14), with a keto group at C3, are also active AChE inhibitors, but about 130-times less than galanthamine (Thomsen et al., 1998). The loss of the methyl group at the N atom, as in Ndemethylgalanthamine (15), decreases the activity 10-fold. On the other hand, sanguinine (18), which has a hydroxylgroup at C9 instead of a methoxyl group, is ca. 10 times more active than galanthamine. Hydrogenation of the C4-C4a, as in lycoramine (16), results in a complete loss of AChE inhibitory activity (López et al., 2002). It is suggested that in plants AChE inhibitors act as pesticides. The synthetic pesticides such as phosphoorganic compounds are non-reversible AChE inhibitors (Hougton et al., 2006).

Tyramine-type
Compounds 1-4 can be attributed to the group of the phenolic amines that impact the hypothalamic-pituitary-adrenal axis (Vera-Avila et al., 1996) due to their structural similarity to adrenaline (epinefrine). The consequent release of adrenocorticotropic hormone and cortisol results in sympathomimetic action with toxic effects in animals (Clement et al., 1998). Hordenine (3) possesses diuretic, disinfectant and antihypotensive properties, and acts as a feeding repellent against grasshoppers (Dictionary of Natural Products).

Tazettine-type
Moderate cytotoxic activity has been reported for tazettine (42), and epimacronine (45) (Weniger et al., 1995). Tazettine, however, is an isolation artefact of chemically labile pretazettine, which is indeed present in plants. This compound has shown remarkable cytotoxicity against a number of tumor cell lines, being therapeutically effective against advanced Rauscher leucemia, Ehrlich ascites carcinoma, spontaneous AKR lymphocytic leukaemia, and Lewis lung carcinoma (Bastida et al., 2006).

Lycorine-type
Lycorine (54), one of the most frequently occurring alkaloids in Amaryllidaceae plants, possesses a vast array of biological properties. It has been reported as a potent inhibitor of ascorbic acid synthesis, cell growth and division and organogenesis in higher plants, algae, and yeasts, inhibiting the cell cycle during the interphase (Bastida et al., 2006). Additionally, lycorine exhibits antiviral (against poliovirus, vaccine smallpox virus and SARS-associated coronavirus), antifungal (Saccharomyces cerevisiae, Candida albicans), and anti-protozoan (Trypanosoma brucei) activities (McNulty et al., 2009), and is more potent than indomethacin www.intechopen.com The Genus Galanthus: A Source of Bioactive Compounds 249 as an anti-inflammatory agent (Citoglu et al., 1998). Lycorine has also been shown to have insect antifeedant activity (Evidente et al., 1986). As a potential chemotherapeutic drug, this compound has been studied as an antiproliferative agent against a number of cancer cell lines (Likhitwitayawuid et al., 1993). The in vitro mode of action in a HL-60 leukemia cell line model is associated with suppressing tumor cell growth and reducing cell survival via cell cycle arrest and induction of apoptosis (Liu et al., 2004). Further investigation showed that it is able to decrease tumor cell growth and increase survival rates with no observable adverse effects in treated animals (Liu et al., 2007), thus being a good candidate for a therapeutic agent against leukaemia (Liu et al., 2009).

Conclusions
Although only some of the species of this phytochemically interesting genus have been studied, it has yielded a considerable number of new structures. Moreover, the high level of intraspecies diversity indicates that new compounds can be expected from already studied taxons. Only a few of the new alkaloids have been screened for their bio-and pharmacological activities, probably due to the small amounts isolated. Consequently, their synthesis or in silico studies will facilitate further bioactivity assessment. Phytochemicals are biologically active compounds present in plants used for food and medicine. A great deal of interest has been generated recently in the isolation, characterization and biological activity of these phytochemicals. This book is in response to the need for more current and global scope of phytochemicals. It contains chapters written by internationally recognized authors. The topics covered in the book range from their occurrence, chemical and physical characteristics, analytical procedures, biological activity, safety and industrial applications. The book has been planned to meet the needs of the researchers, health professionals, government regulatory agencies and industries. This book will serve as a standard reference book in this important and fast growing area of phytochemicals, human nutrition and health.

How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following: http://www.intechopen.com/books/phytochemicals-a-global-perspective-of-their-role-in-nutrition-andhealth/the-genus-galanthus-a-source-of-bioactive-compounds