780 terpenoids Surh, Y.-J. and Lee, S.S. (1996). Capsaicin in Hot Chili Pepper: Carcinogen, Co-carcinogen or Anticarcinogen? Fd. Chem. Toxic, 34, 313-316. Annatto Tree Scotter, M.J., Wilson, L.A., Appleton, G.P. and Castle, L. (1998). Analysis of Anatto (Bixa orelland) Food Coloring Formulations; 1. Determination of Coloring Components and Colored Thermal Degradation Products by High-performance Liquid Chromatography with Photodiode Array Detection, J. Agric. Food Chem., 46, 1031-1038. Saffron Rlos, J.L., Recio, M.C., Giner, R.M. and Manez, S. (1996). An Update Review of Saffron and its Active Constituents, Phytother. Res., 10, 189-193. Part 4 ALKALOIDS Generalities 1. Definition.....................................................................................................................783 2. History.........................................................................................................................787 3. Natural Occurrence, Distribution, and Localization....................................................788 4. Physico-chemical Properties.............................:..........................................................790 5. Detection and Characterization....................................................................................791 6. Extraction of Alkaloids................................................................................................793 A. Extraction per se.............................................................................................793 B. Isolation of the Alkaloids...............................................................................796 7. Quantitation.................................................................................................................793 8. Biosynthetic Origin......................................................................................................797 9. Pharmacological Activity and Uses.............................................................................799 10. Bibliography................................................................................................................799 The number of products that have been described, their structural diversity, and the scope of their pharmacological activities make alkaloids one of the most important groups of naturally occurring substances of therapeutical interest, whose number "is almost unfathomable" (G.Richter *). Considering the structural and biosynthetic variety, this phrase is not as exaggerated as it sounds... 1. DEFINITION The term alkaloid was introduced by W. Meisner at the beginning of the nineteenth century to designate natural substances reacting like bases, in odrer words like alkalis * Richter, G. (1993). Stoffwechselphysiologie der Pflanzen, 1988, Georg Thieme Verlag, Stuttgart. / On- (from the Arabic al kaly, soda, and from the Greek eidos, appearance). There is no simple and precise definition of alkaloids, and it is sometimes difficult to distinguish the thin line between alkaloids and other natural nitrogen-containing metabolites. Initially defined as nitrogen-containing, basic substances of natural origin and of limited distribution, alkaloids have a complex structure. Their nitrogen atom is part of a heterocyclic system and they possess a significant pharmacological activity; according to some authors, they occur only in the vegetable kingdom. They are found as salts, and we may add that they are formed biosynthetically from an amino acid. These elements characterize what may be referred to as true alkaloids. Many authors distinguish, in addition, protoalkaloids and pseudoalkaloids. Pseudoalkaloids most often have all of the characteristics of the true alkaloids, but they are not derived from amino acids. Most of the known examples are isoprenoids and are referred to as terpenoid alkaloids: monoterpenoid alkaloids (e.g., (3-skythantine), sesquiterpenoid alkaloids of the Nymphaeaceae, diterpenoid alkaloids, such as aconitine from the tubercle of the aconite, or steroidal alkaloids (e.g., paravallarine), to name only a few. Also known are heterocyclic nitrogen-containing substances arising from the metabolism of acetate, for example coniine, the toxic principle of hemlock. fi-Skytanthine Paravallarine (+)-Coniine Protoalkaloids are simple amines in which the nitrogen atom is not part of a heterocyclic ring; they are basic and are elaborated in vivo from amino acids. Various substances fulfill this definition: simple amines such as serotonin, mescaline from peyote, or cathinone from Abyssinian tea, but also betains (resulting from the quaternarization of the nitrogen atom of amino acids); some authors include betalains (sometimes referred to as chromoalkaloids) in this group (e.g., betanin). Betanin Although the distinction between true alkaloids, protoalkaloids, and pseudoalkaloids is intellectually appealing, it is not always easy to apply: where does colchicine belong, with its nitrogen atom within an amide function, but not within a heterocyclic ring? Where should caffeine and theophylline be classified? Are amine-containing glycosides such as the chaconines to be considered as no more than nitrogen-containing saponins? In practice, it is widely accepted that the following are not alkaloids: simple amines, peptides*, amino sugars, porphyrins, alkylamines, and arylalkyl-amines, at least those that are widely distributed (in contrast, products with limited distribution such as ephedrine are most often recognized as alkaloids). All other compounds are commonly referred to as alkaloids: it would not occur to anyone to refer otherwise to colchicine, coniine, or aconitine! Thus we can state that an alkaloid is an organic compound of natural origin, which contains a nitrogen atom, is more or less basic, is of limited distribution, and has, at low doses, marked pharmacological properties. That this grouping has a sound basis is confirmed by the fact that these compounds have in common some reactions of precipitation with the "general reagents for alkaloids" (see below). * Again this is often a thin line: there is no major difference between the cyclic polypeptides of amanitas (amatoxins, phallotoxins) and peptide alkaloids such as frangulanine: the latter have a macrocyclic ring attached onto a benzene ring in the 1,3- or 1,4 positions, whereas the former are generally classified as polypeptides in the strict sense of the term. In the same fashion, the macrocycles derived from spermidine or spermine may be considered alkaloids (their ring includes at least one non-peptidic bond). IS' alkaloids To bring this paragraph to a close, note the interesting definition by Pelletier*: "An alkaloid is a cyclic organic compound containing nitrogen in a negative oxidation state which is of limited distribution among living organisms". From this point of view, amines and their oxides, amides, and quaternary ammonium salts are included, but nitrated derivatives (e.g., aristolochic acid) are excluded, and so are acyclic amides and polyamines. This definition allows, according to its author**, the inclusion of caffeine, colchicine, ephedrine, or ricinine in the alkaloids. Note that this definition does not include the activity criterion, and that the author comments that any compound administered at sufficient doses will eventually have an effect on a living organism (this tends to be forgotten, sometimes too easily). 2. HISTORY Although the concept of alkaloid is relatively recent, the knowledge of the toxicity and properties of the plants containing them dates back to ancient times: opium, coca, aconite, belladonna, Colchicum, as well as cinchona, ipecac, and curare have been used for centuries, if not millennia. It was probably Derosne, who, while extracting a mixture of narcotine and morphine from opium in 1803, was the first to isolate a vegetable alkali. In 1806, Serturner recognized the alkaline nature of the somniferous principle of opium, which he named morphine about ten years later. Shortly afterwards, between 1817 and 1820, two French pharmacists, Pelletier and Caventou, "discovered" an impressive series of active compounds: caffeine, emetine from ipecac, strychnine from nux vomica, quinine and cinchonine from cinchona bark, followed a little later by coniine. Chemists attempted to elucidate the structure of these molecules very early on: in the most simple cases, they were successful (coniine, Schiff, 1870), but in other cases they had to wait until the second half of the twentieth century: the polycyclic edifice of strychnine "resisted" the endeavors of chemical investigators for nearly 130 years. Today, advanced NMR techniques and X-ray diffraction spectrometry allow the elucidation of the most complex structures. The synthesis of these compounds also represented a challenge for chemists initially: from the synthesis of coniine at the end of the nineteenth century to that of morphine in 1952, this challenge played—and it continues to play—a key role in the development of organic chemistry. The isolation of reserpine at the beginning of the 1950s and its success in therapeutics inspired phytochemists to systematically explore the immense field of alkaloids: the number of structures described continues to increase, and structural, biosynthetic, synthetic, or pharmacological data have now reached considerable proportions. In a few cases, naturally-occurring compounds have been introduced in * Pelletier, S.W. (1983). The Nature and Definition of an Alkaloid, in "Alkaloids, Chemical and Biological Perspectives", op. cit., 1, p. 26 sq. ** Pelletier is careful to specify, about the term "cyclic": "a cyclic structure in some part of the molecule", which broadens the definition suhstantiallv GENERALITIES ■ n-ch3 Morphine F Nitidine F Harmane G n Pilocarpine H From the simplest... ... to the most complex: Examples of alkaloid structures illustrating the chief heterocyclic system encountered. The basic heterocyclic system is in boldface A: Pyrrolidine, B '.Piperidine, C'. Pyrrolidine, D: Quinolizidine, E: Tropane, F: Isoquinoline, G: Indole H: Imidazole, Quinazoline, j: Quinoline. _ ch30 Leurosidine h : OCOCH3 q ch3 c02ch3 oh ococh3 ch30 clinical trials—for example the binary alkaloids of Catharanthus—and in many other cases, structural analogs have been synthesized and marketed (derivatives of ergot alkaloids); interesting potential pharmacological activities have been uncovered and have inspired developments in a variety of directions (synthesis, structure-activity relationships, receptor experiments, and more). NATURAL OCCURRENCE, AND LOCALIZATION DISTRIBUTIOI Alkaloids occur only exceptionally in bacteria (pyocyanine from Pseudomonas aeruginosa) and rather rarely in fungi (psilocin from the hallucinogenic mushrooms of Central America, ergolines from Claviceps and. other actinomycetes, sporidesmins, roquefortine, and others). The Pteridophytes rarely contain alkaloids, and among them the Lycopodiaceae represent the main exception (alkaloids derived from lysine); the same comment applies to the Gymnosperms (alkaloids from Cephalotaxus). Thus, alkaloids are compounds essentially found in the Angiosperms, and some authors estimate that 10 to 15% of these synthesize those products. Certain families have a marked tendency to elaborate alkaloids: this is true for the Monocotyledons (Amaryllidaceae, Liliaceae) as well as the Dicotyledons (Annonaceae, Apocynaceae, Furnariaceae, Lauraceae, Loganiaceae, Magnoliaceae, Menispermaceae, Papaveraceae, Ranunculaceae, Rubiaceae, Rutaceae, Solanaceae, among others). Within these families, some genera produce alkaloids and others do not. Sometimes, they are found in all of the genera (Papaveraceae), although this is far less common. Certain alkaloids occur in several genera that belong to different families; sometimes these are quite distant taxonomically (caffeine), but most often they are fairly close (reticuline, yohimbine). Other alkaloids are characteristic of a limited number of genera within one family (hyoscyamine), or of a group of species within one genus (thebaine); some are highly specific (morphine). Alkaloid concentrations have a wide range of variation: from a few ppm as in the case of the anticancer alkaloids of the Madagascan periwijfkle {Catharanthus roseus: the level barely reaches 3 g of vinblastine for one nitric ton of leaves) to more than 15% in the bark of the trunk of Cinchona ledgeHana. Only rarely do alkaloid-containing plants contain only one alkaloid: sometimes they do contain virtually only one constituent (e.g., hyoscyamine from the leaves of belladonna), but, most often, they yield a complex mixture, which may be dominated by one major constituent. It is not uncommon to find several dozen alkaloids in one drug (nearly one hundred in the case of the Madagascan periwinkle). As a general rule, all of the alkaloids of a given plant have a common biogenetic origin, even if their structures may at first seem quite different. In a given plant, the concentration of alkaloids can vary widely from part to part, and some parts may contain none. Qualitative variations are also frequent: it is not uncommon for different parts of one plant to contain dissimilar alkaloids. One example is quinine, which accumulates in the trunk bark of Cinchona, but is completely absent from the leaves: similarly, conessine accumulates in the seeds and the bark of kurchi {Holarrhena pubescens), but is absent from the leaves. The occurrence of chemotypes is well known (Rauwolfia, Duboisia). For a long time, alkaloids used to be considered products of die metabolism of plants only. In fact, alkaloids also occur in animals. In some cases, they are products formed from the alkaloids contained in the plants on which the animals feed: examples are castoramine, which arises from the metabolism of the alkaloids of the water-lilies consumed by beavers, and the pyrrolizidine alkaloids found in some butterflies. In other cases, the alkaloids appear to be the products of the metabolism of the animal: this is true for the urodele (salamanders) or anurous Amphibia (Bufo, Phyllobates, Dendrobates, and other toads). Alkaloids occur frequently in the Arthropods, who secrete them in very small quantities in their exocrine glands. They are well known in Hymenoptera (e.g., solenopsine of Myrmicides), but they are also elaborated and used by Coleoptera, Neuroptera, and some of the Myriapoda. They Histrionicotoxine (Dendrobates histrionicus) Glomerine (Glomeris marginata, Myriapodes) Pyocyanine n ""(ch2)10-ch3 h Solenopsine A (ants) hn j Samaderine (salamander) oh 0„ // Castoramine Roquefortine o c6h5 o CMS n oh Taxol o o ?90 alkaloids GENERALITIES have low molecular weights (pyrrolidines, piperidines, pyrroles, indolizidines, pyrazines), and they are volatile enough to act as chemical signals, defense compounds (allomones), or communication compounds (pheromones). In recent years, progress in marine biology has led to the isolation of many nitrogen-containing heterocyclic structures, particularly in the sponges. Localization In the plant, alkaloids occur as soluble salts (citrates, malates, tartrates, meconates, isobutyrates, benzoates), or in combination with tannins. Microchemistry shows that alkaloids are most often localized in the peripheral tissues: external layers of the bark of the stems and roots, or seed tegument. Most of these compounds have a basic character and an antimetabolite activity which make their compartmenta-lization necessary: they are normally stored in the cell vacuoles, which may be specialized (laticiferous) or not. Most often, alkaloid synthesis takes place at specific sites (growing root, laticiferous cells, chloroplasts); the compounds are subsequently transported to the storage site. Function As in the case of many other secondary'metabolites, almost nothing is known of the role of alkaloids in plants. Some may be involved in plant-predator relationships, by protecting the former against the latter: if we accept the notion that structural diversity reflects constant adaptation, it reinforces this hypothesis. Although some authors believe that alkaloids are terminal metabolites, in other words unusable waste, this is highly unlikely: in several cases, they have been shown to act as intermediate metabolites. Storage substances? Growth regulators? The question remains unanswered. 4. PHYSICO-CHEMICAL PROPERTIES Alkaloids have molecular weights ranging from 100 to 900. Although most of the bases that do not contain oxygen atoms are liquid at ordinary temperatures (nicotine, sparteine, coniine), those that do contain oxygen atoms—virtually all of the known structures—are normally crystallizable solids, and in rare cases they are colored (berberine). Almost all of the crystallized bases rotate the plane of polarized light, and have sharp melting points, without decomposition, especially below 200°C. As a general rule, alkaloids as bases are not soluble or are sparingly soluble in water, soluble in apolar or only slightly polar organic solvents, and are soluble in concentrated hydroalcoholic solutions. The basicity of alkaloids varies greatly, since this property depends entirely on the availability of the lone pair of electrons on the nitrogen atom. Electron-withdrawing groups in close proximity to the nitrogen atom decrease the basicity, whereas presence of the carbonyl group on the amide, practically neutral. The basic character of the heterocyclic ring itself varies: in the molecule of pyridine, with 6 n electrons, and in the case of quinoline and isoquinoline, the lone pair of electrons on the nitrogen atom is available and the basicity is clear. In the case of pyrrole or indole, the lone pair of electrons on the nitrogen atom plays a role in the aromatic character, and the compounds are not basic (they are acidic). Another example is pyrrolidine, which is saturated, and is a strong base. The basicity is also influenced by steric constraints (at least in the complex polycyclic molecules). Finally, let us emphasize that their basic character renders these compounds unstable, so that as bases in solution they are sensitive to heat, light, and oxygen. The basic character of alkaloids allows the formation of salts with mineral acids (hydrochlorides, sulfates, nitrates) or organic acids (tartrates, sulfamates, maleates). Alkaloid salts are generally soluble in water and in dilute alcohols, and they are, except in rare cases, not soluble in organic solvents. The crystallized salts can be conserved fairly well and are the common commercial form of these compounds. N I h Pyrrole n 1 h Pyrrolidine n h Piper'.dine Pyridine Mi n Quinoline Isoquinoline Piperine 5. DETECTION AND CHARACTERIZATION A detection technique ought to be, to the extent possible, rapid, simple, reproducible, and sensitive; it must be applicable to a small sample. The detection methods currently in use are preceded by an extraction and consist, most generally, in precipitating the alkaloids by using fairly specific reagents: die "general reagents for alkaloids". The preliminary extraction can be a "classic" alkaloid extraction (see below) or an alcoholic maceration, which takes less time: the alcoholic solution is evaporated and the residue redissolved in acidic water; after filtering, the alkaloids are characterized in the filtrate. The general reactions of precipitation are based on the fact that alkaloids form combinations with metals and metalloids: bismuth, mercury, tungsten, and iodine. In practice, what is used is a solution containing iodine and iodide, or a solution containing potassium iodide and mercuric chloride—known as Mayer's reagent—or a reagent containing bismuth nitrate and potassium iodide, better known as Dragendorff s reagent. It is also possible to use silicotungstic acid (a mixture of / ..,,„/.. ? _./.,_, .'./.. \ " 'urr.i:..,' ;,„/'../T,..V ' £*;iV-i*nViiTJ*'