Sesquiterpenes Sesqui rpenoid Lactones Sesquiterpenoid lactones form a group of substances important by its size — approximately 3,000 known structures—which was described, in the older texts of Materia Medica, under the evocative name "bitter principles *". Sesquiterpenoid lactones have a rather scattered botanical distribution. They occur in the Fungi and Bryophytes, here and there in the Angiosperms (Apiaceae, Lauraceae, Menispermaceae), and chiefly in the Asteraceae. In the latter, the lactones frequently occur in the glandular trichomes located in the leaves, stems, and inflorescence bracts. They are fairly often found in the akenes, but are rare in the subterranean organs (lactucin of chicory roots, helenin of elfdock). * This obsolete terminology covers a very homogeneous group of compounds: with few exceptions (hops, cinchona), the bitterness of the drugs is due to the presence of terpenoid lactones. These are monoterpenoids in the Gentianaceae (p. 604), sesquiterpenoids in chicory (p. 85) or wormwood (p. 524), diterpenoids such as columbin in Jateorrhiza palmata Miers (p. 920), or triterpenoids, such as the limonoids of the Rutaceae and the quassinoids of Simaroubaceae (p. 765). They were formerly used—and, in some cases, continue to be used— 1. Structure.................................................................. 2. Applications of Sesquiterpenoid Lactones............. 3. Chief Drugs Containing Sesquiterpenoid Lactones Feverfew........................................ 4. Sesquiterpenoid Lactones and Allergy 5. Bibliography........................................ Sweet Wormwood (qinghao) Arnica .................................. Elfdock.................................. 620 621 623 .623 627 .629 .631 .633 .634 620 TERPENOIDS SESQUITERPENOID LACTONES 621 1.STRUCTURE The skeleta of sesquiterpenoid lactones vary, but they all arise from the cyclodecadiene-type product of the cyclization of 2E,6E-hmesy\ pyrophosphate. Although experimental evidence is rare, it is generally accepted that the chief skeletons arise from the cyclization of the cyclodecadiene-type cation, via the germacranolides*. Quite logically, the structure of the cyclization product depends on the initial conformation adopted by the macrocycle, and on the position of the double bonds which allow various intramolecular electrophilic cyclizations: the enzyme involved in the reaction acts as a matrix for the precursor and determines the stereospecificity of the reaction. Some of the proposed biosynthetic schemes are considered more plausible because they also correspond to in vitro syntheses. Biogenetic relationships of the chief groups of sesquiterpenoid lactones (the choice of 12,6 lactones is arbitrary) j 15 o Guaianolide 6 Eudesmanolide Eremophilanolide There are many secondary structural variations which affect: • the lactone, which can be cis-6,1, cw-7,8, trans-6,1, or in most cases, transit; it is generally an oc-methylene-y-lactone, and in all cases (except in the Bryophytes), the proton in the 7-position is a; * Proper nomenclature consists in adding the suffix -olide to the name of the sesquiterpenoid skeleton to indicate the presence of a lactone. In the particular case of germacranolides, the configuration of the double bonds determines four subgroups: germacrolides (trans, trans), heliangiolides [lC10)-rmw,4-m], melampolides [l(\0)-cis,4-trans], and m,cw'germa- cranolides. • the methyl groups, which are often functionalized (alcohols, carboxylic acids); • the unsaturations, which may be reduced or oxidized (epoxides, hydroxyl groups); when hydroxyl groups are present, they are frequently esterified. There is no extraction method specific to sesquiterpenoid lactones. They can be extracted with dichloromethane, or with a mixture of diethyl ether, petroleum ether, and methanol, and the extract can be fractionated by the appropriate chromatographic technique. Various reagents are available to visualize the TLC spots, including iodine vapors, a dilute solution of KMn04, vanillin in the presence of hydrochloric acid, and cobalt chloride in an aqueous sulfuric acid solution. 2. APPLICATIONS OF SESQUITERPENOID LACTONES Except in the case of artemisinin derivatives, there are no therapeutic uses for sesquiterpenoid lactones and only a few drugs containing them are used. Although a few drugs with "bitter principles" find some use in folk medicine and in phytotherapy, there is nothing to prove, at least for some of them, that the virtues attributed to them have anything to do with the presence of this type of compound. The potential applications of sesquiterpenoid lactones are, however, far from trivial. This is no surprise, considering their high reactivity: the a-methylene-y-lactone moiety and the epoxides, which are common, are reactive sites for biological nucleophiles, mainly thiol groups and the amines of the active sites of various enzymes (glycogen synthase, DNA polymerase, fhymidylate synthase). As a result, these enzymes are irreversibly alkylated, hence sesquiterpenoid lactones have a broad range of biological activities. Tenulin o o Vernolepin (+)-a-Santonin Ambrosin Many lactones are antibacterial agents, especially against Gram positive bacteria, for examDle. the lactones of the elfdock, or cnicm from the blessed thistle, Cmcus 627 TERPENOIDS to treat wounds and ulcers. Certain compounds in this series are also antifungal agents. Some structures are antiparasitic agents (artemisinin is an anti-malarial of proven efficacy in humans), whereas others are anthelminthics * or toxic to molluscs, for example ambrosin and damsin of Ambrosia ** maritima L., an herbaceous Asteraceae of the damp coastal areas and river basins of Africa. Some authors have proposed to use this drug to control the molluscs that are vectors of schistosomiasis and distomatosis (Biomphalaria, Bulinus, Lymnaea). Its efficacy was confirmed by field tests: 90% of the molluscs disappeared within 3 months from the irrigation canals that were treated. The alkylating potential of these compounds has led to the study of their cytotoxicity. Although many of them have a strong activity (vernolepin, eupatoriopicrin, tenulin), none has undergone clinical trials, in great part because of their marked toxicity. Some of these compounds are interesting tools for biologists: thapsigargin (of Thapsia garganica L., Apiaceae) mobilizes intracellular calcium according to a very specific mechanism. Note that sesquiterpenoid lactone-containing plants represent a serious danger for cattle (Helenium, Centaurea, Geigeria, Hymenoxys) but also, on rare occasions, for humans. Examples include polycyclic compounds such as picrotoxinin from a fish * Santonin belongs to this category: it is the active principle of a drug long used to treat worm infestations, namely wormseed {Artemisia cina Berg. Ex Polj., Asteraceae). Its capitulums were used, and contain, in addition to 10-20 mL/kg essential oil with cineole, 2-3% sesquiterpenoid lactones: santonin and other eudesmanolides of closely related structure. Santonin, long prescribed as a nematicide, has been abandoned in human therapy due to its non-trivial toxicity (gastrointestinal distress, visual disturbances, headaches, dizziness). ** While on the topic of Ambrosia, and although sesquiterpenoid lactones are not at fault, this is a good place to note that several species in the genus, which are not medicinal, are responsible for allergies to pollen. Examples include A. trifida L. and A. artemisiifolia L. (common ragweed). The latter is a weed that originally grew on dirt mounds, embankments, and unimproved lands in western Europe, and has been spreading to cultivated land. Worse, the European Union agricultural policy, by requiring that land remain unexploited according to a given schedule, can only promote its dissemination; however, legislation effective on 21 February 1994 requires farmers to maintain the vegetation without allowing it to produce seeds. In France, the spread of common ragweed is particularly rapid along the river Rhone: 6% of the population of the major city of Lyon is believed to be affected by this allergy. Some of the departements (counties) in the Lyon region have recently launched educational campaigns against this species; since 1995, following the Canadian example, various levels of French local government have passed measures to encourage the eradication of this species. Allergy to Ambrosia pollens is a well-known problem in North America where common ragweed and giant ragweed (A. artemisiifolia and A. trifida, respectively) constitute one of the principal causes of allergy to pollen: in eastern Canada, this plant causes health problems to an estimated 10-12% of the population. Cf., inter alia, Dechamp, C, Rimet, M.L., Meon, H. and Deviller, P. (1997). Quatorze annees de comptes polliniques d'ambroisies fcantmw vulnerarius: wound healing). This plant is rare and protected, and in several European countries, a closely related species is used as a substitute: A. chamissonis Less. SSp. foliosa (Nutt.) Maguire (DAB 10). The Plant, the Drug. Arnica is a species indigenous to mountain areas, easy to identify by its large (60-80 mm) orangy-yellow capitulums, on a 2-3-cm peduncle, surrounded by an involucre of 18-24 acute bracts covered with yellow hairs. The flowers are ligulate (20-30-mm long) with 7-10 veins and 3 teeth. In the tubular flowers (15-mm long) as well as the ligulate flowers, the ovary has a pappus of whitish bristles that are not feathery. Chemical Composition. The color of arnica drug is due to carotenoids. Its odor is due to 5 mL/kg essential oil of pasty consistency because of a high concentration of fatty acids; it contains, among others, terpenoid hydrocarbons and thymol derivatives. The bitterness is due to sesquiterpenoid lactones (0.2-0.5% helenalm, ■.•.dihydrohelenalin, and their esters: acetates, isobutyrates, tiglates, isovalerates, and more): their proportions vary depending on the geographic origin (central Europe and Spain). Note also the presence of triterpenoids, phytosterols, fatty acids, polysaccharides, phenolic acids, coumarins, polyalkynes, and of 0.2-0.3% flavonoids (glycosides of hispidulin, patuletin, betuletol, spinacetin, quercetagetm). A. chamissonis can contain more lactones (up to 1.5%) and in addition to the above compounds, it can contain their 2,3-dihydro-2a-hydroxylated homologs (arnifolins) and their 2,3-dihydro-2a,4a-dihydroxylated homologs (chamissonolides). Again, the proportions of the different compounds vary widely, with some batches closely resembling A. montana. The two species also differ by their flavonoid composition: A. chamissonis is characterized by glycosides of flavonoids acylated by acetic acid or 2-methylbutyric acid. h3cocq h Yingzhaosu Arteflene (Ro 42-1611) Helenatin R2°. 6r Chamissonolides 628 TERPENOIDS Pharmacological Properties - Toxicity. Anti-inflammatory, analgesic, and anti-ecchymotic properties are traditionally attributed to arnica. We cannot exclude that the sesquiterpenoid lactones are in part responsible for this activity: they are known to inhibit the migration of polymorphonuclear leukocytes and the rupture of lysosomal membranes. An inhibitory activity on platelet aggregation has also been reported. Experiments have shown that arnica preparations and constituents are antimicrobial, antifungal, anti-inflammatory, and cytotoxic for various cell lines. All of the galenical forms of arnica cause allergic reactions, and in sensitive subjects, cross-reactions with Asteraceae and other lactone-containing species are frequently observed. The systemic toxicity of the lactones is also known: in his review of the literature, Hausen [1992] lists case reports of vomiting, respiratory and cardiac difficulties, bloody sputum, cerebral problems, and even deaths following the administration of arnica preparations for the purpose of abortion. Tests. Arnica is identified by its microscopic characteristics, particularly by paired covering trichomes with a punctate common wall. The drug is official only if it contains <2% akenes (total foreign matter <4%). Although the French Pharmacopoeial monograph only requires one TLC analysis of phenolics and the verification of the absence of marigold (absence of rutin), it is perfectly feasible, by TLC as well as by HPLC, to distinguish A. montana, A. chamissonis, and other species (e.g., Heterotheca inuloides Cass., but also marigold), based on their flavonoid and/or sesquiterpenoid lactone content. Uses. Arnica and its preparations are reserved for external use, because when they are taken orally they can cause headaches, abdominal pains, as well as vasomotor problems (palpitations) and breathing difficulties. A common form of utilization is the tincture, diluted with water or a dilute alcohol, and applied in a compress onto bruises, ecchymoses, petechiae, sprains, and strains; this is acknowledged by the 1998 French Explanatory Note, which authorizes "traditional use for the symptomatic treatment of ecchymoses". Arnica and its preparations are also traditionally used for sunburns, superficial and limited burns, and diaper rashes. Arnica preparations should not be applied on open wounds or near the eyes or mouth. The German Commission E monograph on arnica specifies that application to damaged skin can induce edematous dermatitis, that prolonged use can induce eczema, and that the most concentrated forms can cause the formation of vesicles and even necrosis. In Germany, the common indications are the same as in France. Package inserts must list contraindications (hypersensitivity to Asteraceae), side effects (allergy), and the need to use the product only for external application. In homeopathy, arnica passes as an agent that promotes the resorption of extravasated blood and prevents hemorrhages. A 1993 Canadian clinical trial showed the lack of statistically significant effect of arnica 5 CH on bleeding time and other blood clotting parameters [Baillargeon et ai, 1993]. SESQUITERPENOID LACTONES ill • Elfdock, Inula helenium L., Asteraceae fl The dried root and rhizome of this perennial plant with large (6-8 cm) yellow capitulums are said to be diuretics, bechics, and anthelminthics (Fr. Ph., 10th Ed.). The drug contains eudesmanolides (alantolactone, isoalantolactone, and their more or less hydrogenated derivatives), a germacranolide, triterpenes, sterols, and depending on the season, 20-45% inulin. Steam distillation of the subterranean parts yields 1-3% of an odoriferous fraction which partially solidifies at ambient temperature. The elfdock rhizome and root consist of irregular fragments with a balsamic odor, and an aromatic and bitter taste. They are frequently excoriated, and this reveals a grayish cortical parenchyma. Under the microscope, the cut and the powder show inulin grains and resin-containing cells. Three TLC analyses allow: 1. the verification of the absence of alkaloids (contamination by belladonna roots); 2. the visualization of the sulfuric acid hydrolysis products of inulin; 3. the study of the sesquiterpenoid lactones after steam distillation, including the characterization of alantolactone. Alantolactone Isoalantolactone Alantolactone and isoalantolactone are cytotoxic and display antibacterial and antifungal properties in vitro: at concentrations of 10 (Xg/mL they inhibit the growth of Microsporum cookei, Trichophyton mentagrophytes, Trichothecium roseum. and other Fungi which are pathogenic for humans {Trichophyton, Epidermophyton). These lactones are also reputed to be anthelminthic and hypotensive: their anthelminthic properties are weak and vary widely, maybe because of a rapid absorption in the digestive tract. In the absence of truly demonstrated properties, the rhizome and roots of the elfdock, as well as their preparations, may be "traditionally" used in France (French Expl. Note, 1998) by the oral route: 1. to enhance urinary and digestive elimination functions, and 2. to treat the symptoms of cough (forms based on total drug powder must pass basic safety tests). The German Commission E monograph lists uses for respiratory, gastrointestinal, renal, and urinary disorders, and states that none of these indications has been completely validated. After a reminder that lactones can cause allergic reactions and that high doses of elfdock can induce vomiting, diarrhea, and other problems, Commission E concludes that the therapeutic use of elfdock cannot be justified. In the United Kingdom, where the drug is used (BHP 1990) for bronchitis and cough, pregnancy and breast-feeding constitute contraindications. Several authors also discourage abusive or prolonged use. Alantolactone and isoalantolactone are responsible for the allergic dermatitis observed with this Asteraceae. • Feverfew, Tanacetum parthenium (L.) Schultz-Bip., Asteraceae The drug (Fr. Ph., 10th Ed.) consists of the dried aerial parts, whole or in fragments. In France, this drug was introduced in phytotherapy relatively recently. The Plant, the Drug. Feverfew is a tall (70-80 cm) perennial herb, originally indigenous to Asia Minor, and fairly common in the neglected fields of Europe. The stems, almost quadrangular and more or less ramified, bear polymorphic leaves, which are pinnatisect or bipinnatisect, and divided into 5-9 segments with a crenate margin. The capitulums of yellow tubular flowers and white ligulate flowers are grouped in corymbs of 5-30 capitulums on long pedicels, with a diameter ranging from 12 to 22 mm. The fruit is a brown akene with 5-10 white ribs, surmounted by a membranous crenate crown. Under the microscope, the pulverized drug is characterized by numerous covering trichomes, 5-7-cellular, uniseriate, with a trapezoidal basal cell, ending with an element often bent at a right angle, very long, flat, tapered, and with a thin wall. Chemical Composition. The strong odor of feverfew is due to an essential oil (3-8 mL/kg in the leaves) containing camphor and chrysanthemyl acetate as major constituents (the latter can be found partially hydrolyzed to chrysanthemol in the dried drug). Flavonoids, water soluble flavone glucuronates, and lipophilic di-3,7-and tri-3,7,3'-methylated flavonols are also found. A sesquiterpenoid lactone, parthenolide, is thought to be the active principle. This is a germacranolide that has been isolated from European and North American batches, but has not been found in Canin Tanaparthin a-peroxyde Seco-tanapartholide A Parthenolide oh oh oh 632 TERPENOIDS SESQUITERPENOID LACTONES 633 Mexican samples in which a eudesmanolide occurs, namely santamarin (santamarin is also found alongside parthenolide in North American plants). In practically all of the batches analyzed, other germacranolides are also found (costunolide and derivatives, artemorin), as well as eudesmanolides (reynosin), and guaianolides (canin, artecanin, anaparthin, a derivative of cumambrin B). Some varieties are particularly rich in parthenolide (f. floculosum) and the concentration of this lactone varies as a function of the vegetative cycle (maximal at the time of blooming) and also as a function of the organ: it is concentrated in the flowering tops (1.38%) and leaves (0.95%), but not in the stems (0.08%), and it is virtually absent from the roots (0.01%; values determined on a batch of plants from the United Kingdom). It has been shown empirically that the parthenolide concentration drops by nearly 50% in 9 months in the crushed drug stored with no particular precaution. Pharmacological Properties. Feverfew extracts inhibit platelet aggregation and the ADP- or adrenalin-induced release of serotonin (=5-HT), which would explain the activity against migraines traditionally attribued to the drug. It has been shown that parthenolide has a—weak—affinity for 5H2A receptors. There is also inhibition of the degranulation of polymorphonuclear leukocytes, of the release of enzymes involved in the inflammatory process, of phospholipase A2 and of prostaglandin synthesis, as well as a protective effect on vascular endothelial cells. The observed activity seems to result from the Michael-type addition of the methylene lactone onto the enzyme thiols groups (cysteine inactivates parthenolide and cancels its effects). A very recent study of the effect of the extracts on smooth vascular muscle has shown that although the chloroform extract of the fresh plant does inhibit contractions induced by various agonists in a non selective and irreversible fashion, extracts prepared from the dried drug do not, in fact their action is opposite—but their analysis shows that they contain no parthenolide. This reversible stimulation of smooth vascular muscle fiber contraction appears to be linked to a potassium channel blockade induced by a unidentified substance. Is this vasoconstriction involved in the antimigraine effect, as it is in the case of ergot alkaloids or sumatriptan? This is one hypothesis. It is worth noting that the properties of parthenolide have been studied in vitro, but that the bioavailability of this very reactive compound remains to be determined. We cannot exclude that other constituents contribute to the activity, for example the flavonoids (?) or chrysanthemyl acetate, which is known to inhibit prostaglandin synthetase. The initial clinical trials tended to show that feverfew leaf powder had an interesting potential to prevent the onset of acute attacks of migraine headache, but these trials were criticized for their patient selection methods. A more recent double blind trial on about 40 patients treated with ethanolic extract capsules titrated to contain 0.5 mg of parthenolide led to the conclusion that the capsules had no beneficial effect in preventing the attacks. Therefore, further research is needed. The only study conducted to evaluate the effect of feverfew on rheumatoid arthritis showed that it had none. Tests. Feverfew is identified by macro- and microscopic examination, and by characterizing parthenolide by TLC analysis of a methanol extract with visualization by spraying with vanillin in the presence of sulfuric acid. The assay per se includes foreign matter (<12%; stems of diameter >5 mm: <10%), total ashes (<12%), and parthenolide quantitation (HPLC, methanolic extract, not less than 0.2% of the drug weight). Uses. In France [French Expl. Note, 1998], the flowering tops of feverfew are used orally for the following indications: traditionally used 1. to prevent headaches *; 2. to relieve menstrual pain. Medicines based on total drug powder and hydroalcoholic extracts of alcoholic titer >30% must pass basic safety tests. In Canada, the marketing of a non-prescription (over-the-counter = OTC) product claiming efficacy in the prophylaxis of migraine headaches has been authorized. Feverfew causes no serious side effects in regular users (ulcerations of the mouth, abdominal pain). Experts from English-speaking countries agree that pregnancy is a contraindication and in Canada, the recommendation is to not prolong treatment beyond 4 months without medical advice. It is rare for this Asteraceae to cause allergic dermatitis. Yet the risk exists, especially for all persons who are hypersensitive to sesquiterpenoid lactones (cross reactions). Comment. A systematic analysis of commercially available leaves, powders, capsules, tinctures, and other forms showed that their quality varied widely (in 1991): parthenolide could not be detected in about one-third of the preparations for the oral route; its concentration ranged from 0.01 to 1% in the other two-thirds. Several types of adulteration were documented (Matricaria maritima L., M. recutita [L.] Rausch., Tanacetum vulgare L.). The standards that are now in place (France, Canada) should help improve the quality of commercial products. 4. SESQUITERPENOID LACTONES AND ALLERGY Asteraceae containing sesquiterpenoid lactones are frequently responsible for dermatitis of allergic origin. These compounds act as haptens and bind to proteins to form allergens, which in turn induce the sensitization of lymphocytes. Of course, this is due to the reactivity of the a-methylene-y-lactone. Produce species (e.g., artichoke, cardoon [cynaropicrin], endive [lactucopicrin]), ornamental species (e.g., mums [arteglasin], daisies, asters, cosmos, gaillardia, rudbeckia), sunflowers [niveusin], and certain medicinal species (e.g., Roman camomile [p. 335], matricaria [p. 520], yarrow [p. 337], feverfew, arnica, or inula [see above]) have caused various cases of occupational papulous dermatitis and conjunctivitis in farmers, horticulturists, and florists. * Until February 1998, the wording of the French regulation was "to prevent acute attacks of migraine headaches". 0J4 TERPENOIDS SESQUITERPENOID LACTONES 635 Asteraceae are not the only species that contain allergenic lactones. Dermatitis can also be observed after contact with laurel (costunolide and other lactones) or hepaticas of the genus Frullania (F. dilatata [L.] Dum., F. tamarisci [L.] Dum., and others [frullanolide and other lactones, eudesmanolides, eremophilanolides]). Also responsible for allergic dermatitis are perfumery products based on Asteraceae (e.g., arnica, camomiles). 5. BIBLIOGRAPHY Generalities Fischer, N.H. (1991). Sesquiterpenoid Lactones, in "Methods in Plant Biochemistry, vol. 7, Terpenoids", (Charfwood, B.V. and Banthorpe, D.V., eds.), p. 187-212, Academic Press, London. Geerts, S., Belot, J., Sabbe, F., Triest, L. and Sidhom, M. (1991). Ambrosia maritima: Effects on Molluscs and Non-target Organisms,/. Ethnopharmacol., 33, 1-12. Alonso Castell, P., Moreno Galdo, A., Sospedra Martinez, E., Roqueta Mas, J., Hidalgo Albert, E. and Iglesias Berengue, J. (1997). Intoxicacion grave por Coriaria myrtifolia: a proposito de un caso, An. Esp. Pediatr., 46, 81-82. Maries, R.J., Pazos-Sanou, L., Compadre, CM., Pezzuto, J.M., Bloszyk, E. and Arnason, J.T. (1995). Sesquiterpene Lactones Revisited: Recent Developments in the Assessment of Bio-logical Activities and Stucture Relationships, in "Phytochemistry of Medicinal Plants", (Arnason, J.T., Mata, R. and Romeo, J.T., Eds.), p. 333-356, Plenum Press, New York. Sweet wormwood Avery, M.A., Mehrotra, S., Johnson, T.L., Bonk, J.D., Vroman, J.A. and Miller, R. (1996). Structure-Activity Relationships of the Antimalarial Agent Artemisinin. 5. Analogs of 10-Deoxoartemisinin Substituted at C-3 and C-9, /. Med. Chem., 39, 4149-4155. Brown, G. (1994). Cadinanes from Artemisia annua that may be Intermediates in the Biosynthesis of Artemisinine, Phytochemistry, 36, 637-641. Ferreira, J.F.S., Charles, D.J., Wood, K., Janick, J. and Simon, J.E. (1994). A Comparison of Gas Chromatography and High Performance Chromatography for Artemisinin Analyses, Phytochem. Analysis, 5, 116-120. 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The Treatment of Malaria, New Engl. J. Med., 335, 800-805. Woerdenbag, H.J., Bos, R., Salomons, M.C., Hendriks, H., Pras, N. and Malingre, T.H. (1993). fl Volatile Constituents of Artemisia annua L. (Asteraceae), Flavour Fragr. J., 8, 131 + 137. Woerdenbag, H.J., Pras, N., van Uden, W., Wallart, T.E., Beekman, A.C. and Lugt, C.B. (1994). Progress in the Research of Artemisinin-related Antimalarials: an Update, Pharmacy World & Science, 16, 169-180. Woerdenbag, H.J., Pras, N., Chan, N.G., Bang, B.T., Bos, R., van Uden, W., Y„ P.V., Boi, N.V. and Lugt, C.B. (1994). Artemisinin, Related Sesquiterpenes, and Essential Oil in Artemisia annua During a Vegetation Period in Vietnam, Planta Med., 60, 272-275. Ziffer, H., Highet, R.J. and Klayman, D.L. (1997). Artemisinin: An Endoperoxidic Antimalarial from Artemisia annua, Fortschr. Chem. Org. Naturst., 72, 121-214. Arnica Baillargeon, L., Drouin, J., Desjardins, L., Leroux, D. and Audet, D. (1993). Les effets de XArnica montana sur la coagulation sanguine. Essai clinique randomise, Can. Fain. Physician (Le Medecin defamide canadien), 39, 2362-2367. Hausen, B.M. (1992). Sesquiterpene Lactones - Arnica montana, in "Adverse Effects of Herbal Drugs", ( De Smet, P.A.G.M., Keller, K., Hansel, R. and Chandler, R.F., eds.), p. 237-241, Springer-Verlag, Berlin. Merfort, I., Pietta, P.G., Mauri, P.L., Zini, L., Catalano, G. and Willuhn, G. (1997). Separation of Sesquiterpene Lactones from Arnicae Flos DAB 10 by Micellar Electrokinetic Chromatography, Phytochem. Analysis, 8, 5-8. Wijnsma, R.,Woerdenbag, H.J. and Busse, W. (1995). Die Bedeutung von Amika-Arten in der Phytotherapie, Z. Phytother., 16, 48-62. Willuhn, G, Leven, W. and Luley, C. (1994). Arnikabliiten DAB 10. Untersuchungen zur qualitativen und quantitativen Variabilität des Sesquiterpenlactongehaltes der officinellen Arnrkadrogen, Dtsch. Apoth.-Ztg., 134, 4077-4085. Feverfew Barsby, R.W.J., Knight, D.W. and McFadzean, I. (1993). A Chloroform Extract of Üie Herb Feverfew Blocks Voltage-dependent Potassium Currents Recorded from Single Smooth Muscle Cells, / Pharm. Pharmacol. 45, 641-645. Barsby, R.W.J., Salan, U., Knight, D.W. and Hoult, J.R.S. (1993). Feverfew and Vascular Smooth Muscle: Extracts from Fresh and Dried Plants Show Opposing Pharmacological Profiles, Dependent upon Sesquiterpene Lactone Content, Planta Med., 59, 20-25. Brown, A.M.G., Lowe, K.C., Davey, M.R., Power, J.B., Knight, D.W. and Heptinstall, S. (1996). Comparison of Extraction Procedures for Parthenolide in Tanacetum parthenium, Phytochem. Analysis, 7, 86-91. Hay, A.J.B., Hamburger, M., Hostettmann, K. et Hoult, J.R.S. (1994). Toxic Inhibition of Smooth Muscle Contractility by Plant-derived Sesquiterpenes Caused by their Chemically Reactive a-Methylenebutyrolactone Functions, Br. J. Pharmacol., 112. 9-12.