Arch. Derm. Res. 255, 111 -- 121 (1976)
Archives for
Dermatological Kesearcn © by Springer-Verlag 1976
ChrysanthemumAllergy III. Identification of the allergens * ** B. M. Hausen and K. H. Schulz Department of Dermatology, Division of Allergology, University Hospital, Hamburg (Federal Republic of Germany)
Summary. Experimental and chemical studies in guinea pigs on contact allergy caused by Chrysanthemum indicum L. (Chrysanthemum of the florists) led to the isolation of several sesquiterpene lactones. From the crude extract of dried flowers 12 fractions were obtained of which four gave strong reactions on epicutaneous application to guinea pigs sensitized with an extract of C. indicum L. One of these allergens was identified as a sesquiterpene lactone of the guaianolide type. It is identical to Arteglasin-A derived from Artemisia douglasiana Bess. The occurrence of Arteglasin-A in Chrysanthemum indieum L. as well as its allergenic properties are described for the first time. These findings give rise to the assumption that the other, as yet unidentified sesquiterpene lactones of this flower also belong to the guaianolide type.
Zusammenfassung. Chemische Untersuchungen und experimentelle Teste an Meerschweinchen bezfiglich der dutch Chrysanthemen verursachten Kontaktallergie fiihrten zur Isolierung mehrerer Sesquiterpenlaktone. Aus dem Rohextrakt getrockneter Chrysanthemen wurden 12 Fraktionen isoliert, yon denen vier im Epikutantest bei sensibilisierten Meerschweinchen eine stark positive Reaktion ergaben. Eins der Allergene wurde als Sesquiterpenlakton vom Guaianolid-Typ identifiziert. Es ist identisch mit Arteglasin-A aus Artemisia douglasiana Bess. Sowohl das Vorkommen von Arteglasin-A in Chrysanthemum indieum L. als auch seine allergene Wirkung werden bier das erste Mal beschrieben. Die Untersuchungsbefunde lassen vermuten, dab die anderen bisher nicht identifizierten Sesquiterpenlaktone ebenfalls dem Guaianolid-Typ angeh6ren, * Part I and II of this series are references 1 and 2. ** A preliminary report of this article is reference 34. Reprint requests to: Dr. B. M. Hausen, Department of Dermatology, University Hospital Hamburg, Martinistr. 52, D-2000 Hamburg 20, Federal Republic of Germany. 8 Arch. Derm. Res,, '9"ol.255
112
B.M. Hausen and K. H. Schulz
INTRODUCTION
Chrysanthemum indicum L. (Chrysanthemum of the florists) is one of the most c o m m o n causes of occupational contact dermatitis among gardeners and florists in the northern part of West Germany. A survey of the history of Chrysanthemum culture, of the plant constituents hitherto known and of the manifestations of Chrysanthemum dermatitis is given in Part I of this series [1]. Part II deals with studies on the isolation and identification of sensitizing components [2]. In the present paper the identification and chemical characterization of one of the contact allergens of Chrysanthemum indieum L. is described. Since 1960 more than 600 compounds of sesquiterpene lactone structure have been found in the family of Compositae, to which C. indieum L. belongs [3--5]. Studies by Mitchell et al. [6,7] in 1970 revealed that patients with contact dermatitis due to Chrysanthemum flowers developed positive patch test reactions to certain sesquiterpene lactones. Further experiments disclosed that the presence of an a-methylene group exocyclic to the 7-1actone appeared to be the immunochemical requisite for the contact allergy [7]. These results are in accordance with our findings obtained with the sesquiterpene lactones alantolactone and parthenolide in sensitized patients and sensitized guinea pigs [2]. MATERIAL AND METHODS Melting points were taken on a Kofler aparatus equipped with an electrical unit "TC 400" (Reichert, Austria) and are uncorrected. Thin-layer chromatography (t.l.c.) (analytical and preparative) was performed on silica gel 60 with and without fluorescence indicator F254 and F866 (Merck). Infra-red spectra were taken in KBr pellets on a Beckman Model IR 4.
Isolation of Constituents Chemical investigation of the raw extract of Chrysanthemum flowers (benzene, chloroform, ethanol extract) by analytical thin-layer chromatography (t.l.c.) showed that positive color reactions could be obtained by 6 different lactone reagents. Out of these reagents spraying with H~SO4--H20 (1 +1) and heating of the plates for 2 min at 200 ° C indicated to be the best indicator for sesquiterpene lactones: they appeared as red, green, blue, and graygreen spots [8--10]. Two spots of the separated extracts also gave color reactions with a special chlorine reagent (diphenylamine-zincchloride, heating 5 min at 200 ° C--red spots) [8]. After these preliminary studies 3280 g powdered dried flowers of Chrysanthemum indieum L. were percolated to exhaustion at room temperature with benzene (50--70 ° C). The residue left after evaporation of the solvent at reduced pressure (30 ° C) was separated into more than 100 portions. Each portion was partitioned between 650 ml benzene (50/70) (upper fraction) and five 130ml portions of 107o aqueous methanol (this fraction was named: s-fraction) [11 ]. The s-fraction, left after evaporation of the solvent, was dissolved in ether (for storage) and used for further chromatographic studies and for sensitization of guinea pigs.
Sensitization Experiments in Guinea Pigs 0.05 ml of a 10 70 solution of the s-fraction (in acetone) was applied daily for 1 week to the clipped and shaved skin of guinea pigs of the Pir-bright-white strain. Challenge of
Chrysanthemum Allergy
113
the allergic reactions was performed with the same solution 8--14 days later, together with controls on untreated animals. As the animals were used more than 1 year for testing of the isolated fractions they had to be boostered 3 times in intervals of 4 months. This was done by three epicutaneous applications of 0.05 ml of the s-fraction (10 ~ in acetone).
Preparative Thin-Layer Chromatography (Prep.t.Le.) Separation of the s-fraction using twodimensional preparative t.l.c, on silica gel was carried out in the following manner: one drop of the s-fraction was applied in one corner of the plate (0.75 mm thick) and chromatographed 3 times in chloroform. After turning the plate by 90° it was run twice in cyclohexane-ethyl acetate (8+2) and once in cyclohexane-ethyl acetate (7 + 3). Every day 20 plates were able to be prepared. One of the plates was sprayed with H2SO4--H20 (1 +1) and heated for 2 min to give typically colored spots. The spots were drawn on transparent paper which then was used as a pattern for marking off the area on the untreated plates. The silica gel material in this area was then scraped off and eluted with ether and acetone.
RESULTS After repeated prep.t.l.c. 12 fractions were obtained in amounts large enough for skin testing in the sensitized guinea pigs. Fig. 1 shows a typical chromatogram of the s-fraction after chromatography in the above mentioned different solvent systems. The results of the skin tests are summarized in Table 1. As can be seen from Tab. 1 fractions IV--VIII produced strong skin responses in the sensitized animals. The lactone compounds of the fractions I - - X I I occurring in the flowers could be detected by prep.t.l.c, in the leaves and stems of C. indicum L. too. Skin tests with the fractions of these green parts of the plant gave the same results as with those of the flowers. Besides the chemical studies epicutaneous tests with different sesquiterpene lactones and other compounds isolated from other Chrysanthemum species or plants of the Compositae family were performed on the sensitized guinea pigs. The results are summarized in Table 2. Four of the seven tested compounds produced allergic contact reactions; parthenolide gave strong reactions, whereas alantolactone, frullania-lactone and chrysartemin-B produced weaker ones. Frullania-lactone and alantolactone have not been found in Chrysanthemum species. Further separation of fractions IV--VIII each yielded amounts of at least 20 rag. To remove other impurities repeated chromatography of these fractions in benzene-acetone (100+5) and cyclohexane-ethyl acetate (8 +2) was carried out. Finally fraction V and fraction VI yielded colorless needles after crystallization from n-hexane-ethyl acetate (V) and acetone (VI) respectively. 8*
114
B.M. Hausen and K. H. Schulz
'5
c~
t'-
,J.., cO x
¢z)
r-'
o
>.,
(,p
°C)
0Oo
,0 0 o
o
,:t.
% ~ 0
Cht0r0f0rm
•r
(3x)
~Start
Fig. 1. Thin-layer chromatogram of the s-fraction after repeated twodimensional chromatography in different solvent systems; treatment with H~SO4--H20 (1 +1) and heating to 200° C for 2min: (All non-hatched areas are visible under UV, 366 nm and 254nm) ~1~ orange; [ ~ red; l ~ greygreen; ~ green; [] redbrown; ~ yellow; blue Table 1. Results of skin tests with different fractions of Chrysanthemum flowers separated by preparative thin-layer chromatography. 5 guinea pigs sensitized with the raw extract (s-fraction) were tested by application of 0.05 ml of each fraction (10 ~o in acetone) Fraction I II III IV V VI VII
24 h
48 h
72 h
96 h
(+) (+)
(+) (+)
o (+)
o o
¢
¢
o
o
++ +++ ++ ++
++ ++ ++ ++
+ ++ ++ ++
o ++ + +
++
+
viii
++
IX X
+
(+)
++
(+)
o
¢
¢
¢
o
XI
¢
¢
o
¢
Xll
¢
¢
¢
o
Chrysanthemum Allergy
115
Table 2. Results of epicutaneous tests with sesquiterpene lactones and other compounds of compositae species (in acetone) in 5 guinea pigs sensitized with Chrysanthemum indieum L. raw extract Compound (sesquiterpene lactone type)
Formula
Occurrence
Test Skin concen- reaction tration 24h 48h
Chrysartemin B (guaianolide)
~~.1J ~ 3 H0.-L-...~ / UH3)-----(
Chrys. parthenium (L.) Bernh. [12] Chrys. morifolium Ram. [13]
1
O~=CH2 0 H0 CIq~ ~
Reynosin (eudesmanolide)
Numbing principle (no sesquiterpene lactone)
(+) q-
Chrys. vulgare (L.) 1 Bernh. [36] Ambrosia eonferti~,, ~- ,)=CH2 floraDC. [14] n2 0~, 0Chrys.parthenium (L.) Bernh. [3]
neg. neg.
Chrys. anethifolium 1 Brouss. [15]
neg. neg.
-~
R
/CH3 R: CH2-CHzCHCHzCHCNHCH2-CH 0 \CH3 CH3
Frullania-lactone (eudesmanolide)
~ ~
Frullania tamarisci 1 (L.) Dum. [16]
+
+
Inulahelenium(L.) 0.1 [17,18]
+
-F
q--k -k-k ++ ++
=CH2
Chrys.parthenium 0.1 (L.) Bernh. [19] 0.05 Micheliachampaea L. (Magnoliceae) [20]
0 =0
Chrys.cinerariaefolium Vis. [21]
neg. neg.
_ CH2
Alantolactone (helenin) a (eudesmanolide)
0
CH3
=0
CH2
CH3 Parthenolide (germacranolide)
Pyrethrosin (germacranolide)
I!
2 a Sigma Chem. St. Louis Miss. U.S.A.
1
116
B. M. Hausen and K. H. Schulz 0(5)
C(17)
c(2)
CO)
Fig. 2. ORTEP drawing of compound V derived from Chrysanthemum indicum L. [33] The spectroscopic data of compound V were: m.p. 203--204.5° C (uncorr.); yield: 8.5 rag; i.r. v,~ax 1760 cm -1 (?-lactone), 1740, 1240 cm -1 (~,/3-unsaturated ketone). 4 mg of compound V were used for skin tests in 3 sensitized guinea pigs; they gave strong allergic contact reactions in the 24 and 48 h readings. Since deduction of the molecular structure was not possible because of the low yield, an X-ray crystallographic analysis was attempted for compound V and VI in the Institute of Mineralogy and Petrography at the University of Hamburg by Drs. Jarchow, Klaska and Schmalle. Recrystallization of compound V from n-hexane-ethyl acetate yielded well formed crystals of which one was investigated. The configuration of the structure obtained by this analysis was that of a sesquiterpene lactone of the guaianolide type equipped with an epoxy group (Figs. 2 and 3). This compound is similar to yeyuhua-lactone found in Chrysanthemum indicum L. by Chien et al. [22] in 1963. It differs only by lacking the hydroxy- and acetate group (Fig.4). The analytical data of compound V were calculated for CtTHe0Os. Particular details of the X-ray analysis are to be published in Acta crystallographica1. In 1971 Lee, Matsueda and Geissman [23] isolated a sesquiterpene lactone from Artemisia douglasiana Bess. (tribe Anthemidae, family Compositae) which 1 Jarchow, O., Klaska, K.H., and Schmalle, H.: Acta crystallographica, in preparation.
Chrysanthemum Allergy Fig. 3. Structure of Arteglasin-A = compound V Fig. 4. Yeyuhua-lactone from C. indicumL. (Structure not completed) [22]
117 C H ~
14-Z, CH3
0V ) =CH 2 I
I
0 Fig. 3
.CH3
~,
-OH
°/CH3~=CH 2 II 0
c-bridge
Fig. 4
they named Arteglasin-A. Comparison of all physical and chemical data led to the conclusion that Arteglasin-A from A. douglasiana Bess. and compound V from C. indicum L. must be identical. The isolation and identification of the sesquiterpene lactone Arteglasin-A as an allergen of Chrysanthemum indicum L. was thus shown for the first time.
DISCUSSION The interesting group of sesquiterpene lactones to which the above described allergen belongs, was rediscovered in 1960 during search for tumor-inhibiting substances from natural sources [24, 25]. Although sesquiterpene lactones seem to be characteristic constituents of Compositae, their occurrence in other plant families has come to light in the last few years, especially in the families of Magnoliaceae and Umbelliferae [26, 27]. Of the different tribes of the Compositae the Anthemidae with more than 45 species, including Chrysanthemums, contain sesquiterpene lactones of the following three types: santanolides, germacranolides and guaianolides [3,28] (Fig.5). Today there is general acceptance of the view that these sesquiterpenoid compounds are the end product of a metabolic pathway which starts from farnesol [3,23, 28, 29]. In this context it is interesting to see that some plants of the Compositae family contain different sesquiterpene lactones in different seasons. Lee, Matsueda and Geissman [23] have shown that Artemisia douglasiana Bess. examined in early spring only contains compounds of the santanolide type, whereas a study of the fall plant in the flowering stage revealed that only constituents with the characteristic of guaianolides could be found. Furthermore it seems to be possible that sesquiterpene lactones of the germacranolide type may be transformed into those of the guaianolide type. By catalytic reduction of parthenolide (a germacranolide isolated from Chrysanthemum parthenium (L). Bernh.) Matsueda [30] succeeded in obtaining a guaianolide (Fig. 6). Arteglasin-A ( = compound V) belongs to the sesquiterpene lactones of the guaianolide type bearing an epoxy group. At least 16 similar constituents (including compounds with two epoxy groups) have been found in species of the
118
B.M. Hausen and K. H. Schulz
0 eudesmanolide (santanolide) germacranolide guaianolide Fig. 5. Sesquiterpene lactone types of the tribus Anthemidae
CH3 0 Reduct o
CTH3~0,_~,=CH2 b
CH3
~r
HO CH3
CH 3
0
-OAe CH3
parthenolide (germacranolide) ClsH~08 (guaianolide) 0 Fig. 6 Fig. 7 Fig. 6. Catalytic reduction of parthenolide to a guaianolide (C15H22Oz) Fig. 7. Matricarin from Matricaria chamomilla L. tribe Anthemidae up to now [3, 28, 29]. As Arteglasin-A is very similar to yeyuhualactone (found in C. indicum L. in 1963) it may be expected that compounds of this type would be more widely distributed in Chrysanthemum species. Our findings with Arteglasin-A in sensitized guinea pigs demonstrate once more that some sesquiterpene lactones also possess a strong sensitizing capacity. The identification of compound V as an active allergen of C. indicum L. gives further evidence for the theory of Mitchell et al. [6,7] that the presence of an 0¢-methylene-~-lactone group appears to be an essential requisite for the allergenic activity of sesquiterpene lactones. C. indicum L. contains perhaps one or two chlorinated sesquiterpene lactones, as found in 1973 by Osawa et al. [35] in Chrysanthemum morifolium Ram. (chlorochrymorin). Our chemical studies disclosed two compounds in the s-fraction, which developed color reactions on t.l.c, after treatment with a special chlorine reagent. It is not yet clear whether these constituents are sensitizers. The occurrence of Arteglasin-A in C. indicum L. and Artemisia douglasiana Bess. provides an explanation for the observation of several authors that Artemisia and Chrysanthemum, as well as Achillea (e.g. Achillea millefolium L.--yarrow) and Matriearia species (e.g. Matricaria chamomilla L--camomile) are closely related as regards contact allergy. There exist several case reports on cross reactions to Matricaria in patients suffering from Chrysanthemum dermatitis and vice versa [1,31]. Some of the sesquiterpene lactones derived from these four species belong to the same group of guaianolide compounds [3,28,29]. However, all sesquiterpene lactones isolated so far from Matricaria chamomilla L. (matricin, matricarin, desacetylmatricarin) (Fig.7) possess no o~-methylene group [3,29]; therefore our findings of positive skin response to the s-fraction of camomile in
Chrysanthemum Allergy
119
guinea pigs sensitized with Chrysanthemum extract may indicate that there must be another sesquiterpenoid compound which bears an a-methylene group at the lactone ring. A further attempt to interpret the observation of cross reactions in chrysanthemum-sensitized guinea pigs to some sesquiterpene lactones is the experimentally obtained isomerization of parthenolide to a guaianolide-skeleton, as postulated by Matsueda [30], (Fig. 6). In a previous publication [2] we have shown skin tests with parthenolide and alantolactone in guinea pigs sensitized with a raw extract of C. indicum L. to be positive. Parthenolide produced stronger reactions than alantolactone. As parthenolide may be considered as a precursor of compounds belonging to the guaianolide type, these findings may serve as evidence for the near biogenetic and allergenic relationship of these sesquiterpene lactones. This interpretation may also be valid for alantolactone, as this sesquiterpene lactone of the eudesmanolide type is formed as an intermediate product in the biosynthesis of guaianolides from the primary precursor farnesol.
Acknowledgement. This study was supported by grant from Deutsche Forschungsgemeinschaft. The authors are grateful to Mrs. Lawatscheck for her technical assistance. They are also grateful to Dr. Dalchow (Stiptitz & Co.) and to Veiling (flower market) Hamburg for providing great amounts of Chrysanthemum flowers. For a supply of different sesquiterpene lactones and other compounds the authors wish to thank Dr. Romo de Vivar, Mexico (chrysartemin-B); Dr. Yoshioka, Japan (reynosin); Dr. Doskotch, Columbus, Ohio (numbing factor); Dr. Muller, France (frullanialactone); Dr. Geissmann, Los Angeles (parthenolide) and Dr. Herz, Tallahassee, Florida (pyrethrosin).
REFERENCES 1. Hausen, B.M., Schulz, K. H. : Chrysanthemen-Allergie 1. Mitteilung. BerufsDermatosen 21, 199--214 (1973) 2. Schulz, K. H., Hausen, B. M., Wallh6fer, L., Schrnidt-L6ffler, P. : ChrysanthemenAllergic. 2. Experimentelle Untersuchungen zur Identifizierung der Allergene. Arch. Derm. Forsch. 251,235--244 (1975) 3. Yoshioka, H., Mabry, T.J., Timmermann, B.N.: Sesquiterpene lactones. University of Tokyo Press, 1973 4. Herz, W. : Pseudoguaianolides in Compositae. Nobel Symp. 25, 153--172 (1974) 5. Romo, J., Romo de Vivar, A., Ortega, A. : Novel sesquiterpenes isolated in compositae. Recent Advanc. Phytochem. 6, 21 --29 (1973) 6. Mitchell, J. C. : Allergic contact dermatitis from compositae. Trans. St John's Hosp. derm. Soc. (Lond.) 55, 174--183 (1969) 7. Mitchell, J. C., Fritig, B., Singh, B., Towers, H. N. : Allergic contact dermatitis from frullania and compositae. J. invest. Derm. 54, 233--239 (1970) 8. Stahl, E.: Dtinnschichtchromatographie. Berlin-Heidelberg-New York: Springer 1967 9. Matsueda, S. : Studies on sesquiterpene lactones. II. Chemical constitution of young growth of Artemisia sp. Yakugaku Zasshi 90, 1024-- 1027 (1970) 10. Geissman, T.A., Griffin, T. S. : Sesquiterpene lactones: acid-catalysed color reactions as an aid in structure determination. Phytochem. 10, 2475--2485 (1971)
120
B.M. Hausen and K. H. Schulz
11. Doskotch, R. W., El-Feraly, T. S.: Isolation and characterization of (q-)-sesamin and /%cyclopyrethrosin from pyrethrum flowers. Can. J. Chem. 47, 1139--1142 (1969) 12. Romo, J., Romo de Vivar, A., Trevino, R., Joseph-Nathan, P., Diaz, E. : Constituents of artemisia and chrysanthemum species--the structure of chrysartemins A and B. Phytochem. 9, 1615--1621 (1970) 13. Osawa, T., Suzuki, A., Tamura, S. : Isolation of chrysartemins A and B as rooting cofactors. Agr. Biol. Chem. 35. 1966--1972 (1971) 14. Yoshioka, H., Renold, W., Fischer, N. H., Higo, A., Mabry, T. J. : Sesquiterpene lactones from Ambrosia confertiflora D.C. Phytochem. 9, 823--832 (1970) 15. Doskotch, R.W., Beal, J. L.: The isolation and identification of the numbing principle in Chrysanthemum anethifolium. Lloydia 33, 393--394 (1970) 16. Knoche, H., Ourisson, G., Perold, G. W., Foussereau, J., Maleville, J. : Allergenic component of a liverwort: a sesquiterpene lactone. Science 166, 239--240 (1969) 17. Tsuda, K., Tanabe, T., Iwai, I., Funakoshi, K. : The structure of alantolactone. J. Amer. Chem. Soc. 79, 5721--5724 (1957) 18. Marshall, J.A., Cohen, N.: The structure of alantolactone. J. Org. Chem. 29, 3727-- 3729 (1964) 19. Soucek, M., Herout, V., Sorm, F.: On terpenes. CXVIII. Constitution of parthenolide. Coll. Czech. Chem. Comm. 26, 803--809 (1961) 20. Govindachari, T. R., Joshi, B. S., Kamat, V. N. : Revised structure of parthenolide. Tetrahedron Lett., 3927--3933 (1964) Heft 52 21. Barton, D. H. R., Mayo, P. de: Sesquiterpenoids. VIII. The constitution of pyrethrosin. J. chem. Soc. 150--158 (1957) 22. Chien, M.-K., Cheng, Z.-X., Tseng, K.-F. : Studies on the constituents of yeyuhua, the flower of Chrysanthemum indicum L. Yao Hsueh Hsueh Pao 10, 129--134 (1963) 23. Lee, K. H., Matsueda, S., Geissman, T. A.: Sesquiterpene lactones of artemisia: new guaianolides from fall growth of A. douglasiana. Phytochem. 10, 405--410 (1971) 24. Doskotch, R. W., E1-Feraly, T. S. : Antitumor agents. II. Tulipinolide, a new germacranolide sesquiterpene, and costunolide. J. pharm. Sci. 58, 877-880 (1969) 25. Kupchan, S. M., Kelsey, J.E., Maruyama, M., Cassady, J. M., Hemingway, J. C., Knox, J. R.: Tumor inhibitors. XLI. Structural elucidation of tumor-inhibitors sesquiterpene lactones from Eupatorium rotundifolium. J. Org. Chem. 34, 3876--3883 (1969) 26. Doskotch, R.W., Hufford, C.D., E1-Feraly, T. S.: Further studies on the sesquiterpene lactones tulipinolide and epitulipinolide from Liriodendron tulipifera L. J. Org. Chem. 37, 2740--2744 (1972) 27. Govindachari, T. R., Joshi, B. S., Kamat, V. N.: Structure of parthenolide. Tetrahedron 21, 1509 -- 1519 (1965) 28. Herout, V. : Sesquiterpenische Lactone -- ein neues taxonomisches Merkmal der Compositae. Planta med. (Suppl.) 1966, pp. 97--106 29. Geissman, T.A.: The biogenesis of sesquiterpene lactones of the compositae. Recent Advanc. Phytochem. 6, 65--95 (1973) 30. Matsueda, S.: Studies on sesquiterpenelactones. VI. Guaiane type sesquiterpenelactone formation from germacranolide by chemical procedure. Sci. Rep. Hirosaki Uni. 18, 8--14(1971) 31. Leipold, H.: Dermatitis der Hfinde und des Gesichts infolge Oberempfindlichkeit gegen Chrysanthemen und Kamillen. Zbl. Haut- u. Geschl.-Kr. 57, 248 (1938) 32. Mitchell, J. C., Geissman, T. A., Dupuis, G., Towers, G. H. N. : Allergic contact dermatitis caused by Artemisia and Chrysanthemum species. J. invest. Derm. 56, 98--101 (1971)
Chrysanthemum Allergy
121
33. Johnson, C. K. : ORTEP, OR NL-3794 Revised Oak Ridge National Laboratory, Oak Ridge, Tennessee, 1965 34. Hausen, B. M., Schulz, K. H., Jarchow, O., Klaska, K. H., Schmalle, H.: A first allergenic sesquiterpene lactone from Chrysanthemum indicum L.: Arteglasin-A. Naturwissenschaften 62, 585 (1975) 35. Osawa, T., Suzuki, A., Tamura, S., Ohashi, Y., Sasada, Y.: Structure of chlorochrymorin, a novel sesquiterpene lactone from Chrysanthemum morifolium. Tetrahedron Lett., 5135--5138 (1973) Heft 51 36. Samek, Z., Holub, M., Grabarczyk, H., Drozdz, B., Herout, V. : On terpenes. CCXXIX. Structure of sesquiterpene lactones from Tanaeetum vulgareL. Coll. Czech. Chem. Comm. 38, 1971 --1976 (1973)
Received January 13, 1976 Dr. B. M. Hausen, Prof. Dr. K. H. Schulz Department of Dermatology University Hospital Martinistr. 52 D-2000 Hamburg 20 Federal Republic of Germany
Archives for
Dermatological Kesearcn © by Springer-Verlag 1976
ChrysanthemumAllergy III. Identification of the allergens * ** B. M. Hausen and K. H. Schulz Department of Dermatology, Division of Allergology, University Hospital, Hamburg (Federal Republic of Germany)
Summary. Experimental and chemical studies in guinea pigs on contact allergy caused by Chrysanthemum indicum L. (Chrysanthemum of the florists) led to the isolation of several sesquiterpene lactones. From the crude extract of dried flowers 12 fractions were obtained of which four gave strong reactions on epicutaneous application to guinea pigs sensitized with an extract of C. indicum L. One of these allergens was identified as a sesquiterpene lactone of the guaianolide type. It is identical to Arteglasin-A derived from Artemisia douglasiana Bess. The occurrence of Arteglasin-A in Chrysanthemum indieum L. as well as its allergenic properties are described for the first time. These findings give rise to the assumption that the other, as yet unidentified sesquiterpene lactones of this flower also belong to the guaianolide type.
Zusammenfassung. Chemische Untersuchungen und experimentelle Teste an Meerschweinchen bezfiglich der dutch Chrysanthemen verursachten Kontaktallergie fiihrten zur Isolierung mehrerer Sesquiterpenlaktone. Aus dem Rohextrakt getrockneter Chrysanthemen wurden 12 Fraktionen isoliert, yon denen vier im Epikutantest bei sensibilisierten Meerschweinchen eine stark positive Reaktion ergaben. Eins der Allergene wurde als Sesquiterpenlakton vom Guaianolid-Typ identifiziert. Es ist identisch mit Arteglasin-A aus Artemisia douglasiana Bess. Sowohl das Vorkommen von Arteglasin-A in Chrysanthemum indieum L. als auch seine allergene Wirkung werden bier das erste Mal beschrieben. Die Untersuchungsbefunde lassen vermuten, dab die anderen bisher nicht identifizierten Sesquiterpenlaktone ebenfalls dem Guaianolid-Typ angeh6ren, * Part I and II of this series are references 1 and 2. ** A preliminary report of this article is reference 34. Reprint requests to: Dr. B. M. Hausen, Department of Dermatology, University Hospital Hamburg, Martinistr. 52, D-2000 Hamburg 20, Federal Republic of Germany. 8 Arch. Derm. Res,, '9"ol.255
112
B.M. Hausen and K. H. Schulz
INTRODUCTION
Chrysanthemum indicum L. (Chrysanthemum of the florists) is one of the most c o m m o n causes of occupational contact dermatitis among gardeners and florists in the northern part of West Germany. A survey of the history of Chrysanthemum culture, of the plant constituents hitherto known and of the manifestations of Chrysanthemum dermatitis is given in Part I of this series [1]. Part II deals with studies on the isolation and identification of sensitizing components [2]. In the present paper the identification and chemical characterization of one of the contact allergens of Chrysanthemum indieum L. is described. Since 1960 more than 600 compounds of sesquiterpene lactone structure have been found in the family of Compositae, to which C. indieum L. belongs [3--5]. Studies by Mitchell et al. [6,7] in 1970 revealed that patients with contact dermatitis due to Chrysanthemum flowers developed positive patch test reactions to certain sesquiterpene lactones. Further experiments disclosed that the presence of an a-methylene group exocyclic to the 7-1actone appeared to be the immunochemical requisite for the contact allergy [7]. These results are in accordance with our findings obtained with the sesquiterpene lactones alantolactone and parthenolide in sensitized patients and sensitized guinea pigs [2]. MATERIAL AND METHODS Melting points were taken on a Kofler aparatus equipped with an electrical unit "TC 400" (Reichert, Austria) and are uncorrected. Thin-layer chromatography (t.l.c.) (analytical and preparative) was performed on silica gel 60 with and without fluorescence indicator F254 and F866 (Merck). Infra-red spectra were taken in KBr pellets on a Beckman Model IR 4.
Isolation of Constituents Chemical investigation of the raw extract of Chrysanthemum flowers (benzene, chloroform, ethanol extract) by analytical thin-layer chromatography (t.l.c.) showed that positive color reactions could be obtained by 6 different lactone reagents. Out of these reagents spraying with H~SO4--H20 (1 +1) and heating of the plates for 2 min at 200 ° C indicated to be the best indicator for sesquiterpene lactones: they appeared as red, green, blue, and graygreen spots [8--10]. Two spots of the separated extracts also gave color reactions with a special chlorine reagent (diphenylamine-zincchloride, heating 5 min at 200 ° C--red spots) [8]. After these preliminary studies 3280 g powdered dried flowers of Chrysanthemum indieum L. were percolated to exhaustion at room temperature with benzene (50--70 ° C). The residue left after evaporation of the solvent at reduced pressure (30 ° C) was separated into more than 100 portions. Each portion was partitioned between 650 ml benzene (50/70) (upper fraction) and five 130ml portions of 107o aqueous methanol (this fraction was named: s-fraction) [11 ]. The s-fraction, left after evaporation of the solvent, was dissolved in ether (for storage) and used for further chromatographic studies and for sensitization of guinea pigs.
Sensitization Experiments in Guinea Pigs 0.05 ml of a 10 70 solution of the s-fraction (in acetone) was applied daily for 1 week to the clipped and shaved skin of guinea pigs of the Pir-bright-white strain. Challenge of
Chrysanthemum Allergy
113
the allergic reactions was performed with the same solution 8--14 days later, together with controls on untreated animals. As the animals were used more than 1 year for testing of the isolated fractions they had to be boostered 3 times in intervals of 4 months. This was done by three epicutaneous applications of 0.05 ml of the s-fraction (10 ~ in acetone).
Preparative Thin-Layer Chromatography (Prep.t.Le.) Separation of the s-fraction using twodimensional preparative t.l.c, on silica gel was carried out in the following manner: one drop of the s-fraction was applied in one corner of the plate (0.75 mm thick) and chromatographed 3 times in chloroform. After turning the plate by 90° it was run twice in cyclohexane-ethyl acetate (8+2) and once in cyclohexane-ethyl acetate (7 + 3). Every day 20 plates were able to be prepared. One of the plates was sprayed with H2SO4--H20 (1 +1) and heated for 2 min to give typically colored spots. The spots were drawn on transparent paper which then was used as a pattern for marking off the area on the untreated plates. The silica gel material in this area was then scraped off and eluted with ether and acetone.
RESULTS After repeated prep.t.l.c. 12 fractions were obtained in amounts large enough for skin testing in the sensitized guinea pigs. Fig. 1 shows a typical chromatogram of the s-fraction after chromatography in the above mentioned different solvent systems. The results of the skin tests are summarized in Table 1. As can be seen from Tab. 1 fractions IV--VIII produced strong skin responses in the sensitized animals. The lactone compounds of the fractions I - - X I I occurring in the flowers could be detected by prep.t.l.c, in the leaves and stems of C. indicum L. too. Skin tests with the fractions of these green parts of the plant gave the same results as with those of the flowers. Besides the chemical studies epicutaneous tests with different sesquiterpene lactones and other compounds isolated from other Chrysanthemum species or plants of the Compositae family were performed on the sensitized guinea pigs. The results are summarized in Table 2. Four of the seven tested compounds produced allergic contact reactions; parthenolide gave strong reactions, whereas alantolactone, frullania-lactone and chrysartemin-B produced weaker ones. Frullania-lactone and alantolactone have not been found in Chrysanthemum species. Further separation of fractions IV--VIII each yielded amounts of at least 20 rag. To remove other impurities repeated chromatography of these fractions in benzene-acetone (100+5) and cyclohexane-ethyl acetate (8 +2) was carried out. Finally fraction V and fraction VI yielded colorless needles after crystallization from n-hexane-ethyl acetate (V) and acetone (VI) respectively. 8*
114
B.M. Hausen and K. H. Schulz
'5
c~
t'-
,J.., cO x
¢z)
r-'
o
>.,
(,p
°C)
0Oo
,0 0 o
o
,:t.
% ~ 0
Cht0r0f0rm
•r
(3x)
~Start
Fig. 1. Thin-layer chromatogram of the s-fraction after repeated twodimensional chromatography in different solvent systems; treatment with H~SO4--H20 (1 +1) and heating to 200° C for 2min: (All non-hatched areas are visible under UV, 366 nm and 254nm) ~1~ orange; [ ~ red; l ~ greygreen; ~ green; [] redbrown; ~ yellow; blue Table 1. Results of skin tests with different fractions of Chrysanthemum flowers separated by preparative thin-layer chromatography. 5 guinea pigs sensitized with the raw extract (s-fraction) were tested by application of 0.05 ml of each fraction (10 ~o in acetone) Fraction I II III IV V VI VII
24 h
48 h
72 h
96 h
(+) (+)
(+) (+)
o (+)
o o
¢
¢
o
o
++ +++ ++ ++
++ ++ ++ ++
+ ++ ++ ++
o ++ + +
++
+
viii
++
IX X
+
(+)
++
(+)
o
¢
¢
¢
o
XI
¢
¢
o
¢
Xll
¢
¢
¢
o
Chrysanthemum Allergy
115
Table 2. Results of epicutaneous tests with sesquiterpene lactones and other compounds of compositae species (in acetone) in 5 guinea pigs sensitized with Chrysanthemum indieum L. raw extract Compound (sesquiterpene lactone type)
Formula
Occurrence
Test Skin concen- reaction tration 24h 48h
Chrysartemin B (guaianolide)
~~.1J ~ 3 H0.-L-...~ / UH3)-----(
Chrys. parthenium (L.) Bernh. [12] Chrys. morifolium Ram. [13]
1
O~=CH2 0 H0 CIq~ ~
Reynosin (eudesmanolide)
Numbing principle (no sesquiterpene lactone)
(+) q-
Chrys. vulgare (L.) 1 Bernh. [36] Ambrosia eonferti~,, ~- ,)=CH2 floraDC. [14] n2 0~, 0Chrys.parthenium (L.) Bernh. [3]
neg. neg.
Chrys. anethifolium 1 Brouss. [15]
neg. neg.
-~
R
/CH3 R: CH2-CHzCHCHzCHCNHCH2-CH 0 \CH3 CH3
Frullania-lactone (eudesmanolide)
~ ~
Frullania tamarisci 1 (L.) Dum. [16]
+
+
Inulahelenium(L.) 0.1 [17,18]
+
-F
q--k -k-k ++ ++
=CH2
Chrys.parthenium 0.1 (L.) Bernh. [19] 0.05 Micheliachampaea L. (Magnoliceae) [20]
0 =0
Chrys.cinerariaefolium Vis. [21]
neg. neg.
_ CH2
Alantolactone (helenin) a (eudesmanolide)
0
CH3
=0
CH2
CH3 Parthenolide (germacranolide)
Pyrethrosin (germacranolide)
I!
2 a Sigma Chem. St. Louis Miss. U.S.A.
1
116
B. M. Hausen and K. H. Schulz 0(5)
C(17)
c(2)
CO)
Fig. 2. ORTEP drawing of compound V derived from Chrysanthemum indicum L. [33] The spectroscopic data of compound V were: m.p. 203--204.5° C (uncorr.); yield: 8.5 rag; i.r. v,~ax 1760 cm -1 (?-lactone), 1740, 1240 cm -1 (~,/3-unsaturated ketone). 4 mg of compound V were used for skin tests in 3 sensitized guinea pigs; they gave strong allergic contact reactions in the 24 and 48 h readings. Since deduction of the molecular structure was not possible because of the low yield, an X-ray crystallographic analysis was attempted for compound V and VI in the Institute of Mineralogy and Petrography at the University of Hamburg by Drs. Jarchow, Klaska and Schmalle. Recrystallization of compound V from n-hexane-ethyl acetate yielded well formed crystals of which one was investigated. The configuration of the structure obtained by this analysis was that of a sesquiterpene lactone of the guaianolide type equipped with an epoxy group (Figs. 2 and 3). This compound is similar to yeyuhua-lactone found in Chrysanthemum indicum L. by Chien et al. [22] in 1963. It differs only by lacking the hydroxy- and acetate group (Fig.4). The analytical data of compound V were calculated for CtTHe0Os. Particular details of the X-ray analysis are to be published in Acta crystallographica1. In 1971 Lee, Matsueda and Geissman [23] isolated a sesquiterpene lactone from Artemisia douglasiana Bess. (tribe Anthemidae, family Compositae) which 1 Jarchow, O., Klaska, K.H., and Schmalle, H.: Acta crystallographica, in preparation.
Chrysanthemum Allergy Fig. 3. Structure of Arteglasin-A = compound V Fig. 4. Yeyuhua-lactone from C. indicumL. (Structure not completed) [22]
117 C H ~
14-Z, CH3
0V ) =CH 2 I
I
0 Fig. 3
.CH3
~,
-OH
°/CH3~=CH 2 II 0
c-bridge
Fig. 4
they named Arteglasin-A. Comparison of all physical and chemical data led to the conclusion that Arteglasin-A from A. douglasiana Bess. and compound V from C. indicum L. must be identical. The isolation and identification of the sesquiterpene lactone Arteglasin-A as an allergen of Chrysanthemum indicum L. was thus shown for the first time.
DISCUSSION The interesting group of sesquiterpene lactones to which the above described allergen belongs, was rediscovered in 1960 during search for tumor-inhibiting substances from natural sources [24, 25]. Although sesquiterpene lactones seem to be characteristic constituents of Compositae, their occurrence in other plant families has come to light in the last few years, especially in the families of Magnoliaceae and Umbelliferae [26, 27]. Of the different tribes of the Compositae the Anthemidae with more than 45 species, including Chrysanthemums, contain sesquiterpene lactones of the following three types: santanolides, germacranolides and guaianolides [3,28] (Fig.5). Today there is general acceptance of the view that these sesquiterpenoid compounds are the end product of a metabolic pathway which starts from farnesol [3,23, 28, 29]. In this context it is interesting to see that some plants of the Compositae family contain different sesquiterpene lactones in different seasons. Lee, Matsueda and Geissman [23] have shown that Artemisia douglasiana Bess. examined in early spring only contains compounds of the santanolide type, whereas a study of the fall plant in the flowering stage revealed that only constituents with the characteristic of guaianolides could be found. Furthermore it seems to be possible that sesquiterpene lactones of the germacranolide type may be transformed into those of the guaianolide type. By catalytic reduction of parthenolide (a germacranolide isolated from Chrysanthemum parthenium (L). Bernh.) Matsueda [30] succeeded in obtaining a guaianolide (Fig. 6). Arteglasin-A ( = compound V) belongs to the sesquiterpene lactones of the guaianolide type bearing an epoxy group. At least 16 similar constituents (including compounds with two epoxy groups) have been found in species of the
118
B.M. Hausen and K. H. Schulz
0 eudesmanolide (santanolide) germacranolide guaianolide Fig. 5. Sesquiterpene lactone types of the tribus Anthemidae
CH3 0 Reduct o
CTH3~0,_~,=CH2 b
CH3
~r
HO CH3
CH 3
0
-OAe CH3
parthenolide (germacranolide) ClsH~08 (guaianolide) 0 Fig. 6 Fig. 7 Fig. 6. Catalytic reduction of parthenolide to a guaianolide (C15H22Oz) Fig. 7. Matricarin from Matricaria chamomilla L. tribe Anthemidae up to now [3, 28, 29]. As Arteglasin-A is very similar to yeyuhualactone (found in C. indicum L. in 1963) it may be expected that compounds of this type would be more widely distributed in Chrysanthemum species. Our findings with Arteglasin-A in sensitized guinea pigs demonstrate once more that some sesquiterpene lactones also possess a strong sensitizing capacity. The identification of compound V as an active allergen of C. indicum L. gives further evidence for the theory of Mitchell et al. [6,7] that the presence of an 0¢-methylene-~-lactone group appears to be an essential requisite for the allergenic activity of sesquiterpene lactones. C. indicum L. contains perhaps one or two chlorinated sesquiterpene lactones, as found in 1973 by Osawa et al. [35] in Chrysanthemum morifolium Ram. (chlorochrymorin). Our chemical studies disclosed two compounds in the s-fraction, which developed color reactions on t.l.c, after treatment with a special chlorine reagent. It is not yet clear whether these constituents are sensitizers. The occurrence of Arteglasin-A in C. indicum L. and Artemisia douglasiana Bess. provides an explanation for the observation of several authors that Artemisia and Chrysanthemum, as well as Achillea (e.g. Achillea millefolium L.--yarrow) and Matriearia species (e.g. Matricaria chamomilla L--camomile) are closely related as regards contact allergy. There exist several case reports on cross reactions to Matricaria in patients suffering from Chrysanthemum dermatitis and vice versa [1,31]. Some of the sesquiterpene lactones derived from these four species belong to the same group of guaianolide compounds [3,28,29]. However, all sesquiterpene lactones isolated so far from Matricaria chamomilla L. (matricin, matricarin, desacetylmatricarin) (Fig.7) possess no o~-methylene group [3,29]; therefore our findings of positive skin response to the s-fraction of camomile in
Chrysanthemum Allergy
119
guinea pigs sensitized with Chrysanthemum extract may indicate that there must be another sesquiterpenoid compound which bears an a-methylene group at the lactone ring. A further attempt to interpret the observation of cross reactions in chrysanthemum-sensitized guinea pigs to some sesquiterpene lactones is the experimentally obtained isomerization of parthenolide to a guaianolide-skeleton, as postulated by Matsueda [30], (Fig. 6). In a previous publication [2] we have shown skin tests with parthenolide and alantolactone in guinea pigs sensitized with a raw extract of C. indicum L. to be positive. Parthenolide produced stronger reactions than alantolactone. As parthenolide may be considered as a precursor of compounds belonging to the guaianolide type, these findings may serve as evidence for the near biogenetic and allergenic relationship of these sesquiterpene lactones. This interpretation may also be valid for alantolactone, as this sesquiterpene lactone of the eudesmanolide type is formed as an intermediate product in the biosynthesis of guaianolides from the primary precursor farnesol.
Acknowledgement. This study was supported by grant from Deutsche Forschungsgemeinschaft. The authors are grateful to Mrs. Lawatscheck for her technical assistance. They are also grateful to Dr. Dalchow (Stiptitz & Co.) and to Veiling (flower market) Hamburg for providing great amounts of Chrysanthemum flowers. For a supply of different sesquiterpene lactones and other compounds the authors wish to thank Dr. Romo de Vivar, Mexico (chrysartemin-B); Dr. Yoshioka, Japan (reynosin); Dr. Doskotch, Columbus, Ohio (numbing factor); Dr. Muller, France (frullanialactone); Dr. Geissmann, Los Angeles (parthenolide) and Dr. Herz, Tallahassee, Florida (pyrethrosin).
REFERENCES 1. Hausen, B.M., Schulz, K. H. : Chrysanthemen-Allergie 1. Mitteilung. BerufsDermatosen 21, 199--214 (1973) 2. Schulz, K. H., Hausen, B. M., Wallh6fer, L., Schrnidt-L6ffler, P. : ChrysanthemenAllergic. 2. Experimentelle Untersuchungen zur Identifizierung der Allergene. Arch. Derm. Forsch. 251,235--244 (1975) 3. Yoshioka, H., Mabry, T.J., Timmermann, B.N.: Sesquiterpene lactones. University of Tokyo Press, 1973 4. Herz, W. : Pseudoguaianolides in Compositae. Nobel Symp. 25, 153--172 (1974) 5. Romo, J., Romo de Vivar, A., Ortega, A. : Novel sesquiterpenes isolated in compositae. Recent Advanc. Phytochem. 6, 21 --29 (1973) 6. Mitchell, J. C. : Allergic contact dermatitis from compositae. Trans. St John's Hosp. derm. Soc. (Lond.) 55, 174--183 (1969) 7. Mitchell, J. C., Fritig, B., Singh, B., Towers, H. N. : Allergic contact dermatitis from frullania and compositae. J. invest. Derm. 54, 233--239 (1970) 8. Stahl, E.: Dtinnschichtchromatographie. Berlin-Heidelberg-New York: Springer 1967 9. Matsueda, S. : Studies on sesquiterpene lactones. II. Chemical constitution of young growth of Artemisia sp. Yakugaku Zasshi 90, 1024-- 1027 (1970) 10. Geissman, T.A., Griffin, T. S. : Sesquiterpene lactones: acid-catalysed color reactions as an aid in structure determination. Phytochem. 10, 2475--2485 (1971)
120
B.M. Hausen and K. H. Schulz
11. Doskotch, R. W., El-Feraly, T. S.: Isolation and characterization of (q-)-sesamin and /%cyclopyrethrosin from pyrethrum flowers. Can. J. Chem. 47, 1139--1142 (1969) 12. Romo, J., Romo de Vivar, A., Trevino, R., Joseph-Nathan, P., Diaz, E. : Constituents of artemisia and chrysanthemum species--the structure of chrysartemins A and B. Phytochem. 9, 1615--1621 (1970) 13. Osawa, T., Suzuki, A., Tamura, S. : Isolation of chrysartemins A and B as rooting cofactors. Agr. Biol. Chem. 35. 1966--1972 (1971) 14. Yoshioka, H., Renold, W., Fischer, N. H., Higo, A., Mabry, T. J. : Sesquiterpene lactones from Ambrosia confertiflora D.C. Phytochem. 9, 823--832 (1970) 15. Doskotch, R.W., Beal, J. L.: The isolation and identification of the numbing principle in Chrysanthemum anethifolium. Lloydia 33, 393--394 (1970) 16. Knoche, H., Ourisson, G., Perold, G. W., Foussereau, J., Maleville, J. : Allergenic component of a liverwort: a sesquiterpene lactone. Science 166, 239--240 (1969) 17. Tsuda, K., Tanabe, T., Iwai, I., Funakoshi, K. : The structure of alantolactone. J. Amer. Chem. Soc. 79, 5721--5724 (1957) 18. Marshall, J.A., Cohen, N.: The structure of alantolactone. J. Org. Chem. 29, 3727-- 3729 (1964) 19. Soucek, M., Herout, V., Sorm, F.: On terpenes. CXVIII. Constitution of parthenolide. Coll. Czech. Chem. Comm. 26, 803--809 (1961) 20. Govindachari, T. R., Joshi, B. S., Kamat, V. N. : Revised structure of parthenolide. Tetrahedron Lett., 3927--3933 (1964) Heft 52 21. Barton, D. H. R., Mayo, P. de: Sesquiterpenoids. VIII. The constitution of pyrethrosin. J. chem. Soc. 150--158 (1957) 22. Chien, M.-K., Cheng, Z.-X., Tseng, K.-F. : Studies on the constituents of yeyuhua, the flower of Chrysanthemum indicum L. Yao Hsueh Hsueh Pao 10, 129--134 (1963) 23. Lee, K. H., Matsueda, S., Geissman, T. A.: Sesquiterpene lactones of artemisia: new guaianolides from fall growth of A. douglasiana. Phytochem. 10, 405--410 (1971) 24. Doskotch, R. W., E1-Feraly, T. S. : Antitumor agents. II. Tulipinolide, a new germacranolide sesquiterpene, and costunolide. J. pharm. Sci. 58, 877-880 (1969) 25. Kupchan, S. M., Kelsey, J.E., Maruyama, M., Cassady, J. M., Hemingway, J. C., Knox, J. R.: Tumor inhibitors. XLI. Structural elucidation of tumor-inhibitors sesquiterpene lactones from Eupatorium rotundifolium. J. Org. Chem. 34, 3876--3883 (1969) 26. Doskotch, R.W., Hufford, C.D., E1-Feraly, T. S.: Further studies on the sesquiterpene lactones tulipinolide and epitulipinolide from Liriodendron tulipifera L. J. Org. Chem. 37, 2740--2744 (1972) 27. Govindachari, T. R., Joshi, B. S., Kamat, V. N.: Structure of parthenolide. Tetrahedron 21, 1509 -- 1519 (1965) 28. Herout, V. : Sesquiterpenische Lactone -- ein neues taxonomisches Merkmal der Compositae. Planta med. (Suppl.) 1966, pp. 97--106 29. Geissman, T.A.: The biogenesis of sesquiterpene lactones of the compositae. Recent Advanc. Phytochem. 6, 65--95 (1973) 30. Matsueda, S.: Studies on sesquiterpenelactones. VI. Guaiane type sesquiterpenelactone formation from germacranolide by chemical procedure. Sci. Rep. Hirosaki Uni. 18, 8--14(1971) 31. Leipold, H.: Dermatitis der Hfinde und des Gesichts infolge Oberempfindlichkeit gegen Chrysanthemen und Kamillen. Zbl. Haut- u. Geschl.-Kr. 57, 248 (1938) 32. Mitchell, J. C., Geissman, T. A., Dupuis, G., Towers, G. H. N. : Allergic contact dermatitis caused by Artemisia and Chrysanthemum species. J. invest. Derm. 56, 98--101 (1971)
Chrysanthemum Allergy
121
33. Johnson, C. K. : ORTEP, OR NL-3794 Revised Oak Ridge National Laboratory, Oak Ridge, Tennessee, 1965 34. Hausen, B. M., Schulz, K. H., Jarchow, O., Klaska, K. H., Schmalle, H.: A first allergenic sesquiterpene lactone from Chrysanthemum indicum L.: Arteglasin-A. Naturwissenschaften 62, 585 (1975) 35. Osawa, T., Suzuki, A., Tamura, S., Ohashi, Y., Sasada, Y.: Structure of chlorochrymorin, a novel sesquiterpene lactone from Chrysanthemum morifolium. Tetrahedron Lett., 5135--5138 (1973) Heft 51 36. Samek, Z., Holub, M., Grabarczyk, H., Drozdz, B., Herout, V. : On terpenes. CCXXIX. Structure of sesquiterpene lactones from Tanaeetum vulgareL. Coll. Czech. Chem. Comm. 38, 1971 --1976 (1973)
Received January 13, 1976 Dr. B. M. Hausen, Prof. Dr. K. H. Schulz Department of Dermatology University Hospital Martinistr. 52 D-2000 Hamburg 20 Federal Republic of Germany
