Chem.-BioL Interactions, 28 (1979) 83--89 © Elsevier/North-Holland Scientific Publishers Ltd.
83
FORMATION OF ADDUCTS OF PARTHENIN AND RELATED SESQUITERPENE LACTONES WITH CYSTEINE AND GLUTATHIONE
ANNA K. PICMANa, ELOY RODRIGUEZ b and G.H.N. TOWERSa aDepartment of Botany, University of British Columbia, Vancouver, B.C. V6T 1 W5 (Canada) and bDepartment of Ecology and Evolutionary Biology, University of California, Irvine, CA (U.S.A.) (Received May 18th, 1978) (Revision received June 4th, 1979) (Accepted June 19th , 1979)
SUMMARY
Parthenin, the major sesquiterpene lactone of Parthenium hysterophorus, a weed responsible for dermatitis in man is primarily restricted to leaf and stem trichomes. Parthenin forms a m o n o a d d u c t with L-cysteine through the a-methylene group of the 7-1actone and a biadduct with the endocyclic double bond on the cyclopentenone ring. Studies with other sesquiterpene lactones support the view that the types of adducts formed are correlated with the biological activity of the sesquiterpene lactones. INTRODUCTION Parthenium hysterophorus L., a widespread composite of the Southern U.S.A., the Caribbean, Australia and parts of South America, has become a serious agricultural pest as well as a medical hazard in India where it is the cause of an epidemic of allergic contact dermatitis [1]. It is generally recognized that partbenin, the major sesquiterpene lactone of this species, is responsible for the dermatological reaction associated with this plant [1]. Sesquiterpene lactones, characteristic of the Compositae, are an important group of terpenoids which exhibit cytotoxic, antineoplastic, allergenic and other biological activities [2]. These compounds, by Michael-type addition, form adducts with amino acids containing SH groups such as cysteine [3,4] or with lysyl or histidyl residues [5]. It has been suggested that the conjugated a-methylene~/-lactone moiety of sesquiterpene lactones is responsible for cytotoxicity [6,7] as well as for allergenicity [8,9]. A structural requirem e n t for significant cytotoxic activity is the O=C--C--CH2 moiety which could be part of an ester, ketone or lactone [10]. Results of tests for aUergenicity with different sesquiterpene lactones indicated differences in response between patients [11]. The fact that
84
A
B
b H
a I HS--C½--#--COO+NH3
C
.~.~
D HOOC
°'-c°/ I s - ~
NH2
COOH
~
'-- N H2
coon Fig. 1. Parthenin, cysteine and their adducts (A-parthenin, B-cysteine, C,monoadduct, D-biadduct).
parthenin (Fig. 1A) possesses two potentially active sites (an exocyclic methylene C13 and an C2 C3 endocyclic double bond allylic to C4 carbonyl group) at which Michael-type additions could occur, might explain toxic effects on cattle and the potent allergenicity of this compound [1,12]. We have examined the reaction of parthenin and of several closely related sesquiterpene lactones w i t h cysteine and:g!utathione in order to determine whether more than one type of adduct is formed. We also have reexamined the distribution of parthenin in aerial parts of P. hysterophorus. MATERIALS AND METHODS
Plant material. P. hysterophorus plants were grown from achenes obtained from Austin, TX, U.S.A. Achenes germinated in wet sand In 1--3 weeks and when leaves had formed, seedlings were transfered to pots and maintained at 30°C during the day and 20°C at night. Light period was 16 h. Detection o f parthenin in plant material. Parthenin was isolated from Parthenium hysterophorus by the method of Rodriguez [12]. Plant material (achenes or seedlings) were extracted with chloroform for 24 hrs and the presence of parthenin checked by thin-layer chromatography (TLC) with a known sample (Eastman Si gel plates with benzene--acetone, 1 : 4, fumed with iodine vapors, Rf = 0.57). Crushed or intact plant material gave identical results. Detection o f parthenin in trichomes and stems o f P. hysterophorus. From
85 ten fresh stems 10 mg of trichomes were removed with a spatula and transfered to distilled methanol (10 ml). The stems, with all trichomes removed (1 g), were cut into small pieces and extracted with distilled methanol (25 ml) overnight. Both extracts were concentrated to minimum volumes and checked by TLC for presence of parthenin. TLC of sesquiterpene lactone-thiol adducts. Eastman cellulose plates, without fluorescent indicator, and the upper phase of n-butanol/acetic acid/ water (4 : 1 : 5) were used. Plates were sprayed with 0.25% ninhydrin (acetone), heated for several minutes at 80°C and then exposed to iodine vapors to detect sesquiterpene lactones w~hich give brown spots. The monoand biadducts of parthenin give violet colored ninhydrin spots with Rf = 0.55 and Rf = 0.19, respectively. (Rf cysteine = 0.36, Rf cystine = 0.13). Reactions o f sesquiterpene lactones with L-cysteine and reduced glutathione. To 0.01 mmol of each of the sesquiterpene lactones shown in Fig. 2 in aqueous ethanol 0.04 mmol of an aqueous solution of L~ysteine (Sigma Chemical Company) was added and the mixture allowed to stand at room temperature. The formation of adducts was checked by TLC. Parthenin and helenalin (0.01 mmol and 0.04 mmol of each of them in water) were allowed to react with 0.04 mmol of an aqueous solution of glutathione (Sigma Chemical Company) in the same way. Isolation and identification of: (a) Monoadduct -- equimolar aqueous solutions of parthenin (5.84 mg/0.58 ml) and L-cysteine (2.42 mg/0.24 ml) were mixed and allowed to stand at room temperature. After 6 h, when TLC showed only one positive spot with ninhydrin, the solution was shaken with Chloroform to remove remaining parthenin. The aqueous phase when freezedried yielded 8.2 mg of a white powder soluble in water, methanol, ethanol, n-butanol, acetone but insoluble in chloroform. It turned brown at 210--212°C and decomposed at 247°C. NMR {15.4 MHz, D20 with DSS): 8 7.67 (H-2, d), 6.17 (H-3, d), 5.06 (H-6, d), 4.03 (Hb, s), 3.10 (Ha, s), 1.27 (C5-Me, s), 1.10 (C10-Me, s). IR (KBr disc): kmax 3300--2900 (aminoacid, s), 1755 (vs), 1715 (cyclopentenone, vs), 1650--1580 (COOH, s), cm -1. (b) Biadduct-aqueous solution of parthenin (17.5 mg representing 0.06 mmol, in 1.75 ml) and L-cysteine (14.5 mg, representing 0.12 mmol, in 1.45 ml) were mixed and allowed to react at room temperature for 6 h and then freeze~lried. The residue, dissolved in water, was chromatographed on a column (25 × 2 cm) of Whatman cellulose powder which was packed and eluted with the upper phase of the solvent system n-butanol/water (4 : 5). After fast moving compounds were removed from the column, methanol/water (1 : 1) was used to elute the cysteine-parthenin biadduct. The fractions containing this product were pooled and freeze
83
FORMATION OF ADDUCTS OF PARTHENIN AND RELATED SESQUITERPENE LACTONES WITH CYSTEINE AND GLUTATHIONE
ANNA K. PICMANa, ELOY RODRIGUEZ b and G.H.N. TOWERSa aDepartment of Botany, University of British Columbia, Vancouver, B.C. V6T 1 W5 (Canada) and bDepartment of Ecology and Evolutionary Biology, University of California, Irvine, CA (U.S.A.) (Received May 18th, 1978) (Revision received June 4th, 1979) (Accepted June 19th , 1979)
SUMMARY
Parthenin, the major sesquiterpene lactone of Parthenium hysterophorus, a weed responsible for dermatitis in man is primarily restricted to leaf and stem trichomes. Parthenin forms a m o n o a d d u c t with L-cysteine through the a-methylene group of the 7-1actone and a biadduct with the endocyclic double bond on the cyclopentenone ring. Studies with other sesquiterpene lactones support the view that the types of adducts formed are correlated with the biological activity of the sesquiterpene lactones. INTRODUCTION Parthenium hysterophorus L., a widespread composite of the Southern U.S.A., the Caribbean, Australia and parts of South America, has become a serious agricultural pest as well as a medical hazard in India where it is the cause of an epidemic of allergic contact dermatitis [1]. It is generally recognized that partbenin, the major sesquiterpene lactone of this species, is responsible for the dermatological reaction associated with this plant [1]. Sesquiterpene lactones, characteristic of the Compositae, are an important group of terpenoids which exhibit cytotoxic, antineoplastic, allergenic and other biological activities [2]. These compounds, by Michael-type addition, form adducts with amino acids containing SH groups such as cysteine [3,4] or with lysyl or histidyl residues [5]. It has been suggested that the conjugated a-methylene~/-lactone moiety of sesquiterpene lactones is responsible for cytotoxicity [6,7] as well as for allergenicity [8,9]. A structural requirem e n t for significant cytotoxic activity is the O=C--C--CH2 moiety which could be part of an ester, ketone or lactone [10]. Results of tests for aUergenicity with different sesquiterpene lactones indicated differences in response between patients [11]. The fact that
84
A
B
b H
a I HS--C½--#--COO+NH3
C
.~.~
D HOOC
°'-c°/ I s - ~
NH2
COOH
~
'-- N H2
coon Fig. 1. Parthenin, cysteine and their adducts (A-parthenin, B-cysteine, C,monoadduct, D-biadduct).
parthenin (Fig. 1A) possesses two potentially active sites (an exocyclic methylene C13 and an C2 C3 endocyclic double bond allylic to C4 carbonyl group) at which Michael-type additions could occur, might explain toxic effects on cattle and the potent allergenicity of this compound [1,12]. We have examined the reaction of parthenin and of several closely related sesquiterpene lactones w i t h cysteine and:g!utathione in order to determine whether more than one type of adduct is formed. We also have reexamined the distribution of parthenin in aerial parts of P. hysterophorus. MATERIALS AND METHODS
Plant material. P. hysterophorus plants were grown from achenes obtained from Austin, TX, U.S.A. Achenes germinated in wet sand In 1--3 weeks and when leaves had formed, seedlings were transfered to pots and maintained at 30°C during the day and 20°C at night. Light period was 16 h. Detection o f parthenin in plant material. Parthenin was isolated from Parthenium hysterophorus by the method of Rodriguez [12]. Plant material (achenes or seedlings) were extracted with chloroform for 24 hrs and the presence of parthenin checked by thin-layer chromatography (TLC) with a known sample (Eastman Si gel plates with benzene--acetone, 1 : 4, fumed with iodine vapors, Rf = 0.57). Crushed or intact plant material gave identical results. Detection o f parthenin in trichomes and stems o f P. hysterophorus. From
85 ten fresh stems 10 mg of trichomes were removed with a spatula and transfered to distilled methanol (10 ml). The stems, with all trichomes removed (1 g), were cut into small pieces and extracted with distilled methanol (25 ml) overnight. Both extracts were concentrated to minimum volumes and checked by TLC for presence of parthenin. TLC of sesquiterpene lactone-thiol adducts. Eastman cellulose plates, without fluorescent indicator, and the upper phase of n-butanol/acetic acid/ water (4 : 1 : 5) were used. Plates were sprayed with 0.25% ninhydrin (acetone), heated for several minutes at 80°C and then exposed to iodine vapors to detect sesquiterpene lactones w~hich give brown spots. The monoand biadducts of parthenin give violet colored ninhydrin spots with Rf = 0.55 and Rf = 0.19, respectively. (Rf cysteine = 0.36, Rf cystine = 0.13). Reactions o f sesquiterpene lactones with L-cysteine and reduced glutathione. To 0.01 mmol of each of the sesquiterpene lactones shown in Fig. 2 in aqueous ethanol 0.04 mmol of an aqueous solution of L~ysteine (Sigma Chemical Company) was added and the mixture allowed to stand at room temperature. The formation of adducts was checked by TLC. Parthenin and helenalin (0.01 mmol and 0.04 mmol of each of them in water) were allowed to react with 0.04 mmol of an aqueous solution of glutathione (Sigma Chemical Company) in the same way. Isolation and identification of: (a) Monoadduct -- equimolar aqueous solutions of parthenin (5.84 mg/0.58 ml) and L-cysteine (2.42 mg/0.24 ml) were mixed and allowed to stand at room temperature. After 6 h, when TLC showed only one positive spot with ninhydrin, the solution was shaken with Chloroform to remove remaining parthenin. The aqueous phase when freezedried yielded 8.2 mg of a white powder soluble in water, methanol, ethanol, n-butanol, acetone but insoluble in chloroform. It turned brown at 210--212°C and decomposed at 247°C. NMR {15.4 MHz, D20 with DSS): 8 7.67 (H-2, d), 6.17 (H-3, d), 5.06 (H-6, d), 4.03 (Hb, s), 3.10 (Ha, s), 1.27 (C5-Me, s), 1.10 (C10-Me, s). IR (KBr disc): kmax 3300--2900 (aminoacid, s), 1755 (vs), 1715 (cyclopentenone, vs), 1650--1580 (COOH, s), cm -1. (b) Biadduct-aqueous solution of parthenin (17.5 mg representing 0.06 mmol, in 1.75 ml) and L-cysteine (14.5 mg, representing 0.12 mmol, in 1.45 ml) were mixed and allowed to react at room temperature for 6 h and then freeze~lried. The residue, dissolved in water, was chromatographed on a column (25 × 2 cm) of Whatman cellulose powder which was packed and eluted with the upper phase of the solvent system n-butanol/water (4 : 5). After fast moving compounds were removed from the column, methanol/water (1 : 1) was used to elute the cysteine-parthenin biadduct. The fractions containing this product were pooled and freeze
