The Chemistry of Allelopathy - American Chemical Society

9 Allelopathic Agents from Common Weeds Amaranthus palmeri, Ambrosia artemisiifolia,

and Related Weeds

NIKOLAUS H. FISCHER and LEOVIGILDO QUIJANO

Downloaded by NORTH CAROLINA STATE UNIV on August 17, 2013 | http://pubs.acs.org Publication Date: December 17, 1985 | doi: 10.1021/bk-1985-0268.ch009

Department of Chemistry, Louisiana State University, Baton Rouge, L A 70803

In a search for a l l e l o p a t h i c agents from common weeds, Amaranthus palmeri S. Wats (Palmer amaranth) and Ambrosia a r t e m i s i i f o l i a L. (Louisiana annual ragweed) have been analysed for their organic natural products. From A. palmeri phytol, c h o n d r i l l a s t e r o l , vanillin, 3-methoxy-4hydroxynitrobenzene and 2,6-dimethoxybenzoquinone were i s o l a t e d . From the roots of Ambrosia a r t e m i s i i f o l i a four polyacetylenes, a mixture of sesquiterpene hydrocarbons, methyl caffeate, and a mixture of β-sitosterol and stigmasterol were obtained. The germination regulation effects of ten sesquiterpene lactones, the flavonoid artemetin and the diterpene lactone, 17-acetoxyacanthoaustralide, on 12 crop and weed seeds are presented. A mechanism i s proposed by which water-insoluble plant l i p i d s ( s t e r o l s etc.) may act as allelochemicals v i a micelle formation with long-chain fatty acids. By this process plant lipid s o l u b i l i t y and transport i n the aqueous medium are enhanced. This might suggest a reevaluation of water-insoluble plant constituents such as sterols as potential a l l e l o p a t h i c agents.

Amaranthus palmeri S. Wats., commonly referred to as Palmer amaranth, i s a common weed belonging to the family Amaranthaceae. The observation that A_. palmeri i n h i b i t s the growth of onion and carrot and also exhibits self growth-inhibition (communicated by Dr. R.M. Menges, USDA, Wesleco, TX) has led to an i n t e r d i s c i p l i n a r y search for the possible a l l e l o p a t h i c a c t i v i t y of A. palmeri. The study involved Dr. R.M. Menges who also provided the plant material for our natural products investigation. Dr. Judith M. Bradow, USDA, Southern Regional Research Center, New Orleans, LA, performed the bioassays of a l l plant extracts and the pure constituents described i n this paper. Her findings related to Palmer amaranth and the 0097-6156/ 85/0268-0133506.00/ 0 © 1985 American Chemical Society

In The Chemistry of Allelopathy; Thompson, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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THE CHEMISTRY OF ALLELOPATHY

common ragweed, Ambrosia a r t e m l s l l f o l l a L., are described i n another paper of this book. Since f i e l d data indicated that the residues of A^ palmeri as well as Ambrosia a r t e m i s i i f o l i a seemed to cause s i g n i f i c a n t growth i n h i b i t i o n s , i t was our i n i t i a l goal to perform an extensive phytochemical study of Amaranthus palmeri, Ambrosia a r t e m i s i i f o l i a and related weeds i n search for possible growth i n h i b i t o r s and/or promoters.


Materials and Methods 1. Extractions and Chemical Analyses of Amaranthus palmeri S. Wats (Amaranthaceae). The plant material for the chemical studies was supplied by Dr. R. M. Menges, USDA, Weslaco, TX. The a i r - d r i e d a e r i a l parts (518 g) were extracted at ambient temperature with petroleum ether (PE), dichloromethane (O^CL^) and methanol (MeOH) and the solvents were evaporated i n vacuo providing 4.5 g, 4.7 g and 12.lg crude extracts, respectively. Samples of the crude extracts were tested by Dr. J . M. Bradow for germination regulation a c t i v i t y and the b i o l o g i c a l data are described i n her paper. The PE extract (4.5 g) was pre-adsorbed on s i l i c a gel (10 g) and chromatographed over 50 g s i l i c a gel (70-230 mesh) using as eluants PE followed by mixtures of PE/CI^Cl? (1:1) and Cl^CWacetone (10,20,40,80%), 100 ml fractions being taken. The PE fractions contained glycerides (*H NMR, MS). Fraction 5 (PE/CH^C^) was further p u r i f i e d by preparative TLC (PE/ether, 3:1, 2 x ) . The low p o l a r i t y band contained glycerides and a more polar band provided phytol (la) which was shown to be i d e n t i c a l with authentic material by H NMR^and MS comparison. Fractions 6-8 (PE/CI^Cl?) gave after further p u r i f i c a t i o n by preparative TLC (PE/ether, 19:1, 5 x) 116 mg c h o n d r i l l a s t e r o l (2), mp 154-6° [ l i t . (2) 168-9°]. Extraction of 650g of a i r - d r i e d , ground A. palmeri roots with C H 2 C I 2 at ambient temperature provided after solvent evaporation i n vacuo 4.7g of a crude syrup which was chromatographed over 100g of s i l i c a gel (70-230 mesh). Solvents were chloroform (CHClo) and mixtures of CHCl-j and acetone (5, 10, 20, 40%) with 100 ml fractions being taken. Early fractions eluted with CHC1~ contained fats ( H NMR, MS). Fractions 7-10 were further p u r i f i e d by prep. TLC (PE/ether, 2:1, 2 x) to give a mixture of fatty acids and c h o n d r i l l a s t e r o l (2). Fraction 20, a f t e r p u r i f i c a t i o n by prep. TLC (PE/etherj 2:1, 2 χ ) , provided a mixture of c h o n d r i l l a s t e r o l (2) and an aromatic compound. Further separation by preparative TLC (CH2CI2/acetone, 9:1) gave 2mg pure 3-methoxy-4-hydroxynitrobenzenp (6). Since an authentic sample of 6 was not available for comparison the 200 MHz Hi NMR and M§ data are given: H NMR, δ (CDC1 ): 4.02s (OMe), 6.99d (J=9.0 Hz) (H-6), 7.77d (£«2.5 Hz) (H2), 7.89dd (J«9.0 Hz, J-2.5 Hz) (H-5). EIMS (probe) m/z_ ( r e l . i n t . ) : 169 (M**") (100.0), 139 ( M - 0 C H ) (21.5), 123 (M "-N0 ) (19.9), 108 (22.9). Fraction 22 was further separated by repeated preparative TLC (PE/ether, 2:1, 6 x) and CH^CL^/acetone, 9:1) to give, besides 2 and 3, v a n i l l i n (7) which was i d e n t i f i e d by H NMR and MS comparison with authentic material. The low p o l a r i t y TLC bands contained traces of a mixture of aromatic compounds ( H NMR, MS) which were not further investigated due to the lack of material. X

3

+

+

2

2

X


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Allelopathic Agents from Common Weeds

135

T r i t u r a t i o n of f r a c t i o n 26 with CHCl^ provided 45mg of yellow needles which were spectroscopically shown to be 2,6-dimethoxybenzoquinone (8), mp 250-2° [ l i t . (3) 254°]. 200 MHz K NMR, δ (CDCIO: 3.858^(2 OMe), 5.86s (H-3,~H-5); EIMS (probe) m/z_ (re 1. i n t . ) : 168 (M ) (37.3), 138 (10.3), 80 (23.9), 69 (100.0). The CH2CI2 extract (4.7 g) was mainly composed of the more polar constituents found i n the PE extract. The MeOH extract (12.lg) remains to be chemically investigated. l


+

2. Extraction and Chemical Analysis of Ambrosia a r t e m i s i i f o l i a L. For preliminary germination tests on lettuce and carrot seeds, extracts of Ambrosia a r t e m i s i i f o l i a L. were used which had been collected on 13 May, 1979 by Mr. Axel Ohmstede i n the East Baton Rouge Parish near the Louisiana State University Campus. Air-dried ground a e r i a l parts (495g) were s t i r r e d with P.E. for 90 min. and f i l t e r e d by suction. The extraction process of the marc was repeated with CHoC^, MeOH and 1^O. Evaporation of the solvents i n vacuo provided the four crude extracts used for the bioassays. Samples of the crude extracts were prepared such that the r a t i o of the weight of the sample to the t o t a l weight of the crude extract was known. The concentrations of the test solutions are described i n terms of % of dry weight of the plant material. A bulk c o l l e c t i o n of Ambrosia a r t e m i s i i f o l i a L. was made on 1 June, 1983 i n Louisiana, East Baton Rouge Parish on Bluebonnet Road behind Bluebonnet Ridge Apartments. Fresh roots (332 g) were extracted at ambient temperature twice with 1 L each of CHCl^. The combined extracts were evaporated jja vacuo to provide 800 mg of a red o i l which was t r i t u r a t e d with PE and f i l t e r e d . The P.E.-soluble material (630 mg) was chromatographed over 15 g of s i l i c a gel to give eleven fractions of 100 mL each. Fraction 1 was re-chromatographed over a small s i l i c a gel column using P.E. as a solvent, three fractions being taken. Further p u r i f i c a t i o n by preparative TLC (P.E./CH C1 ,4:1) provided, besides t r i g l y c e r i d e s ( U NMR), a mixture of sesquiterpene hydrocarbons. GC-MS analysis of this mixture using a 30m SE-30 c a p i l l a r y column ( i n j e c t i o n temp.: 200°; column temp.: 60° for 30 s e c , then increased by 15°/min. to 250°) showed i n the GC-trace eight peaks which gave mass spectra with parent ions at m/z_ 204 ( i 5 2 4 ^ * J constituents were: α-guaiene, 3-patchoulin, α-bulnesene, bergamotene and 3-bisabolene (14), the l a t t e r being the major constituent. Fraction 2 and 3 (P.E.) provided, besides the sesquiterpene hydrocarbon mixture, a compound which on the basis of H NMR and MS spectral evidence was shown to be the acetylenic hydrocarbon pentayneene (9) C^H^, previously isolated from A. a r t e m i s i l f o l i a l

2

2

c

H

T

n

e

f

i

v

e

m

a

o

r

1 Fraction 4 (P.E./C^C^, 1:1) was a bright red solution the H NMR spectrum of which indicated a mixture of two acetylenic constituents (A and B) which were separated by preparative TLC on 1 mm s i l i c a gel plates. The red band (A), when l e f t i n soin for 15 hr at r . t . , lost i t s color. The H NMR spectrum of the decolored soin was i d e n t i c a l with the spectrum of compound B. Comparison of the physical data ( H NMR and MS) of the two compounds with the spectral data reported for the dithiophene (A) (10) and the thiophene (B) (11) (5,,6) established their i d e n t i t y . Both compounds had been



136 THE CHEMISTRY OF ALLELOPATHY



9. FISCHER A N D QUIJANO


137

previously isolated from A. a r t e m i s i i f o l i a L. (£.)· Fraction 5, (P.E./Ch^C^, 1:1), after further preparative TLC separation, contained a minor amount of an acetylenic compound which on the basis of i t H NMR spectrum was tentatively assigned structure (12). The acetate of a triterpene with H NMR spectral features very similar to those of 3-amyrin acetate was obtained from a more polar TLC band. Re-isolation of more material i s necessary for an unambiguous structural assignment of this triterpene. From fractions 6 and 7 (CHCLj), after further separations by preparative TLC, a mixture of fJ-sitoeterol (4) and stigmasterol (3) was obtained. The i d e n t i t y was established by comparison of H NMR and MS data with those of authentic samples. Another TLC band provided minor amounts of a phenolic compound which on the basis of Hi NMR and MS data was tentatively assigned methyl caffeate (13). The alternative structure of f e r u l i c acid was excluded by spectral comparison with an authentic sample of f e r u l i c a c i d . 3. Germination Regulation by Sesquiterpene Lactones (15-24), 17Acetoxyacanthoaustralide (25) and Arternetin (26). Pure sesquiterpene lactones were tested by Dr. Bradow. Since their i s o l a t i o n and physical data have been previously reported, plant sources and l i t e r a t u r e references are given i n Table I. Table II contains the bioassay results of the sesquiterpene lactones (15-24). The flavonoid artemetin (26) was isolated from Melampodlum cinereum DC, (16) and the diterpene 17-acetoxyacanthoaustralide (25) was obtained from Melampodlum longipHum Robins. (18). The effect of artemetin (26) and 17-acetoxyacanthoaustralide (25) i n seed germination tests are given i n Table I I . Results and Discussion 1. Chemical Studies on Amaranthus palmeri S. Wats. Knowledge related to natural products of the genus Amaranthus (Amaranthaceae) i s l i m i t e d . Therefore, a systematic search for secondary metabolites i n A. palmeri was performed. The a i r - d r i e d a e r i a l parts and the roots were separately investigated since the composition and type of constituents can vary dramatically within different plant parts. The ground a e r i a l parts were extracted with organic solvents of increasing p o l a r i t y (PE, C l ^ C l ^ and MeOH). Chromatography of the low p o l a r i t y extract (PE) provided fats as well as phytol ( l a ) . The structure of the l a t t e r diterpene and i t s acetate (lb) was established by comparison of the H NMR and mass spectra with the data of authentic samples of phytol (la) and the acetate ( l b ) . Intermediate fractions provided c h o n d r i l l a s t e r o l (2) (O.02 of dried leave weight) which was i d e n t i c a l with authentic material as shown by spectral methods ( H NMR, MS). Members of the family Amaranthaceae are known to produce ecdysteroids such as 3-ecdysone (19) and inocosterone (20)· From petroleum ether extracts of Amaranthus spinosus Linn. Behari and Andhiwal (21) obtained 3-sitosterol (4), stigmasterol (3), campesterol and cholesterol. From the roots of the same species, Banerji (22) isolated two new saponins, a diglucoside and a triglucoside of α-spinasterol. More recently, Roy et a l . (23)






9.


139


Table I. Sesquiterpene Lactones and their Plant Sources used for Germination Regulation Tests. Compound

Plant Source

Costunolide (15) lljlS-Dihydro-^ parthenolide (16) C o n f e r t i f l o r i i r ?17) Desacetylconfertîf l o r i n (18) Parthenin"(19) ™ Enhydrin (20) ^ Melampodinin A (21) Melampodin Β ( 2 2 ^ Cinerenin (23)"" Calein (24)°"

Saussurea lappa Clark Ambrosia a r t e m i s i i f o l i a L.

7 8

Ambrosia c o n f e r t i f l o r a DC. Ambrosia c o n f e r t i f l o r a DC.

9 9

Parthenium hysterorophorus Linn. Ambrosia psllostachya Enhydra fluctuans Lour. Melampodlum longipllum Robins. Melampodlum amerlcanum L. Melampodlum cinereum DC. Melampodlum cinereum DC. Calea t e r n l f o l i a var. calyculata

Reference

10 11 12 13 14,15 16 16 17


In The Chemistry of Allelopathy; Thompson, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985. 1

-4

103 111 108 75 91 99 92 121 99 114 82 97

102 92 100 102 116 100 111 113 100 111 93 104

16

[10 ]

4

15

[ίο" ]

17 5

98 87 104 86 111 99 112 97 99 96 111 110

[7xlO~ ]

18 5

94 97 98 114 93 100 96 104 99 102 101 100

[7xlO~ ]

19 A

109 95 95 97 114 107 89 92 100 111 115 101

[io" ]

20 4

101 110 86 93 105 105 109 99 100 99 97 104

[io~ ]

21 A

109 99 105 104 77 99 96 96 99 104 83 100

[io- ]

22 4

101 87 96 119 106 101 101 98 99 96 97 100

[ίο" ]

23 5

96 93 98 88 86 97 97 120 100 106 109 104

[6xl0" ]

24 5

99 90 99 101 105 102 93 101 101 93 75 99

[5xl0~ ]

103 95 104 101 96 99 112 91 100 109 111 100

The numbers i n brackets below the structure numbers represent the molar concentrations of the compounds used i n the tests.

3

^ h e detailed b o t a n i c a l names f o r the seed species used i n the germination test are summarized i n Table I of Dr. J . Bradow's paper.

4

[10 ]

The bioassay germinations were performed by Dr. Judy Bradow at 25° for 72 hr i n thedark, i n 3ml s o l u t i o n s containing O.1% diraethylsulfoxide (DMSO) on a sheet of Whatman paper No.l i n 9cm p e t r i dishes. Comparison was made with a p a r a l l e l O.1% DMSO c o n t r o l s o l u t i o n . Paired Student's a n a l y s i s indicated that only wheat wass i g n i f i c a n t l y affected by the DMSO s o l u t i o n (-6%).

Onion Sorghum Oats Ryegrass Wheat Lettuce Clover Carrot Cress Cucumber P. amaranth Tomato

Species

Compound

2

5

93 98 91 96 105 98 100 116 100 101 113 95

[5x10" ]

Table I I . EFFECTS OF SESQUITERPENE LACTONES (15-24) , 17-ACETOXYACANTHOAUSTRALIDE (25) and ARTEMETIN (26) ON SEED GERMINATION »


x ·
r r m r Ο

H

S So

m

χ

m π

I


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described a new s t e r o l , amasterol (5), from roots of Amaranthus v i r i d i s L., a c u l t i v a t e d crop i n India- The authors reported i n h i b i t i o n of seed germination as well as growth i n h i b i t i o n of lettuce seeds (Lactuca sativa cv White). Amasterol was also shown to be a potent growth i n h i b i t o r of Helmintho sporium oryzi, a pathogenic fungus (23). Dichloromethane extracts of roots of A_. palmeri, a f t e r chromatography over s i l i c a g e l , gave c h o n d r i l l a s t e r o l i n non-polar fractions followed by 3-methoxy-4-hydroxynitrobenzene (6). Since nitrobenzene derivatives of type 6 might have been previously used i n f i e l d experiments, i t i s possible that this compound i s an a r t i f a c t . Later chromatographic fractions contained v a n i l l i n (7) which was i d e n t i f i e d by H NMR and mass spectral comparison witt? an authentic sample. Subsequent fractions afforded 2,6-dimethoxy benzoquinone (8), a constituent i n members of a number of plant families (24). To our best knowledge this i s the f i r s t report of the presence of 8 i n the family Amaranthaceae. It i s noteworthy that the quinone 8 exhibits c y t o t o x i c i t y i n the KB c e l l culture system i n v i t r o ( Ê D - 2.8 yg/ml) (25,26). In P-388 lymphocytic leukemia i n v i t r o tests 2,6-dimethoxybenzoquinone displayed a highly cytotoxic a c t i v i t y of O.0015 yg/ml (24). The ecological function of this widespread natural product i s not known. The effects of the above constituents of A_. palmeri on the germination and growth of a number of plant seeds are discussed by Dr. J. M. Bradow i n her chapter of this book. 5 0

2. The Chemical Constituents of Ambrosia a r t e m i s i l f o l i a L. Most ragweeds are distinguished by their adjustment to d i f f e r e n t ecological environments and a successful i n t e r s p e c i f i c competition. Jackson and Willemsen (1_) were able to demonstrate that A. a r t e m i s i l f o l i a i s a dominant species i n the f i r s t year of old f i e l d succession i n the Piedmont of New Jersey. Root exudates and shoot extracts of ragweed i n h i b i t e d the germination and growth of other early invaders of abandoned f i e l d s . The presence of c a f f e i c acid and chlorogenic acid i n addition to unknown phenolic compounds could be demonstrated. Rice had shown e a r l i e r that similar phenolic acids were present i n A. psilostachya DC. (27). Anaya and del Amo (28) provided evidence that aqueous extracts of leaves and roots of the semi-arid and subtropical ragweed, A_. cumanensis H.B.K., inhibited the germination and growth of several plant species and was also auto-inhibitory. Water washes of the leaves depressed the growth of some species and stimulated others. Decomposition products of leaves and roots were highly i n h i b i t o r y to some seedlings, suggesting that i n these cases microorganisms play a major r o l e i n the a l l e l o p a t h i c process. A l l e l o p a t h i c e f f e c t s of extracts of a e r i a l parts and rhizomes of western ragweed, Ambrosia psilostachya DC., on seed germination and seedling growth of a number of plants was recently shown by Dalrymple and Rogers (29). In preparation for the i s o l a t i o n of a l l e l o p a t h i c s from root exudates of A_. a r t e m i s i l f o l i a using the method by Tang and Young (30) we wished to learn f i r s t about the major root constituents and possibly establish their structures. We describe here our preliminary findings on the root natural products of Louisiana A_. a r t e m i s i l f o l i a populations. Freshly obtained roots of the ragweed,


142


which had been collected i n Baton Rouge, LA, were extracted with chloroform. Extensive chromatography of the petroleum ether-soluble extracts provided, besides t r i g l y c e r i d e s , an o i l which was analysed by gas chromatography-mass spectrometry (GC-MS). The f i r s t eight peaks of the GC-trace showed i n the mass spectrum parent ions at jn/z 204. The mass spectral data together with the 200 MHz H NMR spectrum of the mixture suggest a sesquiterpene hydrocarbon mixture of the empirical formula i5 24» The spectral data were i n good agreement with absorptions t y p i c a l of the 3-bisabolene skeleton (14) and other farnesol c y c l i z a t i o n products. Structural assignments of the f i v e major constituents i n the sesquiterpene hydrocarbons mixture were based on MS data of a GC-MS analysis. The major constituents with increasing retention time were: orguaiene, 3-patchoulin, α-bulnesene, bergamotene and 3-bisabolene (14), the l a t t e r sesquiterpene being the major constituent. Pretreatment of seeds with a C ^ C ^ solution (O.25 wt.%) of the sesquiterpene hydrocarbon mixture caused s i g n i f i c a n t seed germination i n h i b i t i o n s . Onion, ryegrass and Palmer amaranth were p a r t i c u l a r l y sensitive to the hydrocarbon mixture, but cucumber germination was s l i g h t l y increased. Application of the sesquiterpene mixture i nO.1%DMSO aqueous solution inhibited germination of oat, ryegrass and cucumber and, i n p a r t i c u l a r , Palmer amarath. S l i g h t l y more polar chromato graphic fractions contained the three polyacetylenes 9-11 which had been previously isolated from A_. a r t e m i s i l f o l i a and are common root constituents of Ambrosia species (4). Compound 10 i s thermally very unstable and extrudes elemental sulfur under formation of the thiophene derivative 11. The 1,2-epoxide derivative of 11 appears to be present i n very^îow concentration as shown by a. NM£. Subsequent fractions provided very small amounts of a triterpene with H NMR absorptions similar to those of 3-amyrin, followed by a band which contained stigmasterol (3) and 3 - s i t o s t e r o l (4). From f r a c t i o n 7 a phenolic compound was isolated which exhibited H NMR and MS data i n agreement with the methyl caffeate structure (13). Spectral comparison with f e r u l i c acid together with p o l a r i t y considerations excluded the l a t t e r structure. Re-isolation of larger quantities of the root constituents of A_. a r t i m i s l i f o l i a and t h e i r separations w i l l be necessary for detailed bioassays of the sesquiterpene hydrocarbons and triterpenes. In a previous investigation of the a e r i a l parts of the common Louisiana ragweed,


c

H

a r t e m i s i l f o l i a (8), we had isolated the germacranolide 11,13dihydroparthenolide (16) the germination a c t i v i t y effects of which w i l l be discussed i n the following section together with the other sesquiterpene lactones. 3. E f f e c t s of Sesquiterpene Lactones on Seed Germination. Sesquiterpene lactones are common constituents of the Asteraceae but are also found i n other angiosperm families and i n certain liverworts (31,32). These highly b i t t e r substances exhibit a wide spectrum of b i o l o g i c a l a c t i v i t i e s C'^3) which include cytotoxicity, anti-tumor, anti-microbial, i n s e c t i c i d a l (34) and molluscicidal (35) properties. Furthermore, they are known causes for livestock poisoning and contact dermatitis i n humans (33)· Structure-activity relationship studies on sesquiterpene lactones have demonstrated that b i o l o g i c a l a c t i v i t y frequently depend on the presence of the or


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methylene-y-lactone moiety A, which i s a potent receptor for b i o l o g i c a l nucleophiles, ^


l-Pro tein

i n p a r t i c u l a r , t h i o l groups to form the Michael addition product Β which can result i n the i n h i b i t i o n of key enzymes such as phosphofructokinase (36)· The function of sesquiterpene lactones as growth regulators has been reviewed by Gross (37). The lactones heliangine (27), helianginol, pyrethrosin and cyclopyrethrosin acetate promote the adventitious root formation on hypocotyls of cuttings taken from Phaseolus mungo seedlings and i n h i b i t elongation of Avena coleoptile sections (38). Compounds with the methylene-y-lactone group reduced to the saturated lactone showed no effect on the root formation. Since a l l active compounds reacted with cysteine which was not the case with the reduced, saturated lactones, the Japanese authors concluded that the root growth promoting a c t i v i t y must be related to the r e a c t i v i t y of the methylene γ-lactones towards b i o l o g i c a l t h i o l groups such as cysteine which deactivates heliangine i n i t s root formation a c t i v i t y (39). The sesquiterpene dilactone vemolepin obtained from Vernonia hymenolepis A. Rich, i n h i b i t s extension growth of wheat coleoptile sections at a 1.8x10 molar concentration but simultaneous administration of increasing amounts of auxin reduced the i n h i b i t o r y effect of vemolepin (40). Parthenin (19) has at a concentration of 50 ppm no effect on the germination of the bean Phaseolus vulgaris but i n h i b i t s the development of radicles and hypocotyls (41). Similar effects were observed by Kanchan for Parthenium hysteropherus and Eleusine coracana coleoptlles (42) and i t was shown that besides parthenin (19), c a f f e i c acid, v a n i l l i c acid, f e r u l i c acid, chlorogenic acid an3 a n i s i c acid were major constituents i n hysteropherus (43). The germination and growth of velvet leaf (Abutilon theophrasti M e d i c ) , a weed causing severe problems i n corn and soybean f i e l d s , is inhibited by tomentosin (28) and a x i v a l i n (29), two lactones i s o l a t e d from seeds of povertyweed (Iva a x i l l a r i s Pursh) (£4). The availabity of a number of sesquiterpene lactones with d i f f e r e n t skeletal types (31) from our previous structural studies of this group of b i o l o g i c a l l y active compounds suggested their testing for possible germination and growth regulation a c t i v i t i e s . Their plant sources are given i n Table I and the b i o l o g i c a l actions on a number of crop and weed seeds are summarized i n Table I I . A O.1 mM solution of the simplest germacranolide, costunolide (15), s i g n i f i c a n t l y promoted germination of sorghum (111%) carrot (121%), and cucumber (114%) but inhibited ryegrass (75%), wheat (91%) and Palmer amaranth (82%). In contrast, 11,13-dihydroparthenolide (16) from Ambrosia a r t e m i s i l f o l i a , which lacks the methylene moiety i n ' the γ-lactone group, i n h i b i t s sorghum (92%) and Palmer amaranth (93%) but s i g n i f i c a n t l y promotes germination of wheat (116%), clover (111%), carrot (113%) and cucumber (111%). This demonstrates that a compound without a nucleophile receptor such as the methylene






9.



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γ-lactone group i s able to exhibit seed germination a c t i v i t i e s - The above data also support e a r l i e r observations (38) that the same substance can i n h i b i t germination of one species and promote another. The two pseudoguaianolides c o n f e r t i f l o r i n (17) and i t s desacetyl derivative (18), both isolated from A. c o n f e r t i f l o r a , show considerable differences i n a c t i v i t y . Sorghum (87%) and ryegrass (86%) are inhibited and wheat (111%), clover (112%) and Palmer amaranth (111%) are promoted by 17. To the contrary, ryegrass (114%) i s promoted by 18 while most other seeds show l i t t l e e f f e c t s . Future studies with a series of ester derivatives of 18 with increasing chain length w i l l give clues about the influence of chain length and l i p o p h i l i c i t y on the seed germinations. Parthenin (19) had been previously shown to i n h i b i t the development of radicals and hypocotyls of beans (Phaseolus vulgaris) (41) and Eleusine coracana (42). In our tests, clover (89%) and carrot (92%) are also inhibited, but onion (109%), wheat (114%), cucumber (111%) and P. amaranth (115%) are promoted. The a l l e l o p a t h i c effects of the two melampolides enhydrin (20) and melampodinin A (21) d i f f e r i n a number of seed germination t e s t s . Sorghum (110%)* and clover (109%) are promoted and oat (86%) i s inhibited by enhydrin (20)· Compound (21) promotes onion (109%) and s i g n i f i c a n t l y i n h i b i t s wheat (77%) and Calmer amaranth (83%). Melampodinin A (21) i s an a n t i leukemic (P-388) compound i n i n vivo tests on laboratory animals (15) and also exhibits insect allelochemic effects with high l a r v a l mortality on the f a l l armyworm (Spodoptera frugiperda J . E. Smith) (34). Enhydrin (20) which lacks a 2,3-epoxide function shows no a c t i v i t y (34). Based on model reactions with sodium methoxide, nucleophilic attacks occur at the methylene lactone group but predominantly at C-2 under opening of the 2,3-epoxide (45). This suggests that b i o l o g i c a l nucleophiles (R-SH) should react at the same centers. The difference between lactones 20 and 21 with respect to the 2,3-epoxide function might explain their different b i o a c t i v i t i e s . The structural difference between the co-occuring melampodin Β (22) and cinerenin (23) l i e i n the acetoxy- and ethoxyattachment at C-2, respectively, causing major differences i n their p o l a r i t y , 22 being more polar. Significant seed germination effects are shown on ryegrass which i s promoted by 22 (119%) and inhibited by 23 (88%). Sorghum i s depressed by both whereas wheat (86%) i s inhibited by 23 only. At the 6x10 molar l e v e l cinerenin promotes seed germination of carrot (120%) and to a lesser extent Palmer amaranth (109%). Calein A (24) which contains one a, 3~unsaturated ketone, ester and lactone moiety has only minor effects on most seed germinations but strongly i n h i b i t s Palmer amaranth (75%) at theO.05mM l e v e l . The diterpene lactone 17-acetoxyacanthoaustralide (25) has minor e f f e c t s , showing promotion of clover (112%), cucumber (109%) and Palmer amaranth (111%) and i n h i b i t i o n of carrot germination (91%). The flavonoid artemetin (26) at O.05 mM concentrations s l i g h t l y affects onion (93%), oat (91%) and promotes seed germinations i n carrot (116%) and Palmer amaranth (113%). In closing, i t i s noteworthy to point out that lettuce and cress are not s i g n i f i c a n t l y affected by a l l ten sesquiterpene lactones as well as the diterpene (25) and artemetin (26).



146


4. Possible Mechanism of A l l e l o p a t h i c Action of Water-Insoluble Plant L i p i d s . Many non-polar natural products with germination and growth regulation a c t i v i t i e s in laboratory tests are i n pure form not s u f f i c i e n t l y water soluble to account for their a l l e l o p a t h i c a c t i v i t i e s observed i n the f i e l d . For this reason the notion exists that sterols and other non-polar plant constituents are not l i k e l y to play a role i n a l l e l o p a t h i c actions, and i t i s generally concluded that the b i o a c t i v i t y data observed i n the laboratory are therefore coincidental. We have recently observed i n our laboratory that water washes of undamaged leaves i n a number of plants contained sterols and other l i p i d s i n s u f f i c i e n t l y high concentration comparable with concentrations used i n t y p i c a l laboratory bioassays. These aqueous lipid solutions are frequently accompanied by long-chain (C-12 to C18) fatty acids. We therefore suggest that micelle formation between the l i p i d s and fatty acids may occur. By this mechanism the lipid s o l u b i l i t y i n the aqueous medium i s s i g n i f i c a n t l y enhanced, thus allowing the release of otherwise water-insoluble plant constituents into the environment. Presently, experiments are i n progress i n our laboratory to provide further evidence for the "micelle-mechanism" of a l l e l o p a t h i c l i p i d s . Acknowledgments We thank Mr. Kenneth R. Wilzer, J r . Plant Protection I n s t i t u t e , USDA, B e l t s v i l l e , MD for a sample of c h o n d r i l l a s t e r o l and Helga D. Fischer and Joel Carpenter for technical assistance. Support for this work by the USDA-ARS Cooperative Agreement No. 58-7B30-2-399 i s acknowledged· Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Jackson, J.R.; Willemsen, R.W. Amer. J . Bot. 1976, 63, 101523. Bergmann, W.; Feeney, R.J. J . Org. Chem. 1950, 15, 812-4. Richtzenhain, H. Chem. Ber. 1944, 77, 409-17. Bohlmann, F.; Burkhardt, T.; Zdero, C. "Naturally Occuring Acetylenes"; Academic: London, 1973. Bohlmann, F.; Kleine, K.M.; Arndt, C. Chem. Ber. 1964, 97, 2125-34. Bohlmann, F.; Kleine, K.M. Chem. Ber. 1965, 98, 3081-6. Rao, A.S.; Kelkar, G.R.; Bhattacharyya, S.C. Tetrahedron 1960, 9, 275-83. Fischer, N.H.; Wu-Shih, Y.F.; C h i a r i , G.; Fronczek, F.R.; Watkins, S.F. J . Nat. Prod. 1981, 44, 104-10. Fischer, N.H.; Mabry, T.J. Tetrahedron 1967, 23, 2529-38. Herz, W.; Miyazaki, M.; Kishida, Y. Tetrahedron Letters 1961, 1961, 82-7. Geissman, T.A.; G r i f f i n , S.; Waddell, T.G.; Chen, H.H. Phytochemistry 1969, 8, 145-50. Ali, E.; Ghosh Dastidar, P.P.; Pakrashi, S.C.; Durham, L. J . ; D u f f i e l d , A.M. Tetrahedron, 1972, 28, 2285-98. Seaman, F.C.; Fischer, N.H. Phytochemistry 1978, 17, 2131-3. Fischer, N.H.; Wiley, R.A.; Perry, D.L. J. Org. Chem 1976, 1976, 41, 3956-9.


9.

FISCHER

15. 16. 17. 18. 19.


20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

45.

AND

QUIJANO


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The Chemistry of Allelopathy - American Chemical Society

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