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Linear Furocoumarins (Psoralens) from the Seed of Texas Ammi majus L. (Bishop’s Weed) G. Wayne Ivie Twelve linear furocoumarins (psoralens) were isolated from the ripened seed of a Texas collection of the photosensitizing weed Ammi majus L. Four of the compounds, xanthotoxin, bergapten, isopimpinellin, and isoimperatorin, have already been reported from this plant, and six of the eight other psoralens isolated are known plant products but have not been previously obtained from A. majus. Two of the A . majus psoralens, 8-[2-(3-methylbutyroxy)-3-hydroxy-3-methylbutoxy]psoralen and 5-[2(acetoxy)-3-hydroxy-3-methylbutoxy]psoralen, have apparently not been previously isolated from any plant source. Six other synthetic psoralens were not observed in the seed extracts. Most of the major A. majus psoralens are potent photosensitizers, based on bioassays against chick skin, and the presence of these compounds in the seed and other parts of A . majus almost certainly accounts for the high photosensitizing activity of this plant toward cattle and sheep. The Umbelliferous plant Ammi majus L. (Bishop’s weed) has been used medicinally in the Middle East for centuries in the treatment of leukoderma (skin depigmentation, vitiligo) (Pathak et al., 1974). Not until the late 1940’s, however, was it established that the photosensitizing and pigment stimulating agents in this plant were a group of compounds known as linear furocoumarins or psoralens. Egyptian workers isolated several substituted psoralens from A. majus and determined that the purified compounds and light were effective in treating leukoderma (Fahmy and Abu-Shady, 1948). Ammi majus and other photosensitizing plants are cultivated even today in some parts of the world as a source of these medicinally important compounds. Some psoralens are also synthesized for medicinal use in treating leukoderma and, more recently, psoriasis (Van Scott, 1976). A recent, extensive review deals with the biochemical basis for light-induced psoralen reactions with skin (Scott et al., 1976). Ammi majus also induces phototoxic responses in livestock and poultry. Young geese and ducks that have eaten as little as (0.5 g of Israeli A. majus seed) bird-’ day-’ experienced a severe photosensitization syndrome after exposure to sunlight. The symptoms included blistering and subsequent deformation of the beak, feet, wings, and ocular areas (Eilat et al., 1974; Egyed et al., 1974a, 1975a,b). Dairy cattle were also affected after consumption of A. majus contaminated feed and subsequent exposure to sunlight (Egyed et al., 1974b). Although A. majus is primarily an Old World species, it grows in the coastal regions of the southern United States and in some areas as far as 200 km or more inland. In Texas, periodic outbreaks of photosensitization affecting thousands of cattle have been observed for many years (Dollahite et al., 1978). The distribution of these outbreaks coincides with the distribution of A. majus, and recent studies in our laboratory have shown that experimental feeding of the Texas A . majus to cattle or sheep, followed by exposure to sunlight, induces a photosensitization syndrome very similar to that seen in the field (Dollahite et al., 1978; Witzel et al., 1978). The photosensitization involves severe blistering and peeling in light-skinned animals and the development of cloudy corneas that can apparently lead to blindness (Witzel et al., 1978). The studies reported here were undertaken to evaluate the furocoumarin chemistry of Texas A. majus. Although Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research, Science and Education Administration, U S . Department of Agriculture, College Station. Texas 77840.
considerable efforts have been directed toward the isolation and structure elucidation of A. majus furocoumarins by Egyptian and, to a lesser extent, European workers, such work apparently has not been undertaken on North American A. majus. The A. majus psoralens, as well as several additional synthetic compounds, were also tested for photosensitizing activity against the skin of newly hatched chicks. MATERIALS AND METHODS
Analytical Procedures. Mass spectra were recorded by direct insertion probe analysis on a Varian/MAT CH-7 magnetic scan spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded mostly using a JEOL Model JNM-MH-100 spectrometer, but some were done in the Varian NMR Applications Laboratory, Palo Alto, Calif., with a 300-MHz superconducting Fourier transform (FT) NMR system. Samples were run in deuterated chloroform or acetone, using tetramethylsilane (Me4Si)as an internal reference. Chemical shifts are reported in parts per million downfield from Me4Si. Melting points (uncorrected) were recorded in open capillary tubes with a Tottoli-type melting point apparatus. Optical rotation studies were done in chloroform at the D line of sodium with a Cary 60 Spectropolarimeter. Extraction of Seed. Mature seed heads of A. majus were collected during June, 1976, in Wharton County, Texas; plants were identified by J. W. Dollahite of this laboratory. The ripened seeds, which dislodged easily as the plants dried, were collected and held frozen until analysis. Whole seed (100 g) was extracted with 250 mL of chloroform by blending thoroughly with a Willems Polytron Homogenizer. The slurry was filtered under vacuum through a medium porosity fritted glass funnel, and the residue was extracted three more times with chloroform. The combined chloroform extracts were dried over sodium sulfate, and the chloroform was removed by vacuum distillation. The residue was partitioned between acetonitrile and hexane (100 mL each) to remove lipids, and the hexane phase was discarded. The acetonitrile was adjusted to 100 mL and this phase, containing 1 g equiv of A. majus seed extract/mL, was subjected to thin-layer chromatographic (TLC) analysis. Chromatography. TLC was used to resolve and isolate the psoralen constituents from the seed extracts. Precoated silica gel plates (20 X 20 cm, Brinkman Silplate F-22, with fluorescent indicator, 0.25, 0.5, or 2.0 mm gel thickness) were used in all separations. With these plates, the substituted psoralens present were easily detected as yellow or golden bands by viewing the developed plates
This article not subject to U.S. Copyright. Published 1978 by the American Chemical Society
J. Agric. Food Chem., Vol. 26, No. 6, 1978
Psoralens from Ammi majus L.
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Table I. TLC R f Values for Ammi majus Psoralens and Related Compounds
R f in indicated solvent systembSC compounda
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Id
0.42 0.53 0.36 0.57 0.27 0.17 0.01 0.14 0.09 0.05 0.02 0.03 0.05 0.07 0.50 0.00 0.10 0.01
2 0.45 0.47 0.47 0.60 0.45 0.40 0.05 0.35 0.29 0.27 0.22 0.16 0.20 0.22 0.52 0.06 0.32 0.12
3 0.54 0.58 0.55 0.65 0.55 0.51 0.06 0.44 0.38 0.32 0.26 0.19 0.28 0.32 0.60 0.06 0.43 0.15
4 0.17 0.21 0.16 0.28 0.10 0.07 0.01 0.03 0.02 0.01 0.01 0.01 0.03 0.02 0.22 0.00 0.04 0.01
5 0.47 0.50 0.48 0.63 0.54 0.46 0.29 0.54 0.46 0.50 0.41 0.39 0.43 0.36 0.54 0.25 0.45 0.30
6 0.55 0.58 0.57 0.67 0.57 0.53 0.08 0.49 0.43 0.37 0.32 0.23 0.34 0.36 0.62 0.08 0.45 0.19
a For chemical names o f compounds see Table 111. Solvent systems Brinkman Silplate F-22,0.25mm gel thickness. as follows: 1 (methylene chloride); 2 (chloroform-ethyl acetate, 2:1);3 (methylene chloride-ether, 2:1);4 (benzene-ether, Plates developed three times. 20:1);5 (hexane-ethyl acetate-methanol, 5:5:1);and 6 (methylene chloride-ether, 1:l).
under long-wave ultraviolet (UV) light. Psoralens were isolated by applying the extract as bands to TLC plates, developing in appropriate solvent systems, locating the compounds under UV light, and then scraping and eluting with chloroform or acetone. Compounds were crystallized directly from appropriate solvents (usually ether or ether-hexane), or if necessary, the compounds were subjected to further cleanup by TLC. The solvent systems used and the numbers used to designate them throughout this report are as follows: 1, methylene chloride; 2, chloroform-ethyl acetate (2:l); 3, methylene chloride-ether (2:l); 4, benzene-ether (20:l); 5, hexane-ethyl acetate-methanol (551); and 6, methylene chloride-ether (1:l). TLC R, values for each of the psoralens studied are indicated in Table I. Synthesis. In some cases, chemical modifications of the A . majus psoralens were made to verify their structures. Other psoralens were synthesized to confirm the structural assignments of A. majus constituents and to provide TLC standards for determining their possible presence in the plant extracts. All structures were verified by mass spectral and NMR analysis and by comparison with previously published data on the compounds where possible. One of the compounds isolated from the A . majus extracts, xanthotoxin (8-methoxypsoralen), was also obtained in gram quantities commercially (Biochemical Laboratories, Redondo Beach, Calif.) and was used in synthetic studies involving this compound because larger quantities were available. Phototoxicity. Compounds were tested for photosensitizing activity against the skin of 1-to 3-day-old chicks (mixed sex, White Leghorn, Kazmeier Hatchery, Bryan, Tex.). The down was removed from the skin over the skull cap by application of a commercial depilatory (Neet lotion, Whitewall Laboratories, New York, N.Y.), followed 1-3 min later by gentle rubbing with a soft cloth. The various furocoumarins [up to 100 pg in 10 pL of dimethyl sulfoxide (Me2SO)solution] were injected subcutaneously under the bare skin, then the chicks were exposed for 24 h to overhead long-wave UV light (Sylvania blacklite, F 30T8-BL, two 90-cm fluorescent tubes spaced about 7 cm apart). [It is well known that long-wave UV is the primary radiation effecting psoralen photosensitization (Scott et al., 1976).] During the exposure, the chicks were held in a cardboard box 84 cm long x 25 cm wide X 25 cm deep, and the lights were rested lengthwise across the top of the box. Controls
consisted of chicks that were injected with 10 pL of MezSO only and exposed to light. In tests to determine if the responses were light dependent, chicks were also injected with the highest level of photosensitizer tested and then exposed only to indirect room lighting and not to long-wave UV. At least three birds were tested a t each photosensitizer concentration and for both light-exposed and nonexposed controls. After the exposure period, the birds were transferred to incubators, and the phototoxic effects were evaluated 6 days later as follows: (-1 no response; (+) definite thickening of the skin; (++) skin blistered; (+++) severe phototoxicity characterized by darkened, dried, and hard skin. Nomenclature. For the past decade, Chemical Abstracts (CAI has named psoralens as derivatives of 7 H furo[3,2-g][ l]benzopyran-7-one,in which the two aromatic protons are numbered 4 and 9. Almost without exception,
however, investigators in this field continue to use either of two older systems of nomenclature in which the compounds are named as “psoralens”, and the aromatic protons are numbered 5 and 8. In the current report, the
alkoxy psoralens isolated from A . majus or synthesized chemically are named as 5- or 8-substituted derivatives. Thus, xanthotoxin (8-methoxypsoralen in this report) is listed by CA as the 9-methoxy derivative of 7H-furo[3,2-g][ llbenzopyran-7-one. The nomenclature of the alkoxy substituents in this paper is strictly according to that in CA. RESULTS
Preliminary TLC studies with the extract of A . majus seed indicated that multiple development of 0.25-mm plates in methylene chloride (solvent system 1)gave good resolution of several components. On the basis of their appearance under long-wave UV light, each of the bands
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Table 11. Yield, Melting Point, and Optical Rotation Data for Substituted Psoralens from Ammi majus Seed and for Related Synthetic Psoralens specific rotation 1 2 3 4
5 6
7 8 9 10 11 12 13 14 15 16 17 18
compound"
yield, %
mp, " C
(xanthotoxin) (bergapten) (isopimpinellin) (isoimperatorin) (oxypeucedanin) (heraclenin) (oxypeucedanin hydrate) (saxalin) (pabulenol)
0.36 0.11 0.23
144.5-145.0 188.0-189.0 147.0 97.0 102.0 105.0 126.5-128.5 142.5-144.0 121.0-122.0 109.0-110.0 oil 141.0-142.0 236.0-240.0 268.0-270.0 96.0-97.0 127.0-128.0 103.0-104.0 oil
