Psoralens as Phytoalexins in Food Plants of the Family Umbelliferae

19 Psoralens as Phytoalexins in Food Plants of the Family Umbelliferae Significance in Relation to Storage and Processing

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ROSS C. BEIER, G. WAYNE IVIE, and ERNEST H. OERTLI Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77841 Linear furanocoumarins (psoralens) are phototoxic, photomutagenic, and photocarcinogenic compounds that occur as natural constituents of hundreds of plant species, including some food plants of the family Umbelliferae (e.g., parsnip, celery, and parsley). Certain plant stresses, particularly diseases, induce biosynthesis of toxic natural plant products; a phenomenon referred to as a phytoalexin response. Such interactions in food plants of the family Umbelliferae may have toxicological implications for man because of the biological activity of psoralens. The linear furanocoumarin phytoalexin response in celery is discussed, with brief comments concerning carrots and parsley. Historical Psoralens. Many plants contain linear furanocoumarins (psoralens) (J_), which were i d e n t i f i e d i n the l a t e 1940 s as the cause of the p h o t o s e n s i t i z a t i o n properties of these plants (_2"_4) · Plants containing l i n e a r furanocoumarins can cause p h o t o s e n s i t i z a t i o n i n l i v e s t o c k and poultry (_5-9_), r e s u l t i n g i n economic losses and, i n some cases, animal death. Man has also encountered problems with the p h o t o s e n s i t i z i n g properties of l i n e a r furanocoumarins. Celery handlers and f i e l d workers are frequently affected with p h o t o s e n s i t i z a t i o n of the f i n g e r s , hands, and forearms (10,11). These s k i n disorders are r e f e r r e d to as c e l e r y d e r m a t i t i s , c e l e r y i t c h , or c e l e r y b l i s t e r s , and are caused by l i n e a r furanocoumarins i n diseased c e l e r y (12). Some researchers (12,13) have found l i n e a r furanocoumarins only i n diseased p l a n t s , whereas others (14,15) obtained them from healthy celery. f

T h i s chapter not subject t o U . S . copyright. P u b l i s h e d 1983, A m e r i c a n C h e m i c a l S o c i e t y

In Xenobiotics in Foods and Feeds; Finley, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

XENOBIOTICS


296

IN F O O D S A N D

FEEDS

Biological Activities of Linear Furanocoumarins» The b i o l o g i c a l a c t i v i t y of these compounds are extremely d i v e r s e because of t h e i r i n t e r a c t i o n s with DNA (32-35), and RNA (36). L i n e a r furanocoumarins have antifeedant a c t i v i t y toward various insects (37,38) and are phototoxic to others (39). I n t e r e s t i n g l y , the black s w a l l o w t a i l b u t t e r f l y has maximized i t s metabolic d e t o x i f i c a t i o n processes allowing i t s l a r v a to feed on plants with a high l i n e a r furanocoumarin content (40). Psoralen, bergapten, xanthotoxin, and isopimpinellin are a n t i b a c t e r i a l when combined with UV l i g h t , whereas psoralen and xanthotoxin have some a n t i b a c t e r i a l a c t i v i t y without UV l i g h t (41). A mixture of p i m p i n e l l i n , i s o p i m p i n e l l i n , isobergapten, and sphondin was fungi t o x i c at 200 ppm or less (42). The individual linear furanocoumarins, psoralen (43), and xanthotoxin (44,45) are also a n t i f u n g a l . Toxicological Implications f o r Man. Because psoralens are potent photoactive compounds, they have been used m e d i c a l l y f o r treatment of skin depigmentation or vitiligo (16,17), and psoriasis (18). However, there has r e c e n t l y been concern a s s o c i a t e d with the medical use of these compounds (19) · This concern i s due to the observed p h o t o t o x i c i t y during therapeutic use (17), the suspected p h o t o c a r c i n o g e n i c i t y of xanthotoxin (20,21), and the l a t e n t onset of tumors i n treated laboratory animals (22). Acute gout secondary to p s o r i a s i s also was exacerbated by psoralen and UV-A (PUVA) photochemotherapy (23). Psoralens i n Healthy Celery. Healthy c e l e r y contains at l e a s t four l i n e a r furanocoumarins (Figure 1), psoralen, bergapten, xanthotoxin, and i s o p i m p i n e l l i n (14). The observed q u a n t i t i e s of l i n e a r furanocoumarins i n healthy samples of three d i f f e r e n t c e l e r y c u l t i v a r s grown at d i f f e r e n t l o c a t i o n s i n the U.S. are shown i n Table I . Table I . Summary of the L i n e a r Furanocoumarin Content i n Fresh Celery Grown i n C a l i f o r n i a , F l o r i d a , and M i c h i g a n 3

b , c

Compound

A

Cultivar Β

Psoralen Bergapten Xanthotoxin Isopimpinellin

0.15 + 0.06 0.14 + 0.04 0.61 + 0.14 0.08+0.03

. s c l e r o t i o r u m i s often implicated i n the production of linear furanocoumarins i n celery, s t e r i l i z i n g the plant tissues maybe an appropriate first step in many investigations.


XENOBIOTICS

304

IN F O O D S A N D

FEEDS


26°C

il 2°C

Φ

Q

10

Time

15

"Ί 20

(min)

Figure 5. HPLC tracings of detector response vs. retention time for UV-treated celery cv. 5270-R after 72 h at 2 °C and 26 °C in comparison to the linear fur anocoumarin standards: psoralen (p), bergapten (b), xanthotoxin (x), and isopim pinellin (i).


19.

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Phytoalexins

in Food

305

Plants


Celery p e t i o l e s were immersed i n 0.1% sodium h y p o c h l o r i t e f o r 20 min. and incubated f o r 72 hrs at 26° C. Subsequent e x t r a c t i o n and HPLC a n a l y s i s gave the t r a c i n g s shown i n Figure 6. The p s o r a l e n l e v e l s at 2°C are s i m i l a r to the CuSO^ t r e a t e d c e l e r y , but at 26° C the l e v e l s are lower and appear qualitatively different from those observed in the CuSO^-treated plant m a t e r i a l . A composite bar graph showing the l e v e l s of the four linear furanocoumarins in both UV-treated and sodium h y p o c h l o r i t e treated c e l e r y analyzed a f t e r 72 hrs at 26°C i s shown i n Figure 7. The observed l e v e l s i n both treated t i s s u e s were s i g n i f i c a n t l y higher than those i n the c o n t r o l s . Summary The total quantities of psoralens and t h e i r increased concentration i n c e l e r y a f t e r d i f f e r e n t treatments i s described i n Table 3.

Table 3.

Summary of P h y t o a l e x i n Response i n Treated C e l e r y (57)

Treatment

Time ( h r )

T o t a l Psoralens (ppm)

F o l d Increase

104.0 23.0 3.4 2.2 8.8

CuSO^ CuSO^ UV l i g h t Sodium h y p o c h l o r i t e Cold CuS0 *

96 79 72 72 72 48

26.0 21.1 7.4 4.9 2.2 29.0

4-day-old CuSO/,*

48

1.3

4

-23.0

*These experiments were c a r r i e d out on c e l e r y procured a t the same time. Part of the l o t was immediately t r e a t e d , while another p o r t i o n was r e t a i n e d f o r 4 days i n the r e f r i g e r a t o r before treatment.

A l l of the treatments i n Table 3 caused an increase i n the q u a n t i t i e s of l i n e a r furanocoumarins to some degree, with some samples containing as much as 29 ppm of t o t a l psoralens. I t i s a l s o i n t e r e s t i n g that a sample of harvested c e l e r y allowed to age 4 days i n the l a b o r a t o r y prior to CuSO^ treatment e x h i b i t e d a 23 f o l d decrease i n the t o t a l l i n e a r furanocoumarin production when compared to non-aged CuSO^-treated celery (Table 3 ) . This r e s u l t may r e f l e c t a d e t e r i o r a t i o n of the c e l l u l a r c o n d i t i o n i n these samples.



306

XENOBIOTICS

I

1—ι

0

5

10

IN FOODS A N D F E E D S

1—I 15

20

Time (min) Figure 6. HPLC tracings of the detector response vs. retention time for sodium hypochlorite-treated celery cv. 5270-R after 72 h at 2 °C and 26 °C in comparison to the linear furanocoumarin standards: psoralen (p), bergapten (b), xanthotoxin (x), and isopimpinellin (i).

Psoralens from celery cv. 5270-R Figure 7. A bar graph of the observed levels of psoralen, bergapten, xanthotoxin, and isopimpinellin in celery cv. 5270-R 72 h after UV light and sodium hypo chlorite treatment.



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What i s the Impact of the Phytoalexin Response i n Celery. Studies to date i n d i c a t e that c e l e r y purchased at l o c a l markets should contain low l e v e l s of linear furanocoumarins (14). Increased l e v e l s of psoralens as a r e s u l t of a phytoalexin response w i l l probably be of l i t t l e or no s i g n i f i c a n c e to the consumer. The major i n t e r e s t that a r i s e s from t h i s phenomenon will be to the grower, f i e l d worker and celery handler. C l e a r l y , c e r t a i n chemicals and s t r e s s s i t u a t i o n s can cause up to 100 f o l d increases i n the l i n e a r furanocoumarin content of previously excised celery petioles. Therefore, i t seems possible that certain stress situations and/or chemical treatments may indeed elevate l e v e l s of psoralens i n c e l e r y to a point where the r i s k of d e r m a t i t i s i s g r e a t l y enhanced. The a c t u a l r o l e of l i n e a r furanocoumarins i n the disease r e s i s t a n c e of c e l e r y i s unknown; however, i t has been concluded that the phytoalexins studied to date play an important r o l e i n r e s i s t a n c e (47). Phytoalexins

i n Other

umbelliferae?

Carrots. Previous phytochemical studies with carrot (Daucus carota L.) have been unsuccessful i n demonstrating the presence of psoralens, and i t i s g e n e r a l l y accepted that c a r r o t s lack l i n e a r furanocoumarins (58,59). We have r e c e n t l y developed techniques to look f o r psoralens at the sub p a r t s - p e r - m i l l i o n l e v e l i n carrot (60), and were s i m i l a r l y unable to see l i n e a r furanocoumarins i n t h i s p l a n t . Along with our c e l e r y s t u d i e s , we treated c a r r o t s l i c e s with CuS0 (9 Χ 10"" M) f o r 0.5 hr, and made analyses by HPLC a f t e r 72 h r s . Even with t h i s attempted s t i m u l a t i o n , no l i n e a r furanocoumarins were detected. I t has been suggested (46) that phytoalexins can be used i n some cases as taxonomic markers. That a p p l i c a t i o n may indeed be appropriate i n the case of c a r r o t s . 3

4

Parsley. P a r s l e y (Petroselinum sativum) has been known to cause d e r m a t i t i s on the hands and arras. This c o n d i t i o n was accompanied by b l i s t e r s which developed on the back of the hands of s c h o o l g i r l s that picked parsley. Peasants i n a v i l l a g e near S o f i a , B u l g a r i a , are f a m i l i a r with t h i s problem, and some cover t h e i r hands with f a t before p i c k i n g (61). The first linear furanocoumarin to be isolated from p a r s l e y was bergapten (62). Later work provided quantitative data f o r bergapten, xanthotoxin, and i s o p i m p i n e l l i n from d r i e d p a r s l e y grown i n greenhouse conditions (15). We are presently investigating parsley for i t s linear furanocoumarins besides those previously identified. Preliminary studies with CuSO^ suggests that parsley also produces psoralens as phytoalexins.


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Literature Cited 1. Nielsen, Β. E. "The Biology and Chemistry of the Umbelliferae"; Heywood, V. H., Ed.; Academic Press: London, 1971, p. 325. 2. Fahmy, I. R.; Abushady, H.; Schonberg, Α.; Sina, A. Nature 1947, 160, 468-9. 3. Fahmy, I. R.; Abu-Shady, H. Quart. J . Pharm. Pharmacol. 1947, 20, 281-91. 4. Fahmy, I. R.; Abu-Shady, H. Q. J . Pharm. Parmacol. 1948, 21, 499-503. 5. Binns, W.; James, L. F.; Brooksby, W. Vet. Med. Small Anim. Clin. 1964, 59, 375-9. 6. Musajo, L.; Rodighiero, G. Phytophysiology 1972, 7, 115-47. 7. Stegmaier, O. C. J . Invest. Dermatol. 1959, 32, 345-9. 8. Dollahite, J . W.; Younger, R. L.; Hoffman, G. O. Am. J . Vet. Res. 1978, 39, 193-7. 9. Egyed, M. N.; Shlosberg, Α.; Eilat, Α.; Cohen, U.; Beemer, A. Refu. Vet. 1974, 31, 128-31. 10. Birmingham, D. J . ; Key, M. M.; Tubich, G. E.; Perone, V. B. Archs. Derm. 1961, 83, 73-85. 11. Legrain, P. MM.; Barthe, R. Bull. Soc. Fr. Derm. Syph. 1926, 33, 662-4. 12. Scheel, L. D.; Perone, V. B.; Larkin, R. L.; Kupel, R. E. Biochemistry 1963, 2, 1127-31. 13. Wu, C. M.; Koehler, P. E.; Ayres, J . C. Appl. Microbiol. 1972, 23, 852-6. 14. Beier, R. C.; Ivie, G. W.; Oertli, Ε. H.; Holt, D. L. Food Chem. Toxicol. 1983, 21, 163-5. 15. Innocenti, G.; Dall'Acqua, F.; Caporale, G. Planta Medica 1976, 29, 165-70. 16. Scott, B. R.; Pathak, Μ. Α.; Mohn, G. R. Mutat. Res. 1976, 39, 29-74. 17. Pathak, M. Α.; Daniels, F.; Fitzpatrick, T. B. J . Invest. Dermatol. 1962, 39, 225-49. 18. Van Scott, E. J. Am. Med. Assoc. 1976, 235, 197-8. 19. "Psoralens," National Toxicology Program Technical Bulletin, 1982, 6, p. 8. 20. Reed, W. B. Acta Derm.-Venereol. 1976, 56, 315-7. 21. Stern, R. S.; Thibodeau, L. Α.; Kleinerman, R. Α.; Parrish, J . A.; Fitzpatrick, T. B.; 22 participating investigators. N. Eng. J . Med. 1979, 300, 809-13. 22. Zajdela, F.; Bisagni, E. Carcinogenesis 1981, 2, 121-7. 23. Burnett, J . W. Arch. Dermatol. 1982, 118, 211. 24. Johnson, C.; Brannon, D. R.; Kuć, J . Phytochemistry 1973, 12, 2961-2. 25. Müller, K.; Börger, H. Arb. Biol. Reichsanstat. Land-u Forstwirtsch. 1940, 23, 189-231. 26. Müller, Κ. Phytopathol. Ζ. 1956, 27, 237-54.



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1983


Psoralens as Phytoalexins in Food Plants of the Family Umbelliferae

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