J. Agric. Food Chem. 1985, 33, 83-86
The quantity of washed protein was assayed by the BioRad protein reagent (Bradford, 1976). Even though there were only two washings, the nicotine contamination in the protein fell to less than 20 ppb (Table VI). If a food formulation is composed of 10% tobacco F-1-p by weight, the possible contamination of nicotine would be 2 ppb. One nicotine-containing chewing gum contains 1OOO-10OOO ppm of nicotine (Lichtneckert et al., 1975), whereas vegetables from the Solanaceae family such as tomato, potato, pepper, and eggplant have several ppm of nicotine in the edible parts (Sheen and Sheen, 1983). This indicates that tobacco F-1-p is safe for human consumption. ACKNOWLEDGMENT We thank Leaf Protein International, Inc., for the tobacco F-1-p samples and Archer Daniels Midland Co. for the soy protein isolates. Registry No. F-1-p, 9027-23-0. LITERATURE CITED Baker, T. S.; Eisenberg, D.; Eiserling, F. A.; Weisman, L. J. Med. Biol. 1976,91, 391. Betschart, A. A. J. Food Sci. 1974,39,1110. Bradford, M. Anal. Biochem. 1976, 72, 248. Bradstreet, R. B. “The Kjeldahl Method for Organic Nitrogen”; Academic Press: New York, 1965; pp 1-167. Cherry, J. P.; McWatters, K. H.; Beuchat, L. R. “Functionality and Protein Structure”; Pour-El, A., Ed.; American Chemical Society: Washington, DC, 1979; ACS Symp. Ser. No. 92, Chapter 1. Ershoff, B. H.; Wildman, S. G.; Kwanyuen, P. Proc. SOC.Exp. Biol. Med. 1978, 157, 626. Fleming, S. E.; Sosoulski, F. W.; Kilara, A.; Humbert, E. S. J. Food Sci. 1974, 39, 188. Jaffe, N. G. “Toxic Constituents of Plant Foodstuffs”, 2nd ed.; Liener, I. E., Ed.; Academic Press: New York, 1980; Chapter 3.
83
Kawashima, N.; Wildman, S. G. Annu. Rev. Plant Physiol. 1970, 21, 325. Knuckles, B. E.; Kohler, G. O., 91st National Meeting of the AIChE, Detroit, MI, Aug 16-17, 1981, Paper 5b. Knuckles,B. E.; Kohler, G. 0. J. Agric. Food Chem. 1982,30,748. Kung, S. D. Science (washington, D.C.) 1976, 191, 429. Lawhon, J. T.; Cater, C. M. J. Food Sci. 1971, 36, 372. Lichtneckert, S.; Lundgren, C.; Ferno, 0. U.S. Pat. Off. Off. Gaz. 1975, 937, 124. Liener, I. E.; Kakade, M. L. ”Toxic Constituents of Plant Foodstuffs”,2nd ed.; Liener, I. E., Ed.; Academic Press: New York, 1980; Chapter 2. Lin, M. J. Y.; Humbert, E. S. J. Food Sci. 1974,39, 368. Lowe, R. H.; Sheen, S. J. Beitr. Tabakforsch. 1982, 11, 161. Marsho, T. V.; Kung, S. D. Arch. Biochem. Biophys. 1976,173, 341. Pirie, N. W. Chem. Znd. (London) 1942,61, 45. Sheen, S. J., University of Kentucky,Lexington,KY, unpublished data, 1982. Sheen, V. D.; Sheen, S. J. “Unconventional Sources of Dietary Fiber”;Funda,I., Ed.; American Chemical Society: Washington, DC, 1983; ACS Symp. Ser. No. 214, Chapter 18. Weissbach,A.; Smyrniotis, P. Z.; Horecker, B. L. J. Am. Chem. SOC.1954, 76, 5572. Wildman, S. G. ”Plants: The Potentials for Extracting Protein, Medicines, and Other Useful Chemicals-Workshop Proceedings”;U.S. Congress, Office of Technology Assessment: Washington, DC, 1983; OTA-BP-F-23, p 63. Wildman, S. G.; Bonner, J. Arch. Biochem. 1947, 14, 381. Wildman, S. G.; Sheen, S. J. U.S. Patent 4289147, 1981. Yamauchi, K.; Shimizu, M.; Kamiya, T. J . Food Sci. 1980, 45, 1237. Received for review April 30,1984. Accepted September 19,1984. This paper has been presented in part at the 185th National Meeting of the American Chemical Society, Seattle, WA, March 21,1983, AGFD 1.
Structure and Fungicidal Activity of Four Pseudoguaianolides Isolated from Helenium quadridentat um Labill Keisuke Watanabe, Yukio Oguri, Masakazu Miyakado, Nobuo Ohno,* and Tom J. Mabry
A new pseudoguianolide, quadridenin, as well as three known pseudoguaianolides, thurberilin, tenulin, and 6-O-angeloylplenolin, were isolated from the Mexican medicinal plant, Helenium quadridentatum (Compositae),as fungicidal constituents. Quadridenin exhibited the most potent fungicidal effect (89?% disease preventive value at 50-ppm treatment) against Pseudoperonospora cubensis, a causative fungi of cucumber downy mildew. This is the first report of pseudoguaianolides as fungicidal constituents against P. cubensis.
In Veracruz, Mexico, leaves and stems of Helenium quadridentatum Labill (Compositae) have been widely
used as folk medicines for colds, stomachaches, and swelling and as a diuretic or an insecticide for fleas (Rosa and Silvia, 1981). Previous study of this medicinal plant species had led to the isolation of helenalin (VII), a typical pseudoguaianolide having strong antitumor activity Pesticides Research Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., 4-2-1Takatsukasa, Takarazuka, Hyogo 665, Japan (K.W., Y.O., M.M., and N.O.), and The Department of Botany, The University of Texas at Austin, Austin, Texas 78713-7640 (T.J.M.).
(Bierner, 1971). The other investigations on the secondary metabolites of Helenium species indicated that they contain pseudoguaianolides with various kinds of biological activities (Fischer et al., 1979). In the course of our screening program to investigate pesticidally active principles of natural origin, we studied the biological activities of constituents of H.quadridentatum. As a result, a CHC1, extract of aerial parts of the plant showed noticeable fungicidal effects, especially against Pseudoperonospora cubensis, a causative fungi of cucumber downy mildew. We now report the isolation and structure elucidation of a new fungicidal pseudoguaianolide (I) as well as three known sesquiterpene lactones, thurberilin (11),tenulin (111), and 6-0-angeloylplenolin (IV) from H.quadridentatum. 0 1985 American Chemical Society
04
Watanabe et al.
J. Agric. Food Chem., Vol. 33, No. 1, 1985
Table I. 'H NMR Spectral Data of I and 11" chemical shift, ppm I assignment CDCL CaDa 11. CDC1, 1 3.05 2.80 m 3.02 m 2 7.70 7.20 dd 7.67 dd 3 6.06 5.85 dd 6.03 dd 5.70 5.75 d 5.57 d 6 3.15 2.90 m 2.55 m 8 4.63 4.20 d t 4.67 dt 9a 2.55 2.70 m 2.50 m 9b 1.55 1.55 m 1.55 m 6.27 d 1.16 d 13a 6.17 13b 5.96 6.12 d 14 1.26 0.95 d 1.25 d 0.90 s 1.23 s 15 1.20 5.87 m 6.05 m 18 6.00 19 1.92 2.00 br d 1.95 br d 20 1.70 1.80 br s 1.73 br s ~~~
~
3
"Coupling constants (Hz) were as follows: 1, 2 = 3.0; 1, 3 = 2.0; 2, 3 = 6.0; 6, 7 = 8.0; 7, 13a = 3.0; 7, 13b = 3.0; 18, 19 = 2.0; 18, 20 = 2.0; 19, 20 = 1.0.
Fungicidal activities of four pseudoguaianolides against P. cubensis are also discussed. EXPERIMENTAL SECTION Instruments. Spectral and physical data were obtained with the following instruments, respectively: 'H and 13C NMR, Hitachi R-900 (90 and 22.6 MHz, Me4& as an internal standard); MS and HRMS, Hitachi M-003 (E1 at 70 eV, direct inlet); IR, Hitachi 260-10 (KBr disk); UV, Hitachi 220; specific rotation, Jasco DIP-181 polarimeter; melting points, Mitamura Riken micro melting point apparatus. Extraction and Isolation. Air-dried and ground leaves and stems of H. quadridentatum (890 g) collected at Victoria City, Tamaulipas, Mexico, in May 14, 1983 (voucher of F. Gonzalez Medrano y Patricia, No. 13052, deposited in the National Herbarium of Mexico), were twice extracted with CHC1, (2.0 and 3.0 L) at room temperature to give 29.3 g of dark syrup that was chromatographed on a silica gel column (Merck, 70-230 mesh, 600 g), eluted with toluene, toluene-EtOAc (9:1, 51, 3:1, 2:1, 1:1, 1:2, and 1:4 v/v), EtOAc, and MeOH (one fraction, 1000 mL), successively. Two fractions that were eluted with toluene-EtOAc, 2:l and 1:1, showed fungicidal activities against P. cubensis. The former fraction (2.0 g) was purified by preparative silica gel TLC (Merck precoated silica gel 60 GFZu,20 X 20 cm, 2.0 and 0.5 mm) and developed with toluene-EtOAc, 2:l (v/v), to give three fungicidal compounds, quadridenin (I, 96 mg, 0.011% ,Rf 0.35), thurberilin (11, 47 mg, 0.005%, R, 0.30), and 6-0angeloylplenolin (IV, 103 mg, 0.012%, R, 0.40) as white needles, respectively. The latter frr :tion (6.0 g) gave tenulin (111, 4.8 g, 0.55%) as amorphous powders. Quadridenin (I): mp 183-185 "C (ethyl ether); [(r]D$13.2 (c 0.34, CHC1,); C2aH2405for M+ found 344.1099, calcd 344.1112; MS m / z (re1 intensity) 344 (2.0), 245 (12), 83 (loo), 55 (20); UV A,, (EtOH) 218 (e 15200), 320 ( E 65); IR v",",: 1755,1720,1710,1670,1580cm-'; 'H and I3C NMR data are given in Tables I and 11. Thurberilin (11): mp 161-162 "C (ethyl ether) [lit., mp 164-165 "C (Herz and Lakshimakikantham, 1965)];IR, 'H NMR, and MS spectra and [aIDwve identical with those of an authentic specimen that 1s provided by Prof. W. Herz. Tenulin (111): mp 193-i 5 OC (acetone) [lit. mp 191-192 "C (Herz and Rabindran, 19561; IR, 'H NMR, and MS spectra and [(r]D were identical with the published data (Herz et al., 1963). 6-0-Angeloylplenolin(IV): mp. 128-129 "C (ethyl ether) 3
Table 11. NMR Spectral Data of I and I1 carbon no. I I1 1 54.2" db 56.6 d 162.7 d 2 162.6 d 3 130.8 d 130.7 d 4 209.1 s 209.3 s 56.6 s 5 56.4 s 74.0 d 6 72.7 d 51.9 d 7 52.4 d 76.2 d 8 76.3 d 44.5 t 9 44.4 t 27.2 d 10 27.3 d 11 137.4 s 42.2 d 12 169.4 s 177.5 s 13 122.0 t 14.0 q 19.7 q 14 19.7 q 15 22.4 q 23.2 q 16 166.3 s 166.0 s 127.2 s 17 127.3 s 18 139.1 d 139.2 d 19 15.7 q 15.6 q 20.3 q 20 20.3 q 'In CDCl8. Signals were assigned by means of single-frequency off-resonance decoupled spectra. Indicates multiplicities on offresonance decoupled spectra.
[lit. mp 127-128 "C]; IR, 'H NMR, and I3C NMR spectra and [aIDwere identical with the reported data (Itoigawa et al., 1981). Catalytic Hydrogenation of Quadridenin (I). A total of 20.0 mg of quadridenin (1) in EtOH (10 mL) was hydrogenated in the presence of 15 mg of PtOz at room temperature for 2 h. The reactant gave two spots (silica gel TLC, 30% EtOAc in toluene). The major product (Rf 0.37,15.0 mg, mp 183-184 "C) was identical with respect to IR, 'H NMR, and MS spectra and mp with those of tetrahydrothurberilin (V), which was prepared by hydrogenation of thurberilin (II) (Herz and Lakshimakikantham, 1965). The minor product (VI, Rf 0.40,4.0 mg, mp 98-99 "C) gave the following spectral data: MS m / z (re1 intensity) 248 (42, M+ - 102), 220 (20), 85 (72), 57 (100);IR 1770, 1730, 1710 cm-'; 'H NMR a$ F:d 0.90 (3 H, t, J = 7.0 Hz), 1.08 (3 H, s), 1.10 (3 H, d, J = 7.0 Hz), 1.13 (3 H, d, J = 7.0 Hz), 1.55 (3 H, d, J = 7.0 Hz, CI1P-methyl), 4.67 (1H, dt, J = 3.0, 12.0 Hz, H8), and 5.23 (1 H, d, J = 7.0 Hz, H6). Bioassays. Seeds of cucumber (species sagumihanjiro) were sowed in soil filled in plastic pots and cultivated in a greenhouse for 14 days to obtain seedlings of cucumber having cotyledon. A definite amount of the test compounds (500, 200, 50, and 12.5 pg/mL) in the form of emulsified solution with Sorpol and water were applied onto the seedlings by foliar treatment. After the seedlings dried, a spore suspension of P. cubensis (6 X lo6 cells/mL) was sprayed onto the seedlings, which were placed at 20 "C under a humid condition for 1 day and then grown at 20 "C under the irradiation with a fluorescent lamp for 5 days. The infectious state of the plant was observed, and the preventive effect value was calculated according to the following equations. The infectious state was indexed as follows: index 0, no infectious spots on the leaf, index 0.5, infectious spots of less than 5% of the leaf area; index 1, infectious spots of less than 20% of the leaf area; index 2, infectious spots of less than 50% of the leaf area; index 4, infectious spots of more than 50% of the leaf area. degree of infection, % = C[(infectious index) X (no. of leaves)1/4(total no. of leaves) x 100 (1) preventive effect value, % = 100 - (degree of infection in treated plot)/(degree of infection in nontreated plot) x 100 (2)
v2:
Pseudoguaianolides Isolated from H. quadrbntafum
J. Agric. Food Chem., Vol. 33, No. 1, 1985 85
Table 111. Fungicidal Activities of Pseudoguaianolides I-IV against P. cubensis disease control, %
I
I1
compound 500 pg/mL CHC13 extract 57 I 98
I1 I11 IV
111
V RGH, Rz=CH3 V I R F C H ~ Rz=H ,
N
VI1
Figure 1. Sesquitepene lactones and their derivatives from H . quadridentatum L. RESULTS AND DISCUSSION A CHC13extract of H. quadridentatum L. yielded four sesquiterpene lactones (I, 11,111, and IV) upon chromatography on a silica gel. Three known pseudoguaianolides, thurberilin (II),tenulin (III),and 6-0-angeloylplenolin (n3, were identified according to their reported physicochemical properties or direct comparison with an authentic specimen. Previously, Biener reported the isolation of an antitumor sesquiterpene lactone helenalin (VII) from H . quadridentatum L. (Bierner, 1971); however, helenalin (VII) was not detected at all in the extract of the present analysis. Although some Helenium species have been known to contain pseudoguaianolides11-IV (Fischer et al., 1979), this is the first isolation of 11-IV from H.quadridentatum L. (see Figure 1). The structure of the new fungicidal pseudoguaianolide (C&%05, mp 183-185 "C) to which a trivial name quadridenin (I) is proposed here was established as follows. The presence of an a-methylene y-lactone moiety was indicated by its IR (1755,1670 cm-') and 'H NMR (a pair of one-proton doublets a t 6 6.17 and 5.96, J = 3.0 Hz) spectra. The IR absorption at 1720 cm-' coupled with MS (m/z83, base peak) suggested a C5a,&unsaturated acyloxy group attached to a sesquiterpene lactone skeleton. This side chain was assigned to be an angeloyloxy group by ita 'H NMFt signals at 6 6.00 (1H, m), 1.92 (3 H, br d, J = 1.0, 7.5 Hz), and 1.70 (3 H, br s, J = 1.0, 2.0 Hz). Two low-field signals at 6 7.70 (1 H, dd, J = 2.0, 6.0 Hz) and 6.06 (lH, dd, J = 3.0, 6.0 Hz) suggested the presence of an a,@-unsaturated cyclopentenone moiety that is a characteristic functional group of pseudoguaianolidessuch as 11-IV. 13C NMR data (see Table 11)as well as IR absorption at 1710 cm-' also supported the presence of the a,b-unsaturated ketone ring system. On irradiation of a multiplet at 6 3.15, two doublets for H1&and H13b changed into sharp singlets, indicating that this signal should be assigned to H7. A t the same time, one doublet at S 5.70 (1H, J = 8.0 Hz) as well as one double triplet a t 6 4.63 (1H, J = 3.0,3.0,12.0 Hz) were changed into a singlet and
98 98 55
200 pg/mL 67 98 88 92 25
50 pg/mL
19 89 45 31 0
12.5 pg/mL 13 0 12 0 0
broad doublet, respectively. The similarities of the chemical shifts and coupling patterns of I to those of thurberilin (11) from Helenium thurberili (Herz and Lakshimakikantham,1965; Romo et al., 1966) suggested that they have the same stereochemical relationships at C6,C,, and C8. Thus, I was estimated to be a C7,C8 trans-fused lactone, and the signal at 6 5.70 was assigned to the P-methine proton on C6to which an angeloyloxy group was attached. Furthermore, two methyl signals at 6 1.20 (3 H, s) and 1.26 (3 H, d, J = 7.0 Hz) were assigned to the C5 and Clo methyls, respectively. On the basis of these spectral considerations, the structure of quadridenin was formulated as I except for the configurations at the C5 and Clo methyl groups. Catalytic hydrogenation of quadridenin (I) furnished a two-component mixture. The major product (Rf0.37) was identical in all respects (IR, 'H NMR, MS, [a]D, and mp) with tetrahydrothuruberilin (V), which was prepared by us from an authentic sample of I1 (Herz and Lakshimakikantham, 1965). The minor product (VI, Rf 0.40) was estimated to be the CI1 epimer of V (see Figure 1). Accordingly, the structure of quadridenin was unambigously established as I. The co-occurrence of 7,8 trans-fused pseudoguaianolides, 1-111, and a 7,8 cis-fused lactone, IV, from the same plant species is interesting from the viewpoint of the biosynthesis of these sesquiterpenoids. Biological Activities. The fungicidal activities of quadridenin (I), thurberilin (II), tenulin (III), and 6-0angeloylplenolin (IV)against P.cubensis are given in Table 111. Among these four pseudoguaianolides, quadridenin (I) exhibited the most potent activity (at 50 ppm, 89% preventive effect value). Three pseudoguaianolides, I, 11, and IV, each bearing an angelic ester moiety at C6,were more active than tenulin (III),a major sesquiterpene lactone from this species. Thurberilin (11) showed almost the same activity as that of 6-0-angeloylplenolin (IV),indicating that the stereochemistries at C8 and Cl1 are not critical for the activity. Furthermore, the a-exomethylene moiety of y-lactone in I seemed not to be required as well, because thurberilin (11) and 6-0-angeloylthurberilin (IV) retained the fungicidal activities despite the presence of a saturated y-lactone moiety. Studies with respect to additional biological activities of quadridenin (I) are now in progress. ACKNOWLEDGMENT We thank G. Medrano and P. Hiriart for the plant collection. We are indebted to Prof. W. Herz for a gift of an authentic specimen of thurberilin and to H. Nakazawa for obtaining mass spectra. Registry No. 1, 93600-83-0; 2, 3668-12-0; 3, 19202-92-7;4, 93600-84-1;5, 3668-11-9; 6, 63640-22-2. LITERATURE CITED Bierner, M. W. Biochem. Syst. Ecol. 1971, I, 97. Fischer, N. H.; Olivier, E. J.; Fischer, H. D. "Progress in the Chemistry of Organic Natural Product 38"; Springer-Verlag: New York, 1979; pp 223-256.
J. Agric. Food Chem. 1985, 33, 86-89
88
Herz, W.; Lakshimakikantham,M.V. Tetrahedron 1965,21,1711. Herz, W.;Rabindran, K. J . Am. Chem. SOC.1956,78,4423. Herz, W.; Romo de Vivar; Romo, J.; Viswanathan, N. J. Am.
Rosa, M. M.; Silvia, A. R., Ed. "Plantas medicinales del estado de Yucatan"; Compania Editional Continentak Veracruz, Mexico, 1981;p 169.
Chem. SOC.1963,85,19.
Itoigawa, M.; Kumagai, N.; Sekiya, H.; Itoh, K.; Furukawa, H. Yakugaku Zasshi 1981,101, 605.
Romo, A. V.; Rodriguez-Hann,L.; Romo, J.; Lakshimakikantham, M. V.; Mirriigton, R. N.; Kagan, J.;Hen, W. Tetrahedron 1966, 22,3279.
Received for review May 24,1984.Accepted September 19,1984. T.J.M. received financial support from the Robert A. Welch Foundation (Grant F-130)and the National Institutes of Health (Grant HDO-4488).
Behavior of Acylanilide and Dicarboximidic Fungicide Residues on Greenhouse Tomatoes Paolo Cabras, Marco Meloni, Filippo M. Pirisi,* and Franco Cabitza The residual behavior of four dicarboximidicand three acylanilidic fungicides was studied in greenhouse grown tomatoes. In different experiments tomatoes underwent single spraying and five sprayings with an interval of 21 days, at rates of application of 380 and 760 g/ha (acylanilides) and 1500 and 3000 g/ha (dicarboximides). The harvesting was carried out weekly. Residues recovered after repeated sprayings confirmed the well-known trend of the four dicarboximidicfungicides: toxically significant accumulation in fruits several days after recommended preharvest times. Among acylanilides, only furalaxyl and benalaxyl showed the accumulation of residues but always at concentrations lower than legal limits, even before the fixed preharvest times. The possible need is raised to revise preharvest times for both categories of fungicide. When tomatoes were single sprayed, the fungicide disappearance from fruits showed pseudo-first-order rate dependence only for dicarboximides; degradation products of the latter, found in a different matrix (wine), were not detected. The fungal diseases most commonly present in greenhouse grown tomatoes are those generated by Botrytis cinerea and Phytophthora infestans. Dicarboximides, of general formula I, are usually employed to control B.
< = o CbIO10 ,male
"
~
-d/'l.
L
4-
CHI
cinerea, while acylanilides, of general formula 11, are usually employed to control P. infestans (Gozzo, 1979). The behavior of residues of the dicarboximidic fungicides vinclozolin and iprodione has been studied by VaCentro Regionale Agrario Sperimentale, 09100 Cagliari, Italy (P.C. and F.C.), and Facoltl di Farmacia dell'Universitii, Istituto di Tecnica e Tecnologie Farmaceutiche, 09100 Cagliari, Italy (M.M. and F.M.P.). 0021-856118511433-0086$01.50/0
nachter et al. (1979) and Van Wambeke et al. (1980) on greenhouse-grown tomatoes. These authors reported an accumulation effect of fungicide residues, due to repeated applications of the same active ingredient (ai). The same effect has been observed on greenhouse-grown lettuce (Meloni et al., 1984; Dejonckheere et al., 1982) and on field-grown grapes and tomatoes (Cabras et al., 1982a,b) as well as with pesticides belonging to different chemical families. Since this effect could result from different ways of using the fungicides and no data are available on the behavior of the acylanilide residues on greenhouse tomatoes, we decided to compare the behavior of the acylanilides and dicarboximides mentioned above. The specific objectives were (1) to complete, under standardized conditions, the data of Vanachter and Van Wambeke on the dicarboximidic fungicides vinclozolin and iprodione, also studying the behavior of procymidone and chlozolinate, (2) to ascertain if acylanilide fungicide behavior was similar to that of the dicarboximides, (3) to ascertain if the simultaneous spraying of several different a.i., belonging to the same family, could affect the content of their residues, (4) to ascertain if the same dicarboximide degradation products found in wine (Cabras et al., 1984) could also form in tomatoes, (5) to study the degradation kinetics of all the fungicides used in the experiments, and (6) to check if, in usual agronomic handling (repeated applications, weekly harvest, etc.), the preharvest times produce residues corresponding to toxic risk. EXPERIMENTAL SECTION Materials and Methods. The trial was carried out inside a 500-m2glasshouse with galvanized iron framework, air warmed and equipped with a drop irrigation system. The tomatoes employed Were Vemone I (SLU GRO) and 81 T I UPM (Clause). Sowing was done on Nov 12,1982, and transplantation on Dec 10, 1982. A random block 0 1985 American Chemical Society
