C h a p t e r 27
Increased Concentration of Myristicin and 6-Methoxymellein in Carrot Root upon Irradiation with UV Light
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Shelly G. Yates Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604
Myristicin has not been reported to possess antifungal activity, and therefore is not a phytoalexin according to the standard interpretation of this term (1). It does, however, potentiate the activity of the insecticide, paraoxon, in flies by inhibiting its degradation (2), and may in similar manner potentiate the action of phytoalexins of carrot root (falcarinol, falcarindiol, 6-methoxymellein) (3,4). The presence of myristicin in carrots is of interest to nutritionists because of its biological properties (5), and its chemical similarity to safrole, a mild carcinogen (6). Results of Wulf et al (7) show that carrot roots obtained from a supermarket contain myristicin; Imperator variety carrots contain an average of 15 parts per million (ppm). Recently harvested, unprocessed carrots only rarely contain myristicin (8). The presence of myristicin in supermarket carrots and its absence in recently harvested ones indicate that its increased concentration may have been induced by some elicitor following harvest. Solar radiation after harvest, or fluorescent lighting during display, may function as such an elicitor. Light is known to produce ethylene and is an activator of phenylalanine ammonia-lyase, one of the regulatory enzymes responsible for phenylpropanoid biosynthesis in plants (9). In the following experiments carrot roots were exposed to various sources of ultraviolet light in the laboratory and set aside to allow time for enzyme synthesis. Following this period, changes in myristicin and phytoalexin levels were measured. A l l of these components of carrot root are measured in one assay. Myristicin and 6-methoxymellein concentrations increased in some samples after irradiation with ultraviolet light; falcarinol and falcarindiol concentration changes did not appear to be related to the ultraviolet light used in this study. Experimental Plant source. Carrot roots were bought in a supermarket or grown in the Northern Regional Research Center field plot. Carrots from the supermarket were used without further cleaning; carrots harvested from the field plot were brushed lightly to remove soil and debris, and used without further cleaning. Identical samples for UV radiation studies were prepared by halving longitudinally each of two carrots and pairing the different halves to give two samples, or by quartering longitudinally each of This chapter not subject to U.S. copyright. Published 1987 American Chemical Society
Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
296
A L L E L O C H E M I C A L S : R O L E IN A G R I C U L T U R E A N D
FORESTRY
four carrots and combining a quarter of each carrot with others to give four samples. Protocol. Following a 1-h i r r a d i a t i o n period with u l t r a v i o l e t l i g h t , carrots were placed i n a polyethylene bag and kept i n the dark at ambient temperature--along with an unirradiated control sample--for an induction period. Changes i n carrot constituents were measured at the end of the induction period by analysis of p u r i f i e d dichloromethane extracts using gas-liquid chromatography (GLC) (8). Response factors for m y r i s t i c i n , f a l c a r i n o l , and f a l c a r i n d i o l were determined by comparing the detector response of pure standards to that of methyl palmitate. The response factor of 6-methoxymellein was assumed to be one. Data from both low-polarity (SE-52) and intermediate-polarity (OV-17) columns were used for peak i d e n t i f i c a t i o n ; data from the low-polarity column only were used for quantitation. 6-Methoxymellein was also i d e n t i f i e d by gas chromatography/ mass spectrometry. Irradiated samples were compared to an i d e n t i c a l , unirradiated control kept under the same induction conditions. Light source. The most suitable l i g h t system examined was a Chromato-Vue Model C-3 from U l t r a V i o l e t Products, Inc., San Gabriel, CA. The l i g h t source was a GE G15T8, 15-W, Germicidal, 2537 bulb. The mercury lamp emitted radiation maxima at 254, 265, 280, 302, 313, 365, 405, and 436 nm. Radiation from this source passed through a f i l t e r which transmitted radiation only between 230 and 410 nm, with peak transmission at 360 nm. Total u l t r a v i o l e t energy received by the carrots was approximately 800 ergs cm s (Kettering Radiometer, Model 68, Charles F. Kettering Research Laboratory, Yellow Springs, Ohio). 2
Results and
1
Discussion
A four-year study of field-grown commercial carrot roots revealed that recently harvested, unprocessed carrot roots contained 24 ppm f a l c a r i n o l and 65 ppm f a l c a r i n d i o l (8). 6-Methoxymellein (6-MM) had not been i d e n t i f i e d by Yates et a l (8) at that time, and was not measured i n that study. Reexamination of data revealed that 6-MM was absent from most samples, but present i n a few at concentrations of 2 to 8 ppm. M y r i s t i c i n , 1 ppm, was detected i n only one sample. Wulf et a l 1978, reported that m y r i s t i c i n was present i n supermarket carrots. Other studies have shown that certain brands of supermarket carrots contain m y r i s t i c i n while others do not (Yates, unpub.). The presence of m y r i s t i c i n i n some samples from the supermarket and i t s absence i n unprocessed carrots analyzed as soon after harvest as possible suggests that m y r i s t i c i n formation i s induced during some stage of processing. Since l i g h t i s known to be an e l i c i t o r of a plant system that results i n the synthesis of phenylpropanoid compounds, a study of the e f f e c t of l i g h t on harvested carrot roots was undertaken. Preliminary experiments, using a container with r e f l e c t i v e surfaces, employed l i g h t from eight fluorescent bulbs that produced a preponderance of radiation at 310 nm. M y r i s t i c i n increased i n samples that were i r r a d i a t e d 1 h and then kept for a 24-h induction period. However, heat produced by this system and the r e s u l t i n g dehydration of carrot roots may have affected r e s u l t s . Later experiments, employing a Chromato-Vue box used for u l t r a v i o l e t examination of TLC plates, avoided these problems. One-hour l i g h t
Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
ND
ND
ND
ND
ND
.035
ND
ND
ND
.028
ND
ND = not detected, ΝΑ = ιnot analyzed.
2
2
2
2
2
Imperator 58
Imperator 58
Imperator 58
Imperator 58
Imperator 58
ND
NA
NA
NA
NA
NA
NA
.031
ND
.058
.033
.062
.124
2
.193
ND
NA
NA
NA
NA
"^Supermarket carrots.
NA
NA
ND
.070
ND
.068
.014
.014
.008
.008
.017
.021
F i e l d p l o t carrots.
.016
.018
ND
ND
ND
.112
NA
NA
NA
NA
NA
.155
.066
.722
2.186
.202
.065
.049
NA
NA
NA
NA
NA
NA .211
.565
NA .062 .120
NA
NA .944 .965
^Per 25 g fresh carrot root.
NA
NA
.088
.173
ND
.046
.079
ND
.012
NA
NA
ND
ND
ND
ND
Buni Luv*
2
.165
.126
NA
NA
ND
ND
ND
ND
ΝΑ
Shirley Fresh*
Imperator 58
.611
.302
NA
NA
.090
.102
.090
.058
6-Methoxymellein Concentration Dark Induction Period Beginning Control 24 h 48 h 72 h 120 h (mg) (mg) (mg) (mg) (mg) (mg)
ΝΑ
Beginning (mg)
M y r i s t i c i n Concentration Dark Induction Period Control 24 h 48 h 72 h 120 h (mg) (mg) (mg) (mg) (mg)
Changes i n Carrot Root Irradiated One Hour with U l t r a v i o l e t Light: M y r i s t i c i n and 6-Methoxymellein
Buni Luv*
Brand or Variety
Table I.
Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
1
2
2
2
2
2
2
1
.688
.320
.725
.991
.682
.241
1.040
NA
NA
.565
.266
.453
.450
.625
.202
.982
.236
.648
ND = not detected, ΝΑ = not analyzed.
Imperator 58
Imperator 58
Imperator 58
Imperator 58
Imperator 58
Imperator 58
Buni Luv
Shirley F r e s h
Buni Luv*
Brand or Variety
NA
NA
NA
NA
NA
NA
.834
.219
.456
.610
.300
.428
.564
.699
.229
NA
.199
.516
.823
.296
NA
NA
NA
NA
NA
NA
NA
Supermarket carrots.
NA
NA
.608
1.280
.202
.606
.944
.219
.464
1 .614
1 .244
2 .217
2,.274
1 .906
1,.334
1,.078
NA
NA
2,.021
1 .557
1,.526
1,.965
2,.563
1,.477
1,.069
1,.013
1 .272
NA
NA
NA
NA
NA
NA
1.278
.972
1.482
1 .663
1,.459
1 .496
1..923
3,.287
1..799
NA
1,.034
1,.590
3.445
1.372
NA
NA
NA
NA
NA
NA
NA
Per 25 g fresh carrot root.
NA
NA
1 .773
3 .719
1,.315
2,.381
1,.024
.959
1,.246
F a l c a r i n d i o l Concentration Dark Induction Period Beginning Control 24 h 48 h 72 h 120 h (mg) (mg) (mg) (mg) (mg) (mg)
F i e l d p l o t carrots.
F a l c a r i n o l Concentration Dark Induction Period Beginning Control 24 h 48 h 72 h 120 h (mg) (mg) (mg) (mg) (mg) (mg)
Table I I . Changes i n Carrot Root Irradiated One Hour with U l t r a v i o l e t Light: F a l c a r i n o l and F a l c a r i n d i o l
27.
YATES
Myristicin
and 6-Methoxymellein
in Carrot
Root
299
treatments using the Chromato-Vue box were followed by induction periods of 24 to 120 h. The last light-treated sample and nonirradiated control sample were analyzed at the same time. Myristicin content of some carrot samples was increased twoto five-fold over nonirradiated controls (Table I). The increase in concentration of myristicin is presumed to be via the phenylpropanoid pathway; phenylalanine ammonia-lyase, an enzyme of that system, is activated by light (9). Failure of some samples exposed to UV light to synthesize myristicin may be due to the absence or inhibition of a key enzyme needed for myristicin synthesis. The dramatic increase in 6-MM content--90 to 270 fold in two samples--was unexpected. 6-MM is a potent antifungal agent (4) and one of the most important carrot phytoalexins. Usually 6-MM, one of the components that contributes to the bitterness of stored carrots (I0) is not detected in fresh carrots, but develops during storage. Biosynthetic studies indicate that 6-MM is synthesized via the acetate pathway and its production is stimulated by ethylene (11). Thus, UV light may trigger ethylene production in carrots which in turn leads to 6-MM accumulation. Falcarinol and falcarindiol concentration changes are small com pared to those in myristicin and 6-MM content, and do not appear re lated to radiation (Table I I ) . These polyacetylenes are present i n fresh carrots. Immediately upon wounding, they are translocated to the surface through o i l ducts (3), and, therefore, their function as phytoalexins apparently does not depend upon de novo synthesis. Although myristicin is not considered to be an antifungal agent, an increase in myristicin concentration in some carrot roots upon exposure to UV radiation indicates that i t may have some protective function. y
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