- Email: [email protected]
Nordihydroguaiaretic acid: inter- and intrapopulational variation in the sonoran desert creosote bush (Larrea tridentata, zygophyllaceae.
B'~chernical Systerna~c,~and Ecology,Vol. 16, No. 6, pp. 551-555, 1988. Printed in GreatBritain.
0305-1978188$3.00-F0.00 ¢) 1988PergamonPressplc.
Nordihydroguaiaretic Acid: Inter- and Intrapopulational Variation in the Sonoran Desert Creosote Bush (Larrea tridentata, Zygophyllaceae)* K. R. DOWNUM, J. DOLE1" and E. RODRIGUEZt Department of Biological Sciences, Florida International University, Miami, FL 33199, U.S.A. l"Phytochemistry and Toxicology Laboratory, Developmental and Cell Biology, University of Califomia, Irvine, CA 92717, U.S.A.
Key Word Index--Larrea t~'dentata; creosote bush; nordihydroguaiaretic acid; NDGA; lignan catechol; leaf resin; Sonoran desert; interpopulational and intrapopulational phytochemical variation. Abstract--Nordihydroguaiaretic acid (NDGA) is the principal phenolic constituent in the leaf resin of the creosote bush, Larrea tridentata, the dominant perennial shrub throughout most of the warmer arid and semi-arid regions of North America. In an effort to determine the quantitative significance of this biologically active lignan catechol in desert ecosystems, we conducted studies of leaf extracts from geographically distinct populations of Larrea which occur throughout the Sonoran desert. Seasonal influence on NDGA concentration as well as on the hexane- and MeOH-soluble leaf constituents were also examined within two populations of Larrea growing near Palm Desert, California. Interpopulational studies revealed that NDGA concentrations declined from northern to southern sampling sites whereas the levels of hexane- or MeOH-soluble leaf components showed little variation. Intrapopulational studies showed that the mean level of both hexane- and MeOH-soluble leaf components increased significantly between mid-April and late June, but that these increases slowed down or stopped between June and July. The mean concentration of NDGA decreased significantly in plants between April and July suggesting a possible relationship between the resin concentration of NDGA and seasonal (i.e. climatic) changes. These observations may, in part, explain some of the latitudinal variation in NDGA levels found in Sonoran desert representatives of Larrea.
Introduction believed to function as a barrier to prevent water The creosote bush, Larrea tridentata (Seese & loss from leaf surfaces, a solar-UV filter which Moc. ex DC.) Coville, is the most abundant and protects foliage from harmful wavelengths of widely distributed evergreen shrub in the light and an insect feeding deterrent [5]. In warmer arid/semi-arid regions of North America addition to the above activities, Larrea leaf resin extending from southern California and south- has been shown to elicit phototoxic responses western Utah, south through Baja California and from various micro-organisms including E. coli Sonora, and east through Western Texas and and the yeast Saccharomyces cerevisiae [6]. The the states of Chihuahua, Coahuila and San Luis resin contains a complex mixture of phenolics, Potosi, Mexico [1-3]. The abundance of this saponins, terpenoids and wax esters that woody shrub, which may attain densities of up account for 10-20% of the leaf dry weight [7-9], to 1700 shrubs per ha in certain locations [4], although the resin on immature foliage consticombined with its perennial growth habit, tutes a considerably higher percentage [9]. makes Larrea among the most important and Approximately 80% of the leaf resin is predictable plant resources in many desert composed of phenolic compounds of which environments. nordihydroguaiaretic acid (NDGA; Fig. 1) is the The leaves of this highly xerophytic shrub are major component. This lignan catechol covered with a resinous coating which is reportedly accounts for 5-10% of the dry leaf weight [7], is a potent antioxidant [10] and mediates important bactericidal, fungicidal and *Dedication: In memory of the late Professor Tony Swain: antiherbivore activities [5, 8, 11-15]. Mentor, scholar and friend. NDGA may well represent one of the most (Received 29 Apri/1988) quantitatively important and widely distributed 551
552
K.R. D O W N U M , J. DOLE A N D E. RODRIGUEZ
Ha
H
O
~
i
'
~
'
~
California
0 H
~.~
~
',
@
\ ~ O H
Arizona
,~
,
FIG. 1, THE CHEMICAL STRUCTURE OF THE LIGNAN CATECHOL NORDIHYDROGUAIARETIC ACID (NDGA).
8~a
allelochemicals in environments where Larrea occurs. The present studies were initiated to determine the quantitative significance of NDGA in Sonoran Desert populations of Larrea, to evaluate the inter- and intrapopulational variation of hexane- and MeOH-soluble components of the creosote bush leaf resin and to ascertain whether the levels of these components fluctuate over the growing season.
California
~
\, ~,
.... .
)'~"~'~
" ~
~
Pacific ~ Ocean
@
'~
~
J' ~
Results and Discussion Sonoran desert populations of the creosote bush extend from southern California and Arizona at the northern limit (ca 35°N latitude), south through Baja California (to ca 24°N latitude) and east into the state of Sonora on the FIG. 2~ COLLECTION LOCATIONS OF LARREATRIDENT TA A mainland of Mexico. Leaf samples from popula- THROUGHOUT THE SONORAN DESERT, Collections w e r e made at 8 0 tions of L. tridentata were collected for 100 km intervals. phytochemical analysis at 80-100 km intervals throughout the Sonoran limits of this species populations. Values for the hexane- and MeOH(refer to Fig. 2 for collection locations). The soluble components did not vary significantly specific geographic coordinates for each of the throughout the geographic range of the populacollection sites are listed in Table 1. tions sampled. Mean percentages of both The concentration of hexane- and MeOH- hexane-soluble (e.g. saponins, terpenoids, soluble leaf constituents and NDGA were deter- waxes, etc.) and MeOH-soluble (e.g. NDGA and mined from the leaves of these creosote bush ~ other phenolic constituents) leaf components TABLE 1. LONGITUDINAL A N D LATITUDINAL COORDINATES OF COLLECTION SITES 1 - 2 7 Collection
North
West
Collection
North
West
number
(latitude)
(longitude)
number •
(latitude)
(longitude)
01
30 ° 0'
115°15 '
15
28° 27'
111°15 ,
02
29 ° 50'
114 ° 35'
16
28~12 '
111 ° 3'
03 04
29°25 , 28 ° 5'
1 1 ~ 20' 113°36 ,
17 18
28 ~ 24' 28 ° 39'
111 ° 3' 111°36 ,
05
2 ~ 21'
1 1 ~ 6'
19
29 ~ 0'
1 1 ~ 55'
06
27°6 ,
111°54 ,
20
30 ° 0'
111 ° 6'
07 08
26 ° 33' 26 ° 9'
111 ° 45' 111 ° 21'
21 22
31~58 ' 32°26 '
111 ° 0' 111 ° 5'
09
25 ° 3'
111 ° 39'
23
32 ° 52'
112°18 '
10
24°45 ,
111°30 ,
24
32°54 ,
113 ° 0'
11
24°24 ,
111 ° 0'
25
32°10 '
11 ~ 43'
12
24 ° 6'
110 ° 33'
26
32°18 '
114 ° 45'
13
26 ° 57'
110°36 '
27
33°15 '
115 ° 35'
14
28 ° 51'
111 ° 50'
NORDIHYDROGUAIARETICACID VARIATION IN LARREA TRIDENTATA
based on dry weight measurements (,~+ SD; n=75) were 4.9%:1:1.6 and 21.9%+2.5, respectively. The levels of these constituents did not correlate with either the latitude or longitude of the collection locations (data not shown). NDGA, in contrast, showed a significant correlation (r2=0.59) with the latitude of the sampling sites (Fig. 3). The concentration of NDGA in leaf extracts ranged from a mean low of 6.4 mg g-1 at the southernmost boundary of the species (Coll. No. 12; 24°N) to a mean high of 60.2 mg g-1 near the northern limit of the study (Coll. No. 25; 32°N). NDGA was also correlated with study site longitude (r2=0.22; levels increased with increasing longitude), but this correlation was much less significant. The combined hexane- and MeOH-soluble leaf extracts yield values that are intermediate between leaf resin values reported in other studies of Larrea. Rhoades [5, 9], using diethyl ether as an extraction solvent, found leaf resin values ranging from 10 to 26% depending on leaf age (young leaves contained the highest levels) from Arizona populations of L. tridentata. Duisberg [16], in studies wtih L tridentata from New Mexico, published leaf resin values of 30-35% for foliage extracted with 95% EtOH. These latter values are considerably higher than both our values and those reported by Rhoades. These .: 60
•
•
50
! ~
40
~,
30
;
° I~
,~.
•
•
•
•
o ::
20 ~~
,o ,o
0
~ t
I
24
. =
I
26
• l
.~ I
28
I
I
I
~
l
32
t
a
34
Lotitude (=N) F~. 3. C ~ E ~ O N OF N ~ ( ~ mP t) ~RSUS ~ U D E THE S ~ WHERE ~ R R ~ L ~ F ~MPLES WERE COLLE~D.
OF
553
percentages may reflect: (1) differences due to the extraction solvent used in the particular studies; (2) differences in the age of the plant tissues extracted which have varying levels of resin; and/or (3) quantitative phytochemical differences between the diploid populations characteristic of New Mexico and the Chihuahuan desert and the tetraploid populations of the Sonoran desert [17, 18]. The concentration of NDGA in MeOH-soluble extracts decreases from northern to southern sampling sites, in contrast to the leaf resin (hexane- and MeOH-soluble leaf extracts). To our knowledge, a similar phytochemical relationship has never been demonstrated before. Different climatic conditions (e.g. precipitation, solar irradiation, temperature, etc.) experienced by plant populations growing near the latitudinal extremes of the species in the months preceding the study may account for some of ~the observed phytochemical variation. Plants at the northern latitudes generally appeared more healthy and had abundant new growth during the sampling period whereas plants growing near the southern limit of the species were noticeably drier and had considerably less new growth. Between-plant variation and the influence of seasonal change on leaf resin and foliar NDGA levels were investigated to determine the effect that these factors might have on plants from populations growing at different latitudes. Table 2 summarizes data gathered from a total of 97 individuals distributed between adjacent research plots (Plots I and III) of L. tndentata at Boyd Deep Canyon Desert Research Center at two different times of the year. The mean level of hexane- and MeOH-soluble leaf constituents (i.e. the leaf resin) increased significantly (P<0.001) in Plot I between mid-April and late June. The increase in hexane-soluble leaf constituents in Plot Ill was less significant (P<0.01) during late June through July, whereas the level of MeOH-soluble compounds did not change appreciably. In contrast, the mean concentration of NDGA in Plots I and III decreased by 9 and 15% during the same sampling periods, respectively. These studies suggest that the concentration of leaf resin (hexane-soluble+MeOH-soluble extracts) increases during early spring, but that this
554
K.R. DOWNUM,J. DOLEAND E. RODRIGUEZ
TABLE 2. QUANTITATIVECOMPARISONOF THE HEXANE-AND MeOH-SOLUBLELEAF CONSTITUENTSAND NDGAEXTRACTEDFROM LARREA TRIDENTATA LEAVESAT 33NODIFFERENTTIMESOF THE YEAR Plot
Collection date
Hexane-solubte components (%±SD)
MeOH-soluble components (%±SD)
NDGA (mg g-~ dw _+SD)
I (n--49)
16 April 1984 28 June 1984
3.2"}- 1.9 7.2± 1.9"
23.5+3.0 27.6±4.6*
59.7±13,2 54.1+11.5'
III (n-48)
28 June 1984 25 July 1984
7.1± 1.4 8.5±2.0~:
27.4±3.1 27.9±4.5
66.5+ 13,0 56.7±13,1t
*Significantly different (P< 0.001). 1"Levelsof significancebetweencollectiondatesat the two plots were determinedusing the paired t-test. $Significanttydifferent (P< 0.01). i n c r e a s e s l o w s (or s t o p s ) as t h e s u m m e r p r o g r e s s e s . T h e i n c r e a s i n g resin c o n t e n t in Plot I, w h i c h w a s o b s e r v e d b e t w e e n m i d - A p r i l a n d late J u n e , c o i n c i d e d w i t h t h e s p r i n g flush of growth o n L a r r e a at D e e p C a n y o n . It is c o n c e i v a b l e t h a t t h e i n c r e a s e d resin l e v e l s during this period may be due to sampling of n e w g r o w t h w h i c h is c o m p o s e d o f a h i g h e r p e r c e n t a g e o f resin t h a n ' o l d e r ' leaf t i s s u e [5, 9]. N D G A l e v e l s fell o v e r b o t h s a m p l i n g p e r i o d s in Plots I a n d III, s u g g e s t i n g a r e l a t i o n s h i p b e t w e e n t h e resin c o n c e n t r a t i o n o f N D G A a n d s e a s o n a l variation between the months of April and July (e.g. as t e m p e r a t u r e s a n d t h e l e v e l s of s o l a r irradiation increase and as precipitation decreases),
Experimental Interpopu/ational studies. Plant material was collected from 27
sites throughout the Sonoran Desert (see Fig. 1). Multiple branches from three shrubs were obtained at each site during a two week collecting trip in May 1984. The material was allowed to air-dry during the collection trip. To assure uniform dryness, all plant material was thoroughly dried (60° for 5 days) on returning to the laboratory. Fifteen leaf pairs were selected randomly from the dried branches (mostly from second and third intemodes from the spray tips) for solvent extraction. Intrapopulational studies. The Boyd Deep Canyon Desert Research Center, situated approximately 10 km south of Palm Desert, California, was the location of all intrapopulational studies. Shrubs from two adjacent sites were used for these studies (described previously, see ref. [13]); Plots I and III contained 49 and 48 bushes, respectively (several shrubs which were considered as single entities by Greenfield et al. [13] and Shelly et al. [19] for insect behavioural studies were considered separately in the present studies). Fifteen leaf pairs (mostly from the second and third internodes from the spray tips) were selected randomly from each of the shrubs in both plots. The plants in Plot I were sampled on 16 April and 28 June 1984. Plot III plants were sampled on 28 June and 25 July 1984. The leaf material was dried as previously noted and the solvent extracted as discussed below.
Extraction of/eaves. Leaf tissue from the second and third internodes from the spray tips (i.e. ca third and fourth leaf pairs) was chosen for phytochemical analysis because much of the observable insect herbivory seemed to be focused on this tissue and, therefore, it was of particular interest. After determining the dry wt of leaf samples, the intact leaves were extracted with hexane (3 ml; 48 h at 4°) to remove hexane-soluble components. The leaves were then extracted with MeOH (3 ml; 5 days at 4°). The MeOH-soluble leaf extract containing NDGA was decanted, filtered through COtton (to remove particulates) and then stored at --20° until analysis. The percentage of hexane- and MeOH-soluble leaf extracts was determined from leaf dry wt measurements taken before and after each solvent extraction. HPLC. MeOH-soluble leaf extracts were analysed for NDGA by reverse-phase HPLC on Altex Ultrasphere ODS columns (4.6 × 150 mm) using water (solvent A) and acetonitrile (solvent B). HjPO. (1%; v/v) was added to both A and B to suppress ionization during chromatography. NDGA was separated from other MeOH-soluble leaf components using a linear gradient from 40 to 100% B over 12 min and a flow rate of 1 ml min -1. Elution of NDGA was monitored spectrophotometrically at its absorption maximum (285 nm). Quantification was accomplished by peak area integration using authentic NDGA (Sigma No. N-5360) dissolved in MeOH. The line of best fit based on 3-5 injections at varying concentrations of NDGA was determined by linear regression (r ~ = 0.998),
Acknowledgements--We thank AI and Vic Muth for use of the research facilities at the Philip L. Boyd Deep Canyon Desert Research Center, Palm Desert, California and Michael Greenfield for permission to sample Larrea foliage from established field plots at the research center. We also thank J. Downum, A. Quiroz, T. E. Shelly, M. Trevino and S. Villegas for field and technical assistance, M. Trevino for graphic preparation, and R. Chapman and M. Greenfield for critical reading of the manuscript. The grant support of NSF (PCM 82-09100) and NIH (AI 18398) is gratefully acknowledged.
References 1. Shreve, F. (1942) Bo~ Rev. 8, 195. 2. Rzedowski, J. and Medellin Leal, F. (1958) Acta Ci. Potos. 2, 133. 3. Wells, P. V. and Hunziker, J. H. (1976) Ann. Missouri Bot. Gard. 63, 843.
NORDIHYDROGUAIARETICACIDVARIATIONIN LARREA TRIDENTATA 4. Barbour, M. G., MacMahon, J. A., Bamberg, S. A. end Ludwig, J. A. (1977) Creosote Bush--BiologyandChemiMry of Larrea in New World Deserls (Mabry, T. J., Hunziker, J. H. and DiFeo Jr, D. R., eds), p. 48. Dowden, Hutchinson and Ross, Stroudsberg, Pennsylvania. 5. Rhoades, D, F. (1977) Biochem. Sys¢ Ecol. 5, 281. 6. Downum, K. R., Villegas, S., Keil, D. J. and Rodriguez, E. (1988) J. Chem. Ecol. (in press). 7. Mabry, T. J., DiFeo Jr, D. R., Sakakibara, M., Bohnstedt, C. F. and Siegler, D. (1977) Creosote 8ush--B/ology and Chemis~/ of Larrea in New World Deserts (Mabry, T. J., Hunziker, J. H. and DiFeo Jr, D. R., eds.), p. 115. Dowden, Hutchinson and Ross, Stroudsberg, Pennsylvania. 8. Seigler, D., Jakupcsk, J. and Mabry, T. J. (1974) Phymchemistry 13, 983. 9. Rhoades, D. F. (1977) Creosote Bush--Biology and Chemistry of Larrea in New World Deserts (Mabw, T. J., Hunziker, J. H. and DiFeo Jr, D. R., eds), p. 115. Dowden, Hutchinson and Ross, Stmudsberg, Pennsylvania.
555
10. Oliveto, E. (1972) Chem. Ind¢ 677. 11. Fernandez, S., Hurtado, L. and Hemandez, F. (1979) Larrea, p. 327. Serie el Desierto, VoL 2. Centro de Investigacion en Quimice Aplicade, Saltillo, Coah., Mexico. 12. Florencio, J. D. and Pablo, V. G. (1979) Larrea, p. 343. Serie el Desierto. Vol, 2. Centro de Investigacion en Quimica Aplicada, Saltillo, Coah., Mexico. 13. Greenfield, M. D., Shelly, T. E. and Downum, K. R. (1987) Ecology,8, 828. 14. Gonzalez-Coloma, A., Widsom, C. S. and Rundel, P. W. (1988) B~chem. Syst. Ecol. 16, 59. 15. Chapman, R. R, Bemays, E. A. and Wyatt, T. (1988) J. Chem. Ecol. 14, 561. 16. Duisberg, P. C. (1951) P/antPhysiol. 27, 769. 17. Yang, T. W. (1970) J. Affz. Acad. Sc~ 6, 41. 18. Hunziker, J. H., Palecios, R. A., de Vales, A. G. and Poggio, L. (1972) Ann. MissouH Bo~ Gard. F~, 224. 19. Shelly, T. E., Greenfield, M. D. and Downum, K. R. (1987) Anita. Behav. 35, 1200.
0305-1978188$3.00-F0.00 ¢) 1988PergamonPressplc.
Nordihydroguaiaretic Acid: Inter- and Intrapopulational Variation in the Sonoran Desert Creosote Bush (Larrea tridentata, Zygophyllaceae)* K. R. DOWNUM, J. DOLE1" and E. RODRIGUEZt Department of Biological Sciences, Florida International University, Miami, FL 33199, U.S.A. l"Phytochemistry and Toxicology Laboratory, Developmental and Cell Biology, University of Califomia, Irvine, CA 92717, U.S.A.
Key Word Index--Larrea t~'dentata; creosote bush; nordihydroguaiaretic acid; NDGA; lignan catechol; leaf resin; Sonoran desert; interpopulational and intrapopulational phytochemical variation. Abstract--Nordihydroguaiaretic acid (NDGA) is the principal phenolic constituent in the leaf resin of the creosote bush, Larrea tridentata, the dominant perennial shrub throughout most of the warmer arid and semi-arid regions of North America. In an effort to determine the quantitative significance of this biologically active lignan catechol in desert ecosystems, we conducted studies of leaf extracts from geographically distinct populations of Larrea which occur throughout the Sonoran desert. Seasonal influence on NDGA concentration as well as on the hexane- and MeOH-soluble leaf constituents were also examined within two populations of Larrea growing near Palm Desert, California. Interpopulational studies revealed that NDGA concentrations declined from northern to southern sampling sites whereas the levels of hexane- or MeOH-soluble leaf components showed little variation. Intrapopulational studies showed that the mean level of both hexane- and MeOH-soluble leaf components increased significantly between mid-April and late June, but that these increases slowed down or stopped between June and July. The mean concentration of NDGA decreased significantly in plants between April and July suggesting a possible relationship between the resin concentration of NDGA and seasonal (i.e. climatic) changes. These observations may, in part, explain some of the latitudinal variation in NDGA levels found in Sonoran desert representatives of Larrea.
Introduction believed to function as a barrier to prevent water The creosote bush, Larrea tridentata (Seese & loss from leaf surfaces, a solar-UV filter which Moc. ex DC.) Coville, is the most abundant and protects foliage from harmful wavelengths of widely distributed evergreen shrub in the light and an insect feeding deterrent [5]. In warmer arid/semi-arid regions of North America addition to the above activities, Larrea leaf resin extending from southern California and south- has been shown to elicit phototoxic responses western Utah, south through Baja California and from various micro-organisms including E. coli Sonora, and east through Western Texas and and the yeast Saccharomyces cerevisiae [6]. The the states of Chihuahua, Coahuila and San Luis resin contains a complex mixture of phenolics, Potosi, Mexico [1-3]. The abundance of this saponins, terpenoids and wax esters that woody shrub, which may attain densities of up account for 10-20% of the leaf dry weight [7-9], to 1700 shrubs per ha in certain locations [4], although the resin on immature foliage consticombined with its perennial growth habit, tutes a considerably higher percentage [9]. makes Larrea among the most important and Approximately 80% of the leaf resin is predictable plant resources in many desert composed of phenolic compounds of which environments. nordihydroguaiaretic acid (NDGA; Fig. 1) is the The leaves of this highly xerophytic shrub are major component. This lignan catechol covered with a resinous coating which is reportedly accounts for 5-10% of the dry leaf weight [7], is a potent antioxidant [10] and mediates important bactericidal, fungicidal and *Dedication: In memory of the late Professor Tony Swain: antiherbivore activities [5, 8, 11-15]. Mentor, scholar and friend. NDGA may well represent one of the most (Received 29 Apri/1988) quantitatively important and widely distributed 551
552
K.R. D O W N U M , J. DOLE A N D E. RODRIGUEZ
Ha
H
O
~
i
'
~
'
~
California
0 H
~.~
~
',
@
\ ~ O H
Arizona
,~
,
FIG. 1, THE CHEMICAL STRUCTURE OF THE LIGNAN CATECHOL NORDIHYDROGUAIARETIC ACID (NDGA).
8~a
allelochemicals in environments where Larrea occurs. The present studies were initiated to determine the quantitative significance of NDGA in Sonoran Desert populations of Larrea, to evaluate the inter- and intrapopulational variation of hexane- and MeOH-soluble components of the creosote bush leaf resin and to ascertain whether the levels of these components fluctuate over the growing season.
California
~
\, ~,
.... .
)'~"~'~
" ~
~
Pacific ~ Ocean
@
'~
~
J' ~
Results and Discussion Sonoran desert populations of the creosote bush extend from southern California and Arizona at the northern limit (ca 35°N latitude), south through Baja California (to ca 24°N latitude) and east into the state of Sonora on the FIG. 2~ COLLECTION LOCATIONS OF LARREATRIDENT TA A mainland of Mexico. Leaf samples from popula- THROUGHOUT THE SONORAN DESERT, Collections w e r e made at 8 0 tions of L. tridentata were collected for 100 km intervals. phytochemical analysis at 80-100 km intervals throughout the Sonoran limits of this species populations. Values for the hexane- and MeOH(refer to Fig. 2 for collection locations). The soluble components did not vary significantly specific geographic coordinates for each of the throughout the geographic range of the populacollection sites are listed in Table 1. tions sampled. Mean percentages of both The concentration of hexane- and MeOH- hexane-soluble (e.g. saponins, terpenoids, soluble leaf constituents and NDGA were deter- waxes, etc.) and MeOH-soluble (e.g. NDGA and mined from the leaves of these creosote bush ~ other phenolic constituents) leaf components TABLE 1. LONGITUDINAL A N D LATITUDINAL COORDINATES OF COLLECTION SITES 1 - 2 7 Collection
North
West
Collection
North
West
number
(latitude)
(longitude)
number •
(latitude)
(longitude)
01
30 ° 0'
115°15 '
15
28° 27'
111°15 ,
02
29 ° 50'
114 ° 35'
16
28~12 '
111 ° 3'
03 04
29°25 , 28 ° 5'
1 1 ~ 20' 113°36 ,
17 18
28 ~ 24' 28 ° 39'
111 ° 3' 111°36 ,
05
2 ~ 21'
1 1 ~ 6'
19
29 ~ 0'
1 1 ~ 55'
06
27°6 ,
111°54 ,
20
30 ° 0'
111 ° 6'
07 08
26 ° 33' 26 ° 9'
111 ° 45' 111 ° 21'
21 22
31~58 ' 32°26 '
111 ° 0' 111 ° 5'
09
25 ° 3'
111 ° 39'
23
32 ° 52'
112°18 '
10
24°45 ,
111°30 ,
24
32°54 ,
113 ° 0'
11
24°24 ,
111 ° 0'
25
32°10 '
11 ~ 43'
12
24 ° 6'
110 ° 33'
26
32°18 '
114 ° 45'
13
26 ° 57'
110°36 '
27
33°15 '
115 ° 35'
14
28 ° 51'
111 ° 50'
NORDIHYDROGUAIARETICACID VARIATION IN LARREA TRIDENTATA
based on dry weight measurements (,~+ SD; n=75) were 4.9%:1:1.6 and 21.9%+2.5, respectively. The levels of these constituents did not correlate with either the latitude or longitude of the collection locations (data not shown). NDGA, in contrast, showed a significant correlation (r2=0.59) with the latitude of the sampling sites (Fig. 3). The concentration of NDGA in leaf extracts ranged from a mean low of 6.4 mg g-1 at the southernmost boundary of the species (Coll. No. 12; 24°N) to a mean high of 60.2 mg g-1 near the northern limit of the study (Coll. No. 25; 32°N). NDGA was also correlated with study site longitude (r2=0.22; levels increased with increasing longitude), but this correlation was much less significant. The combined hexane- and MeOH-soluble leaf extracts yield values that are intermediate between leaf resin values reported in other studies of Larrea. Rhoades [5, 9], using diethyl ether as an extraction solvent, found leaf resin values ranging from 10 to 26% depending on leaf age (young leaves contained the highest levels) from Arizona populations of L. tridentata. Duisberg [16], in studies wtih L tridentata from New Mexico, published leaf resin values of 30-35% for foliage extracted with 95% EtOH. These latter values are considerably higher than both our values and those reported by Rhoades. These .: 60
•
•
50
! ~
40
~,
30
;
° I~
,~.
•
•
•
•
o ::
20 ~~
,o ,o
0
~ t
I
24
. =
I
26
• l
.~ I
28
I
I
I
~
l
32
t
a
34
Lotitude (=N) F~. 3. C ~ E ~ O N OF N ~ ( ~ mP t) ~RSUS ~ U D E THE S ~ WHERE ~ R R ~ L ~ F ~MPLES WERE COLLE~D.
OF
553
percentages may reflect: (1) differences due to the extraction solvent used in the particular studies; (2) differences in the age of the plant tissues extracted which have varying levels of resin; and/or (3) quantitative phytochemical differences between the diploid populations characteristic of New Mexico and the Chihuahuan desert and the tetraploid populations of the Sonoran desert [17, 18]. The concentration of NDGA in MeOH-soluble extracts decreases from northern to southern sampling sites, in contrast to the leaf resin (hexane- and MeOH-soluble leaf extracts). To our knowledge, a similar phytochemical relationship has never been demonstrated before. Different climatic conditions (e.g. precipitation, solar irradiation, temperature, etc.) experienced by plant populations growing near the latitudinal extremes of the species in the months preceding the study may account for some of ~the observed phytochemical variation. Plants at the northern latitudes generally appeared more healthy and had abundant new growth during the sampling period whereas plants growing near the southern limit of the species were noticeably drier and had considerably less new growth. Between-plant variation and the influence of seasonal change on leaf resin and foliar NDGA levels were investigated to determine the effect that these factors might have on plants from populations growing at different latitudes. Table 2 summarizes data gathered from a total of 97 individuals distributed between adjacent research plots (Plots I and III) of L. tndentata at Boyd Deep Canyon Desert Research Center at two different times of the year. The mean level of hexane- and MeOH-soluble leaf constituents (i.e. the leaf resin) increased significantly (P<0.001) in Plot I between mid-April and late June. The increase in hexane-soluble leaf constituents in Plot Ill was less significant (P<0.01) during late June through July, whereas the level of MeOH-soluble compounds did not change appreciably. In contrast, the mean concentration of NDGA in Plots I and III decreased by 9 and 15% during the same sampling periods, respectively. These studies suggest that the concentration of leaf resin (hexane-soluble+MeOH-soluble extracts) increases during early spring, but that this
554
K.R. DOWNUM,J. DOLEAND E. RODRIGUEZ
TABLE 2. QUANTITATIVECOMPARISONOF THE HEXANE-AND MeOH-SOLUBLELEAF CONSTITUENTSAND NDGAEXTRACTEDFROM LARREA TRIDENTATA LEAVESAT 33NODIFFERENTTIMESOF THE YEAR Plot
Collection date
Hexane-solubte components (%±SD)
MeOH-soluble components (%±SD)
NDGA (mg g-~ dw _+SD)
I (n--49)
16 April 1984 28 June 1984
3.2"}- 1.9 7.2± 1.9"
23.5+3.0 27.6±4.6*
59.7±13,2 54.1+11.5'
III (n-48)
28 June 1984 25 July 1984
7.1± 1.4 8.5±2.0~:
27.4±3.1 27.9±4.5
66.5+ 13,0 56.7±13,1t
*Significantly different (P< 0.001). 1"Levelsof significancebetweencollectiondatesat the two plots were determinedusing the paired t-test. $Significanttydifferent (P< 0.01). i n c r e a s e s l o w s (or s t o p s ) as t h e s u m m e r p r o g r e s s e s . T h e i n c r e a s i n g resin c o n t e n t in Plot I, w h i c h w a s o b s e r v e d b e t w e e n m i d - A p r i l a n d late J u n e , c o i n c i d e d w i t h t h e s p r i n g flush of growth o n L a r r e a at D e e p C a n y o n . It is c o n c e i v a b l e t h a t t h e i n c r e a s e d resin l e v e l s during this period may be due to sampling of n e w g r o w t h w h i c h is c o m p o s e d o f a h i g h e r p e r c e n t a g e o f resin t h a n ' o l d e r ' leaf t i s s u e [5, 9]. N D G A l e v e l s fell o v e r b o t h s a m p l i n g p e r i o d s in Plots I a n d III, s u g g e s t i n g a r e l a t i o n s h i p b e t w e e n t h e resin c o n c e n t r a t i o n o f N D G A a n d s e a s o n a l variation between the months of April and July (e.g. as t e m p e r a t u r e s a n d t h e l e v e l s of s o l a r irradiation increase and as precipitation decreases),
Experimental Interpopu/ational studies. Plant material was collected from 27
sites throughout the Sonoran Desert (see Fig. 1). Multiple branches from three shrubs were obtained at each site during a two week collecting trip in May 1984. The material was allowed to air-dry during the collection trip. To assure uniform dryness, all plant material was thoroughly dried (60° for 5 days) on returning to the laboratory. Fifteen leaf pairs were selected randomly from the dried branches (mostly from second and third intemodes from the spray tips) for solvent extraction. Intrapopulational studies. The Boyd Deep Canyon Desert Research Center, situated approximately 10 km south of Palm Desert, California, was the location of all intrapopulational studies. Shrubs from two adjacent sites were used for these studies (described previously, see ref. [13]); Plots I and III contained 49 and 48 bushes, respectively (several shrubs which were considered as single entities by Greenfield et al. [13] and Shelly et al. [19] for insect behavioural studies were considered separately in the present studies). Fifteen leaf pairs (mostly from the second and third internodes from the spray tips) were selected randomly from each of the shrubs in both plots. The plants in Plot I were sampled on 16 April and 28 June 1984. Plot III plants were sampled on 28 June and 25 July 1984. The leaf material was dried as previously noted and the solvent extracted as discussed below.
Extraction of/eaves. Leaf tissue from the second and third internodes from the spray tips (i.e. ca third and fourth leaf pairs) was chosen for phytochemical analysis because much of the observable insect herbivory seemed to be focused on this tissue and, therefore, it was of particular interest. After determining the dry wt of leaf samples, the intact leaves were extracted with hexane (3 ml; 48 h at 4°) to remove hexane-soluble components. The leaves were then extracted with MeOH (3 ml; 5 days at 4°). The MeOH-soluble leaf extract containing NDGA was decanted, filtered through COtton (to remove particulates) and then stored at --20° until analysis. The percentage of hexane- and MeOH-soluble leaf extracts was determined from leaf dry wt measurements taken before and after each solvent extraction. HPLC. MeOH-soluble leaf extracts were analysed for NDGA by reverse-phase HPLC on Altex Ultrasphere ODS columns (4.6 × 150 mm) using water (solvent A) and acetonitrile (solvent B). HjPO. (1%; v/v) was added to both A and B to suppress ionization during chromatography. NDGA was separated from other MeOH-soluble leaf components using a linear gradient from 40 to 100% B over 12 min and a flow rate of 1 ml min -1. Elution of NDGA was monitored spectrophotometrically at its absorption maximum (285 nm). Quantification was accomplished by peak area integration using authentic NDGA (Sigma No. N-5360) dissolved in MeOH. The line of best fit based on 3-5 injections at varying concentrations of NDGA was determined by linear regression (r ~ = 0.998),
Acknowledgements--We thank AI and Vic Muth for use of the research facilities at the Philip L. Boyd Deep Canyon Desert Research Center, Palm Desert, California and Michael Greenfield for permission to sample Larrea foliage from established field plots at the research center. We also thank J. Downum, A. Quiroz, T. E. Shelly, M. Trevino and S. Villegas for field and technical assistance, M. Trevino for graphic preparation, and R. Chapman and M. Greenfield for critical reading of the manuscript. The grant support of NSF (PCM 82-09100) and NIH (AI 18398) is gratefully acknowledged.
References 1. Shreve, F. (1942) Bo~ Rev. 8, 195. 2. Rzedowski, J. and Medellin Leal, F. (1958) Acta Ci. Potos. 2, 133. 3. Wells, P. V. and Hunziker, J. H. (1976) Ann. Missouri Bot. Gard. 63, 843.
NORDIHYDROGUAIARETICACIDVARIATIONIN LARREA TRIDENTATA 4. Barbour, M. G., MacMahon, J. A., Bamberg, S. A. end Ludwig, J. A. (1977) Creosote Bush--BiologyandChemiMry of Larrea in New World Deserls (Mabry, T. J., Hunziker, J. H. and DiFeo Jr, D. R., eds), p. 48. Dowden, Hutchinson and Ross, Stroudsberg, Pennsylvania. 5. Rhoades, D, F. (1977) Biochem. Sys¢ Ecol. 5, 281. 6. Downum, K. R., Villegas, S., Keil, D. J. and Rodriguez, E. (1988) J. Chem. Ecol. (in press). 7. Mabry, T. J., DiFeo Jr, D. R., Sakakibara, M., Bohnstedt, C. F. and Siegler, D. (1977) Creosote 8ush--B/ology and Chemis~/ of Larrea in New World Deserts (Mabry, T. J., Hunziker, J. H. and DiFeo Jr, D. R., eds.), p. 115. Dowden, Hutchinson and Ross, Stroudsberg, Pennsylvania. 8. Seigler, D., Jakupcsk, J. and Mabry, T. J. (1974) Phymchemistry 13, 983. 9. Rhoades, D. F. (1977) Creosote Bush--Biology and Chemistry of Larrea in New World Deserts (Mabw, T. J., Hunziker, J. H. and DiFeo Jr, D. R., eds), p. 115. Dowden, Hutchinson and Ross, Stmudsberg, Pennsylvania.
555
10. Oliveto, E. (1972) Chem. Ind¢ 677. 11. Fernandez, S., Hurtado, L. and Hemandez, F. (1979) Larrea, p. 327. Serie el Desierto, VoL 2. Centro de Investigacion en Quimice Aplicade, Saltillo, Coah., Mexico. 12. Florencio, J. D. and Pablo, V. G. (1979) Larrea, p. 343. Serie el Desierto. Vol, 2. Centro de Investigacion en Quimica Aplicada, Saltillo, Coah., Mexico. 13. Greenfield, M. D., Shelly, T. E. and Downum, K. R. (1987) Ecology,8, 828. 14. Gonzalez-Coloma, A., Widsom, C. S. and Rundel, P. W. (1988) B~chem. Syst. Ecol. 16, 59. 15. Chapman, R. R, Bemays, E. A. and Wyatt, T. (1988) J. Chem. Ecol. 14, 561. 16. Duisberg, P. C. (1951) P/antPhysiol. 27, 769. 17. Yang, T. W. (1970) J. Affz. Acad. Sc~ 6, 41. 18. Hunziker, J. H., Palecios, R. A., de Vales, A. G. and Poggio, L. (1972) Ann. MissouH Bo~ Gard. F~, 224. 19. Shelly, T. E., Greenfield, M. D. and Downum, K. R. (1987) Anita. Behav. 35, 1200.