Allelochemicals: Role in Agriculture and Forestry - American Chemical

Chapter 30

Isolation and Identification of Plant Growth Inhibitors from Leaves of the American Cranberry (Vaccinium macrocarpon) Peter Wepplo

Downloaded by EAST CAROLINA UNIV on September 27, 2016 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch030

Agricultural Research Division, American Cyanamid Company, Princeton, NJ 08540

An extract of leaves of the American cranberry plant, Vaccinium macrocarpon, was examined for growth inhibitors. No free parasorbic acid, a known growth inhibitor from cranberry leaves, could be isolated in the absence of a preliminary acid or base treatment. The parasorbic acid was isolated from the extract in the form of its glucoside. It was shown that this glucoside could only account for a portion of the growth inhibition of the leaf extract. Cranberry plants produce growth inhibitors requiring cranberry growers to remove fallen leaves and berries from their fields. If this dead material is allowed to remain it will cause reduced yields and growth. If it is allowed to accumulate over a period of years, it will result in dead areas in the bog. Devlin (1) reported that an aqueous extract of cranberry leaves inhibited the germination and growth of wheat. We became interested in identifying this growth inhibitor and thus examined the cranberry leaf extract. Vapor phase chromatographic analysis of the extract, prepared according to the procedure of Devlin, did not indicate the presence of abscisic acid, a growth inhibitor known to have this type of activity. Quercetin, 1, which has been reported to be a plant growth inhibitor (2) and has been found in cranberries (3), precipitated from one of our extracts. It was identified by comparison of the NMR, IR, and chemical ionization mass spectroscopic data with those of an authentic sample. The point of attachment and identity of the sugar residue was not determined. Although quercetin has been reported to be a plant growth inhibitor, we were unable to find any effect of this water insoluble glycoside or its aglycone on wheat seed germination and growth. Parasorbic Acid While seeking to isolate the growth inhibitor from the cranberry leaf extract, Cardellina and Meinwald (4) reported the isolation of 0097-6156/87/0330-0328$06.00/0 © 1987 American Chemical Society

Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

30.

Plant Growth Inhibitors from American Cranberry Leaves

WEPPLO

329

p a r a s o r b i c a c i d ( a c t u a l l y a l a c t o n e ) , 2, from the c r a n b e r r y p l a n t . S i n c e p a r a s o r b i c a c i d i s known to i n h i b i t seed g e r m i n a t i o n 6 ) , we sought to determine what r o l e t h i s a c i d ( l a c t o n e ) might p l a y i n the inhibition.

m

J\y HO

· / \,

r τ τ ·

\J

sy \y

r ?

Non

V

\

I

HO


9

0 ·ν · 0H \ / \ / V / \-„

We p r e p a r e d a sample o f p a r a s o r b i c a c i d by the method o f Stafford (7)· T h i s allowed us to d e v e l o p an a n a l y t i c a l g l c technique to determine the a c i d or i t s p r e c u r s o r i n the leaf extract. Since parasorbic a c i d was previously isolated by steam d i s t i l l a t i o n o f the j u i c e o f mountain ash b e r r i e s ( 8 ) , we steam d i s t i l l e d a sample o f the c r a n b e r r y l e a f e x t r a c t , but obtained l i t t l e 2. The l i t e r a t u r e r e p o r t s t h a t b e f o r e the ash b e r r y j u i c e was d i s t i l l e d , i t was t r e a t e d w i t h c a l c i u m h y d r o x i d e to p r e c i p i t a t e m a l i c a c i d . Tschesche l a t e r showed t h a t such treatment f o l l o w e d by a c i d i f i c a t i o n c o n v e r t e d the g l u c o s i d e o f p a r a s o r b i c a c i d , 3^, i n t o the f r e e a c i d ( l a c t o n e ) , 2 (9). T h i s base treatment e f f e c t s a βe l i m i n a t i o n o f the g l u c o s e fragment. In the absence o f t h i s base t r e a t m e n t , no f r e e p a r a s o r b i c a c i d was l i b e r a t e d from the b e r r i e s . 9

2 1

HO.-o

Î

' OH

o-.^ ·

AH

,

f )o

Ν

· - ·

Π

?

+-

glucose

6 2

Hence we sought to determine whether we simply were o b s e r v i n g the e f f e c t o f p a r a s o r b i c a c i d , a known p l a n t growth i n h i b i t o r , or i t s g l u c o s i d e i n our t e s t s . When the c r a n b e r r y l e a f e x t r a c t was treated with calcium hydroxide f o l l o w e d by a c i d i f i c a t i o n and e x t r a c t i o n w i t h ether, p a r a s o r b i c a c i d was indeed i s o l a t e d . I t was i d e n t i f i e d from the IR and NMR s p e c t r a o f the crude e x t r a c t and by i d e n t i c a l r e t e n t i o n time on two g l c columns w i t h an a u t h e n t i c sample o f p a r a s o r b i c a c i d . We then a n a l y z e d samples o f the c r a n b e r r y l e a f e x t r a c t by g l c b e f o r e and after treatment with calcium hydroxide (followed by acidification), samples o f p a r a s o r b i c a c i d alone, and extract p l u s p a r a s o r b i c a c i d ( i n the p r e s e n c e o f c a l c i u m h y d r o x i d e f o l l o w e d by a c i d i f i c a t i o n ) . Our r e s u l t s ( T a b l e I) show t h a t p a r a s o r b i c a c i d from the extract was obtained only upon calcium hydroxide treatment. T h i s r e s u l t agrees w i t h T s c h e s c h e s o b s e r v a t i o n s on mountain ash b e r r i e s . 1


330 Table

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

I.

P a r a s o r b i c A c i d Found A f t e r C a l c i u m

Sample 4.6 g e x t r a c t 4.6 g e x t r a c t 4.6 g e x t r a c t

Added Parasorbic Acid 42 mg 41 mg


I s o l a t i o n o f Glucoside

H y d r o x i d e Treatment

Ca(OH) Treatment

+ + +

Parasorbic Acid Found 29.5 mg 35.6 mg 28.6 mg 0

of Parasorbic

m

s

Acid

These r e s u l t s r a i s e d the p o s s i b i l i t y t h a t the g l u c o s i d e o f p a r a s o r b i c a c i d was r e s p o n s i b l e f o r the b i o l o g i c a l a c t i v i t y o f the extracts. Therefore, we s e t out to i s o l a t e the g l u c o s i d e , determine i t s b i o l o g i c a l a c t i v i t y , and c a l c u l a t e whether i t c o u l d account f o r the growth i n h i b i t i o n o f the e x t r a c t . I s o l a t i o n o f the g l u c o s i d e was a i d e d by the f a c t t h a t Tschesche had a c c o m p l i s h e d this before and h y d r o l y z i n g the g l u c o s i d e to p a r a s o r b i c acid p r o v i d e d a v e r y s e n s i t i v e and r a p i d g l c method f o r g u i d i n g the i s o l a t i o n . The s u c c e s s f u l i s o l a t i o n p a r a l l e l e d the i s o l a t i o n o f an analogous g l y c o s i d e , r a n u n c u l i n 11). The p r o c e d u r e was as f o l l o w s . The l e a v e s were e x t r a c t e d w i t h 85% aqueous acetone and t h e extract f i l t e r e d . The f i l t r a t e was f r e e z e d r i e d and the r e s i d u e washed w i t h e t h e r . The s o l i d r e s i d u e was d i g e s t e d i n acetone and the m i x t u r e f i l t e r e d . The acetone f i l t r a t e was e v a p o r a t e d and the r e s i d u e d i s s o l v e d i n water. The water was t r e a t e d w i t h c h a r c o a l , which absorbed the g l u c o s i d e . The crude g l u c o s i d e was e l u t e d from the c h a r c o a l w i t h 50% methanol, chromatographed on a s i l i c a g e l column, and e l u t e d w i t h e t h y l a c e t a t e and a c e t o n e . From the t o t a l o f 200-400 g o f l e a v e s , we were a b l e to i s o l a t e about 800 mg o f g l u c o s i d e , mp 131.5-132°C.; an IR band a t 1695*" cm. Comparison o f our g l u c o s i d e w i t h the m a t e r i a l r e p o r t e d by T s c h e s c h e showed d i f f e r e n c e s i n the m e l t i n g p o i n t and i n f r a r e d spectrum. Tschesche r e p o r t e d a mp o f 68-69°C f o r the h y d r a t e and 143-144°C a f t e r h e a t i n g a t 80°C o v e r n i g h t . S i n c e our g l u c o s i d e was i s o l a t e d d i r e c t l y as the anhydrous s o l i d r a t h e r than the h y d r a t e , d i f f e r e n c e s i n the m e l t i n g p o i n t o f the anhydrous s o l i d might be expected. There were a l s o some d i f f e r e n c e s i n c h e m i c a l s h i f t s i n the NMR spectrum, b u t these were due to s o l v e n t e f f e c t s . The m u l t i p l i c i t y o f the spectrum, however, was i n agreement w i t h the r e p o r t e d v a l u e s and w i t h the a g l y c o n e 4 ( T a b l e I I ) . S i n c e we knew

HO 4 t h a t p a r a s o r b i c a c i d c o u l d be l i b e r a t e d upon treatment, the l a c t o n e m o i e t y must be p r e s e n t .

calcium

hydroxide


30.

Table I I .

Glucoside

Proton NMR

M8)

Aglycone 4(8) Found for Glucoside 3


331

Plant Growth Inhibitors from Amencan Cranberry Leaves

WEPPLO

Shifts (ppm) of Parasorbic Acid Glucoside, 3, and i t s Aglycone, 4

H2 2.85 (m,2) 2.56 (m,2) 2.63 (d,2)

H3

4.30 (m,l) 4.28 (m,l)

H4

1.78 (m,2) 1.72 (m,l) 2.12 (m,l)

H5

4.80 (m,l) 4.77 (m,l)

H6

1.33 (d,3) 1.32 (d,3)

HI 4.80 (d)

1

4.34 (d,l)

While differences from the reported values were evident for the glucoside, they were removed upon i t s conversion to the t e t r a acetate. This was effected, i n 74% y i e l d , by treating the glucoside with acetic anhydride and pyridine. The melting point and a l l other properties of the tetra-acetate were in agreement with those reported by Tschesche. Having the glucoside, we could now determine i t s concentration in the leaves. Extract samples of varying sizes were subjected to calcium hydroxide treatment, a c i d i f i c a t i o n , extraction, and glc quantitation. The amounts of parasorbic acid obtained from the samples are shown i n Figure 1 to be proportional to sample size. In order to estimate the amount of parasorbic acid glucoside present, we needed to know the e f f i c i e n c y of recovery of parasorbic acid from the extracts being analyzed. To do so, a second set of samples was taken i n which a constant weight of glucoside was added to each sample. These samples were s i m i l a r l y analyzed. The upper line indicates the amount of glucoside that was added to each sample with the displacement from the lower line being equal to the weight of added glucoside. Since the weight of glucoside was constant, the upper line is p a r a l l e l to the f i r s t l i n e . The center line gives the amounts found for this second set of samples. The difference between the center and the upper line gives the recovery, 63%. Applying this fraction to the cranberry leaf extract gives 31 mg +_ 6.9 mg of glucoside per gram of extract or 6.8 mg + 1.5 mg of glucoside per gram of leaves. Wheat Seed Bioassay The bioassay was performed by placing twenty wheat seeds (Triticum aestivum var. "Olaf" spring wheat) between two pieces of f i l t e r paper in p l a s t i c P e t r i dishes. The extract solutions were poured onto the paper, the dish covered, and the dishes placed i n the dark for about 64 hours for the seeds to germinate. Checks were provided by using de-ionized water. The lengths of the primary roots were measured. Seeds with the five longest and five shortest roots were removed and the lengths of the primary roots of the ten remaining seeds recorded. A range of concentrations were chosen so that the range of root i n h i b i t i o n would be between 50 and 90%. By having several points, a b e s t - f i t line could be found for the test sample and the ED



332

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

mg of Extract Figure

1.

Recovery of Parasorbic Acid from Cranberry Extract

Leaf

determined. The best f i t line (10 g 1/concentration versus root length) was found v i a a least squares analysis for a plot of the root length equal to one-half the water control (check) length. However, i n some bioassays the check lengths were shorter than the test lengths obtained at high d i l u t i o n . In those instances, the calculated length for zero concentration was used i n place of the actual check length i n determining the E D ^ Q » This was most often the case when the check lengths were abnormally short and the root lengths at high dilutions about the same as the check lengths noted in other tests. Results and Conclusions Bioassay of the glucoside gave results as follows: (concentration in mg/mL, root length i n cm) 6.25, 1.18; 3.13, 2.07; 1.56, 2.46; 0.78, 3.88; 0.39, 2.77; 0.00 ( H O ) , 2.56. Thus the root length was 50% inhibited at about 5.75 mg/mL (19.6 millimolar concentration). As the glucoside was approximately 3.1% of the extract and 0.68% of the leaf, i f i t were solely responsible for the growth i n h i b i t i o n , then 193 mg extract/mL or 875 mg leaves/mL would be required to give the same b i o l o g i c a l response. Similar bioassay of residue produced by evaporation of the crude acetone extract of the leaves gave results as follows (concentration i n mg/mL, root length i n cm):



30.

WEPPLO

Plant Growth Inhibitors from American Cranberry Leaves

333

110, 0.14; 66, 0.40; 39.5, 1.15; 24, 1.22; 14, 1.43; 8.5, 1.63; 5, I. 48; 3, 1.66; 1.8, 1.31; 1.1, 1.78; 0.0 (H-O), 2.85. Thus the crude residue has an approximate ED^Q of 14.8 mg extract /mL which corresponds to 68 mg leaves/mL. Therefore, the glucoside can account for only a fraction of the total activity. Although the bioassay of racemic parasorbic acid showed good activity (wheat seed root growth was 50% inhibited with 0.25 mg/mL), the conclusion that little of the growth-retardant activity in cranberry leaves can be accounted for by parasorbic acid was confirmed by examination of Devlin's extract. In this case neither parasorbic acid nor its glucoside was present. Subsequent work by Hussain ( 12) has resulted in the isolation of two plant growth inhibitors identified as cinnamyl alcohol and 3-phenyl-l-propanol (hydrocinnamyl alcohol). Acknowledgments I must acknowledge the contributions of others: Robert Devlin, for alerting us to the problem and providing cranberry leaves and extract; Robert Templeton, for working out the bioassay; Laurie Lutz for doing the bioassay and especially for measuring the root lengths; James Clapp, for being skeptical about the bioassay; Marinus Los, who helped to make i t happen; and Ronie Bilotto and Claire Perniciaro, for typing the manuscript. Finally, I thank American Cyanamid Company, whose support made this possible. Literature Cited 1. Devlin, R. Μ., Plant Growth Regulation Bulletin 1980, 8, 7-8. 2. Einhellig, F. Α.; "CRC Handbook of Natural Pesticides: Methods, Vol. I", CRC Press: Boca Raton, Fl., 1985, p.176. 3. Puski, G.; Francis, F. J.; J. Food Sci., 1967, 32, 527. 4. Cardellina, J.; Meinwald, J.; Phytochemistry, 19, 2199. 5. Buston, H. W.; Koy, S. K.; Hatcher, E. S. J.; Rawes, M. R.; Arch. Biochem., 1949, 22, 269. 6. Moewus, F.; Schrader, Ε.; Z. Naturforsch. 1951, 66, 112. 7. Stafford, A. E.; Black, D. R.; Haddon, W. F.; Waiss, J. R., A. C.; J. Sci. Food Agric., 1972, 23, 771. 8. Kuhn, R.; Jerchel, D.; Chem. Ber., 1943, 76, 1413. 9. Tschesche, R.; Hoppe, H.-J.; Snatzke, G.; Wulff, G.; Fehlhaber, H.-W.; Chem. Ber., 1977, 104, 1420. 10. Hill, R.; Van Heyningen, R.; Biochem. J., 1951, 49, 332. 11. Benn, M. H.; Yelland, L. J.; Can. J. Chem., 1968, 46, 729. 12. Hussain, M.; Lutz, L.; Stout, S.; Orloski, E.; Templeton, Α.; and Devlin, R.; Proc. Plant Growth Reg. Soc. Am., 1983, 10, 151. RECEIVED June 9, 1986


Allelochemicals: Role in Agriculture and Forestry - American Chemical

Recommend Documents