Journal of Chemical Ecology, Vol. 14, No. 2, 1988

EXTRACTION OF TANNIN FROM FRESH AND PRESERVED LEAVES

A N N E. H A G E R M A N Department of Chemistry Miami University Oxford, Ohio 45056 (Received January 7, 1987; accepted February 23, 1987) Abstract--The extractability of tannin from fresh, lyophilized, and dried leaves collected at various times in the growingseason was determinedusing the radial diffusionassay for protein-precipitatingphenolics. The amount of tannin extracted depended on the method of leaf preservation and on the maturity of the leaf. Early in the season, more tannin was extracted from lyophilized leaves than from fresh leaves, but late in the season more tannin was extracted from fresh leaves. At all times, more tannin was extracted with aqueous acetone than with aqueous or acidic methanol. Key Words--Leaf preservation, tannin extraction, phenolicanalysis, protein precipitation.

INTRODUCTION Although tannin is frequently cited as an example of a plant defensive chemical (Swain, 1979), experimental documentation of a defensive role for tannin is limited. Clear demonstration of a defensive function for tannin depends on accurate quantitation of tannin. W h e n adequate analytic methods are available, it will become possible to determine whether there is any correlation between tannin content and patterns of herbivory. The accuracy with which a chemical component in a biological matrix, such as tannin in a plant leaf, can be determined depends on several factors. The tissue of interest must be collected and preserved so that the component is not altered or destroyed. The component must be extracted from the tissue in high yield. The method of assaying the component must be free of interferences from other materials present in the extract. However, there is no consensus on 453 0098-0331/88/0200-0453506.00/0 9 1988 Plenum Publishing Corporation

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the best method for preserving leaves or the most efficient solvents for extracting tannins (Swain, 1979). Definitive studies of the role of tannin have been hampered by the limited knowledge of appropriate methods for sample collection and tannin extraction. Although extraction and analysis of fresh tissues would probably minimize changes to the tannin, most studies of the ecological role of tannin are conducted under conditions where immediate analysis is impossible. Instead, tissues are collected and preserved for later extraction and analysis. Lyophilization is thought to be the gentlest method of preservation, and lyophilized leaves may be equivalent to fresh leaves (Martin and Martin, 1983). However, diminished extractability of tannin after lyophilization has been noted in some cases (Price et al., 1979). Drying the tissue at ambient or at elevated temperature is more convenient than lyophilization, although little tannin can be extracted from samples dried at elevated temperatures (Bate-Smith, 1975; Price et al., 1979). Lower temperature drying is preferable (Swain, 1979), but even drying at room temperature may alter the chemical nature of tannin (Butler, 1982). Three solvents are commonly used to extract tannin from plant samples: boiling aqueous methanol, aqueous acetone, or acidic methanol. Boiling aqueous methanol is thought to be the most effective solvent for condensed tannin (BateSmith, 1975), but the recovery of tannin is estimated to be as low as 30% for some tissues (Bate-Smith, 1973a; Swain, 1979). Since aromatic ester (depside) bonds are hydrolyzed by aqueous alcohols, they are thought to be unsuitable for extraction of hydrolyzable tannin (Haslam et al., 1961; Swain, 1979). Aqueous acetone is routinely used to extract hydrolyzable tannin (Jones et al., 1976; Foo and Porter, 1980), but no quantitative estimates of recovery are available. Some authors believe condensed tannin is extracted quite efficiently with aqueous acetone (Feeny and Bostock, 1968; Fletcher et al., 1977; Lane and Schuster, 1981), but others have found that condensed tannin is recovered in low yields when aqueous acetone is employed (Stafford and Cheng, 1980; Martin and Martin, 1984). Acidic methanol is the best solvent for extracting the condensed tannin from some varieties of sorghum (Price et al., 1978). It is hypothesized that the tannin in those varieties is chemically unique, perhaps covalently attached by an acid-labile bond to some component of the grain. Although aqueous methanol (pH 5-6) causes methanolysis of depside bonds in hydrolyzable tannins, at more acidic pH values (pH < 3) methanolysis does not occur (Haslam et al., 1961). Thus acidic methanol should be appropriate for extraction of hydrolyzable tannin. The applicability of acidic methanol as an extractant of plant tissues other than sorghum has not been demonstrated, but it may be useful in cases of strong interaction between tannin and other components of the tissue. Some plants contain tannin that is completely unextractable with aqueous acetone or alcohol (Bate-Smith, 1973a, 1975). The purpose of this study was to compare several methods of leaf preser-

TANNIN EXTRACTION

455

ration and to quantitate the extractability of tannin with various solvents. The study was conducted throughout one growing season, and the effect of leaf maturity on tannin extractability was also noted. METHODS AND MATERIALS

The leaves used in this study were collected from trees on the Miami University campus throughout the growing season of 1986. A single tree of each type was used for all samples, and all samples were collected from low branches on the north side of the trees. Three species of trees were used: burr oak, Quercus macrocarpa; sugar maple, Acer saccharum; shagbark hickory, Carya ovata. Specimens have been deposited in the Miami University herbarium. The leaves were removed from the trees and immediately put on ice. Major veins were removed with a razor blade and discarded. The leaves were cut into small pieces ( - 2 cm2), frozen in liquid nitrogen, and ground with a mortar and pestle. The powder was either extracted immediately, or lyophilized, or oven dried at 40~ or at 90~ Extractions were performed either in test tubes or in miniature columns. If test tubes were used, the sample was weighed into the tube, mixed with the solvent, and centrifuged after the extraction was completed. The supernatant was removed and the final volume of extract recovered was recorded. In a faster method for extracting samples, 1-ml micropipet tips were used as columns. The end of the plastic pipet tip was sealed by touching it with a hot glass rod. A polyethylene frit cut to the proper size with a cork borer was inserted - 0 . 7 5 mm from the tip to support the plant tissue. The tissue was placed in the tip, on the frit, weighed, and the solvent was added. After the extraction was completed, the sealed end was cut off, the column placed in a large test tube, and centrifuged. The extract was collected in the test tube. This method provided excellent recovery of the extract for small samples (100 mg) of fresh or dried tissue. For all the solvents, the tissue was initially extracted for 30 min at room temperature. If the solvent was to be heated, the sample was placed in an 85 ~ water bath for an additional 10 min of extraction. The extracts were assayed with the radial diffusion assay (Hagerman, 1987). Wells were made in the bovine serum albumin-containing plates with a 4-mm punch, and three 8-#1 aliquots of extract were applied to each well. After 94 hr, the diameter of the ring that developed was measured. The diameter squared is proportional to amount of tannin added to the well (Hagerman, 1987). The data were expressed as centimeters squared per gram dry tissue. These values could be converted to milligrams tannic acid per gram tissue or to milligrams condensed tannin per gram tissue using a standard curve (Hagerman, 1987), but those values would not be more meaningful than the unconverted raw data for the leaves that contained both types of tannin.

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Tannic acid or quebracho tannin (Asquith and Butler, 1985) was added to fresh spinach or young barley leaves, and the tannin recovered from the leaves determined with the Prussian blue assay (Price and Butler, 1977). The leaves (0.34 g) were cut into small pieces, and placed in a mortar with sand, 3.0 ml of the solvent, and a 100-tzl aliquot of a 100 mg/ml tannin solution. The leaves were ground with a pestle, centrifuged, and the supematant assayed for total phenolics. Control samples without added tannin were used to determine the phenolics from the barley or spinach, and those values were subtracted from the values obtained for the spiked leaves. The Prussian blue assay was run on aliquots of tannin, and the recovery from the leaves was then calculated as percent of tannin added to the leaves. RESULTS Leaves of oak and maple contain both condensed and hydrolyzable tannin (Feeny and Bostock, 1968; Bate-Smith, 1977; Schultz and Baldwin, 1982); hickory contains only condensed tannin (Wilken and Cosgrove, 1964). The radial diffusion assay (Hagerman, 1987), which was used to determine the extracted tannin, does not discriminate between condensed and hydrolyzable tannin. Condensed tannin gives a lower response than hydrolyzable tannin with this assay. The response to a mixture of tannins is equal to the sum of the responses to the individual components of the mixture (Hagerman, 1987). The extraction of tannin from lyophilized maple or oak leaves collected in midsummer was most efficient when 70% aqueous acetone was used (Table 1). Aqueous alcohol did not extract as much tannin as did the acetone. Boiling aqueous methanol apparently destroyed some tannin in the maple leaves. Heated aqueous acetone extracted less tannin than did unheated acetone (data not shown). Similar results were obtained in experiments in which condensed or hydrolyzable tannin was added to fresh leaves just before the leaves were homogeTABLE 1. EXTRACTION OF TANNIN a FROM LYOPHILIZED LEAVES WITH VARIOUS SOLVENTS (LEAVES COLLECTED JULY 9, 1986)

Solvent 70% acetone 50% methanol 50% methanol, boiling 1% HC1 in methanol

Oak 546 387 359 343

+ 87a ___21b ___20b + 35b

Maple 1381 + 149c 1117 + 133d 994 ___78e 97 + 177d

~Tannin determined by the radial diffusion assay (Hagerman, 1987) and expressed as cm2/g dry tissue. Each value (_ SD) is the mean of at least three determinations. Values followed by the same letter are the same at the 95 % confidence interval (t test).

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457

TABLE 2. RECOVERY OF ADDED TANNIN FROM FRESH, TANNIN-FREE TISSUE

Recovery (%)a Spinach Solvent 70% acetone 50% methanol 50% methanol, boiling l%HClin methanol

Barley

Condensed tannin

Hydrolyzable tannin

Condensed tannin

Hydrolyzable tannin

70 + 6 _b b

79 + 4 __b b

66 + 1 58 + 1 61 _ 3

68 _+ 3 53 _ 5 b

52___2

79 + 3

47_+ 8

71 + 4

Recovery of tannin added to tannin-free leaves determined with the Prussian blue assay (Price and Butler, 1977). bNot determined. n i z e d in v a r i o u s solvents (Table 2). T h e added tannin was m o s t efficiently r e c o v e r e d with a q u e o u s a c e t o n e as the solvent. Less tannin was r e c o v e r e d with the a l c o h o l - c o n t a i n i n g solvents, a l t h o u g h acidic m e t h a n o l was as effective as a q u e o u s a c e t o n e for r e c o v e r y o f h y d r o l y z a b l e tannin (Table 2). H o w e v e r , w h e n l y o p h i l i z e d t a n n i n - c o n t a i n i n g tissues w e r e extracted, acidic m e t h a n o l was m u c h less efficient than a q u e o u s a c e t o n e (Table 1). T h e extractability o f tannin was d e t e r m i n e d for l e a v e s c o l l e c t e d at different t i m e s during the g r o w i n g season. In the early s u m m e r , m u c h m o r e tannin was extracted f r o m l y o p h i l i z e d m a p l e l e a v e s by a q u e o u s a c e t o n e than was extracted by h e a t e d m e t h a n o l (Table 3). A s the s u m m e r progressed, the a m o u n t o f tannin extracted by a c e t o n e d e c r e a s e d m o r e rapidly than the a m o u n t extracted by alcohol. In the late s u m m e r , equal a m o u n t s o f tannin w e r e extracted f r o m the l y o p h ilized m a p l e l e a v e s by a q u e o u s a c e t o n e o r by heated a q u e o u s m e t h a n o l (Table TABLE 3. EXTRACTIONOF TANNINa PROM LYOPHILIZEDMAPLE LEAVES COLLECTEDON VARIOUS DATES Date Solvent

May 27, 1986

July 9, 1986

August 13, 1986

70% acetone 50% methanol

1794 + 36a 1228 + 54b, d

1381 _ 149b 1117 + 133d

692 + 93c 802 + 77c

aTannin determined by the radial diffusion assay and expressed as cm2/g dry tissue. Each value (+ SD) is the mean of at least three determinations. Values followed by the same letter are the same at the 95 % confidence interval (t test).

458

HAGERMAN

Maple

140 120 100

80~

(,%

'//z

60 40

r IIIJ

20 ILl I'O n" IX ILl

Z

'//I

7I/ .m

.~ 8 o ~60~

Hickory

ol

Z I'-

40O 200 0 Oak

800

-'t.:.:.:.:

? 8/13/86

6/16/86 DATE

Fro. 1. Tannin extracted from fresh, lyophilized, and dried leaves. Leaves were collected on the indicated date and were either extracted immediately ( [ ] ) , lyophilized and then extracted ( D ) , or dried at 40~ and then extracted (E~). The tissue was extracted with 70% aqueous acetone and the extracts analyzed with the radial diffusion assay (Hagerman, 1987). Tannin was calculated on the basis of dry weight. Error bars show 1 SD from the mean of at least three trials.

TANNIN EXTRACTION

459

3). Similar results were obtained with lyophilized oak or hickory leaves and with dried leaves from all three species (Figure 1). It has previously been noted that the amount of tannin in plant tissues diminishes throughout the growing season (Bate-Smith, 1973a; Buffer, 1982). However, the role solvent plays in determining the apparent magnitude of the seasonal change has not been previously noted. The extractability of tannin from leaves preserved by several methods was determined at two times during the growing season (Figure 1). Early in the season, more tannin was extracted from lyophilized leaves than from fresh leaves. Late in the season, the amount of tannin extracted from lyophilized leaves diminished, as noted above. However, the amount of tannin extracted from fresh leaves collected late in the season was greater than the amount that could be extracted from fresh leaves collected early in the season. The extraction of tannin from the dried leaves was quite efficient for some samples and less efficient for other samples. Drying at elevated temperatures was particularly harmful; no tannin could be extracted from hickory leaves that were dried at 90~ and only a small amount of tannin could be extracted from oak or maple leaves dried at 90~ Similar amounts of tannin were extracted from frozen samples of late-season leaves and from fresh samples of the same leaves.

DISCUSSION

It is not possible to recommend a single optimal protocol for extraction of tannin from all plant samples. Each sample has unique characteristics, including the structure of the tissue and the composition of the tannin, that determine tannin extractability (Bate-Smith, 1973a; Swain, 1979). Although a very limited number of samples were examined in the study described here, some general recommendations can be made based on these results. Aqueous acetone appears to be the best solvent for extracting tannin from leaf tissue. Aqueous alcohol or acidic alcohol does not extract more tannin than aqueous acetone. Heated aqueous alcohol may extract less tannin than aqueous acetone. Acetone is an effective solvent because it inhibits interaction between tannin and proteins (Hagerman and Robbins, 1987) and thus prevents tannin from binding to leaf proteins during homogenization. However, acetone inhibits many of the common precipitation assays for tannin (Bate-Smith, 1973b; Hagerman and Butler, 1978; Martin and Martin, 1983), So procedures for analysis of acetone extracts must be chosen carefully. Acetone does not interfere with the radial diffusion assay because the acetone evaporates before the tanninprotein interaction takes place (Hagerman, 1987). Acetone does not interfere with most chemical assays for tannin (Price and Buffer, 1977; Price et al., 1978).

460

HAGERMAN

To minimize artifacts associated with leaf preservation, leaves should be extracted and analyzed immediately after collection. However, if analysis of fresh tissue is impossible, tissue should be lyophilized or dried at low temperature rather than in the sun or in an oven. If tissues must be dried a higher temperature, analysis of at least a few samples of fresh tissue, for comparison with the dried tissue, would provide insight into the changes in tannin extractability associated with drying. Tannin levels and extractability change dramatically through the growing season. Environmental conditions such as light intensity affect phenolic biosynthesis and accumulation (Balsa et al., 1979; Waterman et al., 1984). Seasonal changes in leaf morphology, moisture content, and chemistry may affect tannin extractability (Swain, 1979). Comparisons of tannin levels in plants must be made cautiously since tannin extractability is dependent on many factors. The seasonal variation in tannin extractability observed in this limited study of three temperate-climate species may not resemble the seasonal changes in other plants. Studies of the ecological significance of tannin should include investigations of seasonal variations for the plant of interest, using the plant preservation and tannin extraction techniques that will be used in the ecological study.

REFERENCES ASQUITH,T.N., and BUTLER,L.G. 1985. Use of dye-labeled protein as spectrophotometricassay for protein precipitants such as tannin. J. Chem. Ecol. 11:1535-1544. BALSA,C., ALIBERT,G., BRULFER,J. QUEIROZ,O., and BOUDET,A.M. 1979. Photopefiodiccontrol of phenolic metabolism in Kalanchoe blossfeldiana. Phytochemistry 18:1159-1163. BATE-SMITH,E.C. 1973a. Tannins of herbaceous leguminoseae. Phytochemistry 12:1809-1812. BATE-SMITH,E.C. 1973b. Haemanalysis of tannins: The concept of relative astringency. Phytochemistry 12:970-912. BATE-SMITH,E.C. 1975. Phytochemistryof proanthocyanidins. Phytochemistry 14:1107-1113. BATE-SMITH,E.C. 1977. Astringent tannins of Acer species. Phytochemistry 16:1421-1426. BUTLER,L.G. 1982. Relative degree of polymerizationof sorghumtannin during seed development and maturation. J. Agric. Food Chem. 30:1090-1094. FEENY, P.P., and BOSTOCK,H. 1968. Seasonal changes in the tannin content of oak leaves. Phytochemistry 7:871-880. FLETCHER,A.C., PORTER,L.J., HASLAM,E., and GUPTA,R.K. 1977. Plant proanthocyanidins. Part 3. Conformational and configurational studies of natural procyanidins. J. Chem. Soc. Perk. 1 1977:1628-1637. Foo, L.Y., and PORTER,L.J. 1980. The phytochemistryof proanthocyanidinpolymers. Phtyochemistry 19:1747-1754. HAGERMAN,A.E. 1987. Radial diffusion method for determining tannin in plant extracts. J. Chem. Ecol. 13:437-449. HAGERMAN,A.E., and BUTLER,L.G. 1978. Protein precipitation method for the quantitative determination of tannins. J. Agric. Food Chem. 26:809-812. HAGERMAN,A.E., and ROBBINS,C.T. 1987. Implications of soluble tannin-protein complexes for tannin analysis and plant defense mechanisms. J. Chem. Ecol. 13:1243-1259.

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HASLAM,E., HAWORTH,R.D., MILLS, S.D., ROGERS,H.J., ARMITAGE,R., and SEARLE,T. 1961. Gallotannins. Part II. Some esters and depsides of gallic acid. J. Chem. Soc. 1961: 18361842. JONES, W.T., BROADHURST,R.B., and LYTTLETON,J.W. 1976. The condensed tannins of pasture legume species. Phytochemistry 15:1407-1409. LANE, H.C., and SCHUSTER,M.F. 1981. Condensed tannin of cotton leaves. Phytochemistry 20:425-427. MARTIN, J.S., and MARTIN, M.M. 1983. Tannin assays in ecological studies. Precipitation of ribulose-1,5-bisphosphate carboxylase/oxygenase by tannic acid, quebracho, and oak leaf foliage extracts. J. Chem. Ecol. 9:285-294. MARTIN, M.M., and MARTIN, J.S. 1984. Surfactants: Their role in preventing the precipitation of proteins by tannins in insect guts. Oecologia (Berlin) 61:342-345. PRICE, M.L., and BUTLER, L.G. 1977. Rapid visual estimation and spectrophotometric determination of tannin content of Sorghum grain. J. Agric. Food Chem. 25:1268-1273. PRICE, M.L., VANSCOYOC,S., and BUTLER, L.G. 1978. A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J. Agric. Food Chem. 26:1214-1218. PRICE, M.L., STROMBERG,A.M., and BUTLER, L.G. 1979. Tannin content as a function of grain maturity and drying conditions in several varieties of Sorghum bicolor (L.) Moench. J. Agric. Food Chem. 27:1270-1274. SCHULTZ, J.C., and BALDWIN, I.T. 1982. Oak leaf quality declines in response to defoliation by gypsy moth larvae. Science 217:149-151. STAFFORD,H.A., and CHENG, T.-Y. 1980. The procyanidins of douglas fir seedlings, callus and cell suspension cultures derived from cotyledons. Phytochemistry 19:131-135. SWAIN, T. 1979. Tannins and lignins, pp. 657-682, in G.A. Rosenthal and D.H. Janzen (eds.). Herbivores: Their interaction with Secondary Plant Metabolites. Academic Press, New York. WATERMAN,P.G., Ross, J.A.M., and McKEY, D.B. 1984. Factors affecting levels of some phenolic compounds, digestibility and nitrogen content of the mature leaves of Barteria fistulosa (Passifloraceae). J. Chem. Ecol. 10:387-401. WILKEN,L.O., and COSGROVE, F.P. 1964. Phytochemical investigation of Carya illinoensis. J. Pharm. Sci. 53:364-368.