Journal ofChemicalEcology, Vol. 17, No. 1, 1991

METHODS A N D PITFALLS OF EXTRACTING CONDENSED TANNINS AND OTHER PHENOLICS FROM PLANTS: INSIGHTS FROM INVESTIGATIONS ON Eucalyptus LEAVES

STEVEN

J. C O R K * a n d A N D R E W

K. K R O C K E N B E R G E R

1

CS1RO Division of Wildlife & Ecology PO Box 84 Lyneham, ACT 2601, Australia (Received May 25, 1990; accepted August 21, 1990) Abstract--Optimal conditions for extraction of tannins and other phenolics from tree foliage and their subsequent storage rarely have been investigated. We investigated methods of drying leaves, optimal solvents, and the effects of light and temperature on the extractability and stability of condensed tannins (proanthocyanidins) and total phenolics from leaves of Eucalyptus trees. Aqueous acetone was a better solvent than aqueous methanol for condensed tannins and total phenolics, but condensed tannins were less stable in aqueous acetone than aqueous methanol. Stability of condensed tannins also was decreased substantially by room temperature versus 4~ and by exposure to indirect sunlight, although the assay for total phenolics was unaffected. For quantitative estimation of condensed tannins, extraction with 50% acetone was better than methods of direct analysis of leaf tissue. The highest estimates of total condensed tannins were obtained by exhaustive extraction with 50% acetone followed by direct analysis of the residue. Lyophilization of fresh leaf increased yield of condensed tannin (although usually by less than 10%). Lyophilization and subsequent storage of extracts had little effect on assays for condensed tannins or total phenolics. Key Words--Tannin extraction, plant phenolics, Eucalyptus, browse analysis.

INTRODUCTION M u c h a t t e n t i o n h a s b e e n p a i d to c h e m i c a l a n a l y s i s o f t a n n i n s a n d o t h e r p h e n o l i c s in p l a n t foliage b e c a u s e o f t h e i r p o s t u l a t e d e c o l o g i c a l i m p o r t a n c e in p l a n t plant and plant-animal interactions. Methods for chemical and biochemical

*To whom correspondence should be addressed. Present address: School of Biological Sciences, University of Sydney, N.S.W. 2006, Australia. 123 0098-0331/91/0100-0123506.50/0 9 1991 Plenum PuNishing Corporation

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analysis of tannins have been reviewed in detail (Mole and Waterman, 1987a,b; Hagerman and Butler, 1989). However, methods of preparing leaf samples for chemical analysis, which can introduce greater errors than the analyses themselves, have received little critical attention apart from a recent paper by Hagerman (1988). Frequently it is inconvenient to analyze fresh leaf tissue and some form of drying is needed. It is well established that air- or oven-drying, especially at temperatures above 70~ decreases yield of tannins and other phenolics (BateSmith, 1975; Price et al., 1979; Gartlan et al., 1980; Lindroth and Pajutee, 1987; Hagerman, 1988). Lyophilization (freeze-drying) is the most promising compromise and is adopted uncritically by many workers. However, the few studies that have investigated the effects of lyophilization on yield of phenolics have shown that some loss, no loss, or some enhancement of tannin extractability is possible (Martin and Martin, 1983; Price et al., 1979; Hagerman, 1988). Debate about the best solvent for tannins in plant tissues has been based on a limited number of studies of few plant species, and conclusions are not always consistent with one another. Aqueous methanol (boiling or at room temperature), acidic methanol, and aqueous acetone have been recommended by various authors. Usually one solvent is recommended without comparison with alternatives or without measurement or estimation of the extent of extraction achieved. The suitability of methanol as an extractant for hydrolyzable tannins has been questioned (Haslam et al., 1961; Swain, 1979), and its ability to extract condensed tannins from some plants also is low (Bate-Smith, 1973, 1975; Foo and Porter, 1980). Many workers now use aqueous acetone for extraction of tannins, although it does not give high recoveries with all plants (Martin and Martin, 1984; Stafford and Cheng, 1980). The presence of water in organic extractants can both increase extraction of phenolics and enhance their breakdown after extraction (Swain, 1979; Lindroth and Pajutee, 1987). The choice of water content usually appears to be arbitrary between 0% and 50% and to follow precedents set by other workers analyzing other plant species. Extraction time has been studied even less rigorously. Times from a few minutes to one or more days have been used. Short extraction times aim to minimize chemical degradation of extracted tannins, and long times to maximize extraction, but seldom is the extent of extraction or degree of degradation quantified. Similarly, few published methodologies make any recommendations about exposure to light, which might affect the stability of phenolics. We investigated the best pretreatment, solvent, time, and temperature for extraction of tannins and other phenolics from the foliage of Eucalyptus trees, as a preliminary to a survey of tannins in eucalypt forests in southeast Australia. The foliage of eucalypts contains a wide range of phenolic compounds, including condensed and hydrolyzable tannins and nontannin phenolics (Hillis, 1966;

125

EXTRACTION OF TANNINS

Fox and Macauley, 1977; Cork, 1984; Cork and Pahl, 1984). We observed several trends that probably have general applicability, have been reported seldom or never before in the literature, and could substantially alter conclusions from chemical analyses of tannins in plants unless taken into account. METHODS

AND MATERIALS

Samples and Their Collection. We collected mature leaves from low branches of Eucalyptus spp. trees growing in the grounds of CSIRO Division of Wildlife and Ecology in Canberra. The following codes are used throughout this paper to designate the trees that were sampled: EMAN1 and EMAN2--two individuals of Eucalyptus mannifera; EMEL1 and EMEL2--E. melliodora; EVIM--E. viminalis; EPAUC--E. pauciflora; EHYB--unidentified hybrid eucalypt. The leaves were immediately frozen and ground in liquid nitrogen in a small coffee-grinder or by mortar and pestle. A portion of this ground, undried leaf was stored at - 2 0 ~ and another portion was lyophilized first. During all of the tests described below, the dry matter content of samples (including those lyophilized) was determined by oven-drying a subsample to constant mass at 70~ Total phenolics in acetone or methanol extracts (details of extraction procedure are given below) were analyzed by the Folin-Ciocalteu method (Singleton and Rossi, 1965). Condensed tannins (a subset of total phenolics) in acetone or methanol extracts, solid residues remaining after extraction, and unextracted solid samples were analyzed by the HCl-butanol (proanthocyanidin) method (Reed et al., 1982; Mole and Waterman, 1987a). The HCl-butanol reagent used in all of these analyses (Mole and Waterman, 1987a) contained a transition-metal salt (FeSO4 9 7H20) to ensure consistency of proanthocyanidin yield (Porter et al., 1986). Correction was made for interference from chlorophyll by running unheated reagent + sample blanks (Swain and Hillis, 1959). The extent of interference was small compared with the tannin concentrations, so it was deemed unnecessary to take more comprehensive precautions, such as suggested by Watterson and Butler (1983). Due to the difficulty of obtaining suitable standards for phenolic analyses (Hagerman and Butler, 1989; Mole et al., 1989), we expressed all values relative to quebracho tannin (a condensed tannin). The quebracho tannin was purified (Asquith and Butler, 1985) from crude material supplied by A. E. Hagerman (Hagerman and Butler, 1989). Quebracho clearly produced relatively more color than eucalypt total phenolics in the Folin-Ciocalteu assay and/or less color than eucalypt condensed tannin in the HCl-butanol assay, resulting in estimates of condensed tannin often being greater than total phenolics. This did not, however, affect any of the comparisons made or conclusions drawn. We did not analyze for hydrolyzable tannins because a suitable method was not available, although one has come to our attention since (Inoue and Hagerman, 1988).

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Investigation of Extraction Conditions (Experiment 1). We observed in preliminary experiments that when either acetone- or methanol-based aqueous solvents were used at room temperature with exposure to indirect sunlight (normal daytime room light), the concentration of condensed tannins (although not of total phenolics) in the extract fell rather than rose after about 2-5 hr. This suggested instability due to light and/or temperature. Therefore, experiment 1 was set up to investigate the stability of leaf extracts (in 50 % and 70 % acetone, and 50% methanol for reasons given below) under three storage conditions: (1) unprotected from indirect sunlight (on laboratory bench) at room temperature; (2) in a dark cupboard at room temperature; and (3) in the dark at 4~ The extracts were sampled initially and after three days. Investigation of Solvents (Experiment 2). In this experiment we compared methanol-based with acetone-based solvents because these are most often recommended. Preliminary investigations showed that anhydrous acetone or methanol extracted less than 50% of the total phenolics and condensed tannins that aqueous mixtures extracted, so no further investigation of anhydrous solvents was made. We had found previously that 50 % methanol was superior to higher proportions of methanol in water for extracting phenolics from eucalypts. Fifty percent methanol was compared with 50% and 70% acetone in the experiments reported here. Samples were extracted (0.02 g wet leaf per 5 ml solvent) in test tubes at 4~ in a sonicator (to minimize extraction time) for three successive 30-min periods. Previous tests had shown that negligible amounts of condensed tannins or total phenolics were removed by further extraction. After each period, the tubes were centrifuged (3000 rpm, 15 rain, at 4~ and the supernatant was removed, sampled, and replaced with fresh solvent. As an alternative to sonication, we also tested continuous agitation on a mechanical shaker at the maximum speed possible without loss of solvent by splashing and found it to give identical results (unpublished data). Comparison of Extraction with Analysis of Whole Sample (Experiment3). Because some authors (Bate-Smith, 1973, 1975; McArthur, 1988) have suggested that higher yields of condensed tannin can be obtained by analyzing whole tissue rather than extracts, we compared five alternative ways of estimating the total condensed tannin present in leaf samples: (A) leaf samples were extracted exhaustively in 50% acetone, as above; (B) samples were extracted exhaustively as in (A) and condensed tannins were also measured in the remaining solid residue; (C) condensed tannin was measured directly in unextracted, undried, ground leaf tissue; (D) analysis was performed as in (C) after pretreatment with boiling methanol (1 ml to 20 mg sample) for 5 rain (Bate-Smith, 1973); and (E) analysis was performed as in (C) after sonication in 50% acetone (1 ml to 20 mg sample) for 30 min. Subsamples for all five treatments were taken from the same sample at the same time and processed simultaneously.

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EXTRACTION OF TANNINS

Effects of Methods of Drying (Experiment 4). With leaf samples from the same trees as used in the experiments described above, but sampled two months later, we tested the effects of three drying treatments on extraction and measurement of condensed tannins and total phenolics. Leaves were sampled and immediately ground in liquid nitrogen, then subsamples were analyzed after: (1) no drying; (2) lyophilization; or (3) oven drying at 70~ for 24 hr. We also investigated the feasibility of lyophilizing 50% acetone extracts for storage: subsamples were analyzed either immediately or after lyophilization, storage for up to a week, and reconstitution with water. Statistical Analysis. Treatment means were compared using a two-factor analysis of variance (Zar, 1984) with eucalypt species as one factor and the treatment as the other. Arcsine transformation (Zar, 1984) was applied to percentages before analysis.

RESULTS

Experiment 1. Both light and temperature had pronounced effects on the stability of condensed tannins in leaf extracts (Table 1). Storage at 4~ in the dark, regardless of solvent, resulted in only small reductions in measurable condensed tannin, but room temperature and exposure to indirect sunlight caused much greater reductions. In most cases, the effects of light and temperature were less pronounced for 50% methanol than for the two concentrations of aqueous acetone. There was a statistical trend towards higher recovery of total phenolics after storage at room temperature in the dark than after the other two storage treatments, but the magnitude of this difference seems biologically insignificant (Table 1). Experiment 2. As expected, all three solvents removed very little additional condensed tannin or total phenolics after 2 x 30-min sonications (Table 2). Aqueous acetone extracted considerably more condensed tannin and total phenolics than did aqueous methanol. Fifty percent acetone consistently extracted more condensed tannins than did 70 % acetone and extracted as much or more total phenolics (Table 2). Experiment 3. Of the five extraction-analysis treatments compared in experiment 3, exhaustive extraction with 50% acetone followed by analysis of the residue (treatment B) usually gave the highest estimate of total condensed tannins (Table 3). Direct analysis of ground leaf consistently gave a lower estimate of condensed tannin than did extraction alone. Pretreatment of the ground leaf with either boiling butanol or 50 % acetone and sonication before adding the reagent improved the yield of condensed tannin but still gave an estimate at best 105% of treatment B and at worst 62% (Table 3). In all of these direct analyses the leaf tissue was colored red after boiling in HCl-butanol, indicating

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CORK AND KROCKENBERGER TABLE 1. EFFECT OF LI6nT, TEMPERATURE, AND SOLVENT ON STABILITYOF

CONDENSED TANNINS (CT) AND TOTAL PnENOLICS (TP) (ExPEedMENT 1)a Treatment Room temp. / dark

4~ Species code EMAN2

EMEL1

EVIM

EPAUC

Means

Solvent 50% 70% 50% 50% 70% 50% 50% 70% 50% 50% 70% 50% 50% 70% 50%

Ac Ac Me Ac Ac Me Ac Ac Me Ac Ac Me Ac Ac Me

Room temp./ sunlight

CT

TP

CT

TP

CT

TP

96 101 106 91 99 103 63 107 70 85 104 109 84b 103b 97b

97 100 96 92 96 94 96 98 94 98 101 96 96b 99b 95

83 87 91 77 83 87 40 93 53 69 80 90 67 86 80

99 108 94 96 102 98 100 104 97 104 109 101 100 106 98

63 60 102 39 35 83 31 61 65 42 59 96 44 54 86

94 101 94 90 96 92 96 102 95 99 104 98 95 101 95

a Extracts were analyzed initially and then after storage for three days under the conditions indicated. Data are means of duplicate determinations and are expressed as percentage of the initial concentration. bSignificant treatment effect (P < 0.005) within row. that not all tannin was extracted and m e a s u r e d . This p r o b l e m appeared to be less p r o n o u n c e d for t r e a t m e n t B, w h i c h was c o n c l u d e d to give the best possible e s t i m a t e o f total c o n d e n s e d tannins. Experiment 4. O v e n - d r y i n g greatly reduced the amounts o f c o n d e n s e d tannin and total p h e n o l i c s extractable by solvents and the a m o u n t o f c o n d e n s e d tannin detectable in the residue r e m a i n i n g after extraction with 50% acetone (Table 4). L y o p h i l i z a t i o n caused a consistent increase in extractability and measurability o f c o n d e n s e d tannin and total p h e n o l i c s f r o m leaf, although this increase was small in all but one case (Table 4). L y o p h i l i z a t i o n and reconstitution o f 50 % acetone extracts had no consistent effect on m e a s u r e m e n t s (Table

4). DISCUSSION T o interpret the role o f tannins and o t h e r p h e n o l i c s in p l a n t - p l a n t or p l a n t a n i m a l interactions, it is i m p o r t a n t that at least the total a m o u n t present in plant samples can be e s t i m a t e d confidently and preferably that quantitative extracts

EXTRACTION OF TANNINS

129

TABLE 2. EFFECTS OF SOLVENT ON EXTRACTION OF CONDENSED TANNINS ( c r ) AND TOTAL PHENOLICS (TP) FROM EUCALYPT FOLIAGE (ExPEmMENT 2) a

1st extraction Species code EMAN1

EMAN2

EMEL1

EPAUC

Means b

2rid extraction

3rd extraction

Totals

Solvent

CT

TP

CT

TP

CT

TP

CT

TP

50% Ac 70% Ac 50% Me 50% Ac 70% Ac 50% Me 50% Ac 70%Ac 50% Me 50% Ac 70%Ac 50% Me 50% Ac 70% Ac 50% Me

564 527 487 468 428 416 248 269 233 165 149 117

194 18t 163 202 184 181 97 116 98 90 85 63

69 82 97 85 119 53 102 43 48 31 27 15

26 25 29 34 48 25 42 18 20 18 20 11

4 0 13 13 16 15 17 14 I5 6 13 10

8 6 11 9 9 8 9 7 7 7 13 8

637 609 597 566 563 484 367 326 296 202 189 142 443 421 379

228 212 203 245 241 214 148 141 125 115 118 82 184 178 157

"Extraction was in the dark, at 4~ in a sonicator, for three successive 30-min periods. All data are expressed as quebracho equivalents (mg/g dry leaf). Values are means of duplicate determinations. hSignificant solvent effects (P < 0.005) on total yield of both condensed tannins and total phenolics.

can be obtained for chemical and biochemical analyses (Mole and Waterman, 1987a,b; Hagerrnan, 1988). The present study questions some untested notions prevalent in the literature about how best to estimate total condensed tannins, adds to the meagre data on the relative effectiveness of various solvents for phenolics in plant tissues, and focuses on some major errors that can occur if extractions are not performed, or extracts are not subsequently stored, under appropriate conditions. Bate-Smith (1975) concluded that condensed tannins (proanthocyanidins) seldom are completely extractable from plants. He suggested direct analysis of unextracted leaf to estimate total condensed tannin. McArthur (1988) applied this approach to analysis of eucalypt foliage where it appeared that oven-drying had seriously reduced extractability of phenolics. Reed et al. (1982) analyzed residual phenolics in isolated cell-wall fractions of shrub foliage. However, direct analysis of plant tissue rarely has been compared with other ways of estimating total condensed tannins when fresh samples are used. We found that, for eucalypts, summing the results of analysis of the 50% acetone extracts and of the leaf residues gave 20-40% higher estimates of total condensed tannin

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CORK AND KROCKENBERGER

TABLE 3. EFFECTSOF SOLVENTEXTRACTIONVERSUSDIRECTANALYSISOF LEAF TISSUEON YIELDOF CONDENSEDTANNINS(EXPERIMENT3)a Percent of estimated total CT measured by Species code EMANI EMAN2 EMEL1 EMEL2 EPAUC EHYB Meansb

(A) Exhaustive extraction 95 96 95 89 86 81 90

(B) Solvent + residue 100 100 100 100 100 100 100

(C) Whole sample 73 66 79 67 62 67 69

(D) Boiling methanol 87 80 81 76 66 70 77

(E) Presonication 86 75 105 105 62 83 86

aFive treatments were employed: A, exhaustive extraction in 50% acetone as in Table 2; B, extraction as in A followedby direct analysisof condensedtannin remainingin the extracted residue; C, direct analysisof leaf tissue; D, analysisof leaf tissue after pre-treatmentwith boiling methanol for 5 min; and E, analysis of leaf tissue after sonicatiori in 50% acetone, at 4~ for 30 rain. Values are expressed as percentage of treatmentB, which in all but two instancesgave the highest estimate of total condensed tannins. Data are means of duplicate determinationson extracts and quadruplicate determinationson leaf tissues and residues. oSignificanttreatment effect (P < 0.005).

than direct analysis of unextracted leaf and that extraction in 50% acetone alone also is superior to the direct analysis. Bate-Smith (1973, 1975) recognized the need to pretreat some leaf samples for tissue analysis, but we found that pretreatments gave only small improvements in most cases. A major benefit of combining use of solvent with analysis of the residue is that an estimate of the extractability of tannins (and other phenolics if a total phenolic analysis of the residue can be performed) is obtained. Extractability of tannins might have at least as much physiological and/or ecological significance for herbivores as the total amount present (Mole and Waterman, 1987a). Very few studies that have included analysis of phenolics have stipulated extraction at low temperatures (e.g., Lindroth and Pajutee, 1987; Inoue and Hagerman, 1988), although boiling during extraction has been discouraged (Foo and Porter, 1980; Hagerman, 1988). We know of no studies that have emphasized protection from light during extraction. We presume that many workers perform their extractions without these precautions, as we hawe previously. In the present study, instability of condensed tannins caused by sunlight and room temperature was evident and substantial within 2-5 hr of the start of extraction. Therefore, it probably causes significant underestimates of extracted tannin even in short extraction periods. Inclusion of water in solvents is another source of

EXTRACTIONOF TANNINS

13 1

TABLE4. EFFECTSOF THREE DRYINGTREATMENTS(No DRYING, FREEZE-DRYING, AND OVEN DRYINGAT 70~ ON EXTRACTIONOF TOTALPHENOLICS(TP) AND CONDENSEDTANNINS(CT) IN 50% ACETONE, AND EFFECTSOF TWO SUBSEQUENT TREATMENTSOF 50% ACETONEEXTRACT(IMMEDIATEANALYSISOR ANALYSIS AFTERFREEZE-DRYINGAND RECONST1TUTIONWITHWATER) (EXPERIMENT4) a Fresh leaf

Freeze-dried

Oven-dried

Species code

Extract treatment

TP

CT

%Ex

TP

CT

%Ex

TP

CT

%Ex

EMAN1

Immed. Reconst. Immed. Reconst. Immed. Reconst. Immed. Reconst. Immed. Reconst. ImmedJ' Reconst.'

175 184 148 157 166 169 82 88 142 148 143 149

434 477 358 355 26 28 187 181 289 247 258 258

89

186 195 152 157 163 169 134 88 151 154 157 153

475 488 382 355 29 25 307 289 314 253 301 282

91

165 172 146 153 138 137 73 73 93 95 123 126

383 393 362 349 14 13 120 110 161 136 208 201

87

EMEL1 EVIM EPAU EHYB Means

90 80 73 81 83

87 81 90 81 86

86 69 58 60 72

"Also indicated (%Ex) is the percentage of total condensed tannin (estimated as in treatment B, Table 3) extracted by 50% acetone after the three drying treatments. All data are quebracho equivalents (mg/g dry leaf). 1'Significant effect of drying treatment (P < 0.05) for TP, CT and %Ex. 'No significant effects of extract treatment (P > 0.05).

(hydrolytic) degradation of phenolics (Lindroth and Pajutee, 1987), but there is no alternative if quantitative estimates o f phenolics are needed, because extraction usually is poor in anhydrous solvents. It appears from Table 1 that the structure of Eucalyptus spp. condensed tannins is altered in an aqueous solvent enough to reduce their reactivity with the H C l - b u t a n o l reagent but not sufficiently to reduce the intensity o f color production on reaction with the FolinCiocalteu (total phenolics) reagent. This might be due to light-catalyzed polymerization (oxidative coupling) o f condensed tannin chains that would not affect the phenolic hydroxyls with which the Folin-Ciocalteu reagent reacts (P. G. Waterman, personal communication). Consequently, if extraction and/or storage conditions are inappropriate, analysis o f total phenolics might indicate stability but biochemical tests that rely on the reactivity o f tannins with proteins or other substrates (Mole and Waterman, 1987b) could be seriously in error. The optimal choice between acetone- and methanol-based solvents probably varies with the plant species under study (Hagerman, 1988), but some generalizations are possible. Extractability in methanol decreases with the size

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of the tannin polymer (Goldstein and Swain, 1963; Foo and Porter, 1980), whereas no such trend is apparent for acetone (Jones et al., 1976; Foo and Porter, 1980). Acetone appears to break or prevent tannin-protein associations, whereas methanol does not (Foo and Porter, 1980; Hagerman and Robbins, 1987). Hagerman (1988) found aqueous acetone was superior to aqueous methanol for extracting tannins (mixtures of condensed and hydrolyzable) from several plant species, but the extent of superiority varied seasonally. We conclude that aqueous acetone should be the choice of solvent for extracting phenolics from eucalypts also, because it consistently extracted more condensed tannin and total phenolics than did aqueous methanol in the species we examined. This conclusion is strengthened by the observations that eucalypts contain high concentrations of hydrolyzable as well as condensed tannins (Hillis, 1966; Fox and Macauley, 1977; Cork et al., 1983; Cork and Pahl, 1984) and the aromatic ester (depside) bonds in hydrolyzable tannins are unstable in aqueous alcohols (see Hagerman, 1988). Oven-drying of eucalypt foliage is reported to reduce the yield of total phenolics and condensed tannins by 12-100% (Cork et al., 1981; McArthur, 1988), and the reduction observed in the present study was 0-45 % (Table 4). Reductions in extractability of tannins as a result of oven-drying have been reported for leaves of hickory, oak, and maple (Hagerman, 1988) and 20 African rain-forest species (Gartlan et al., 1980). Sun-drying produces similar reductions to oven-drying (Gartlan et al., 1980). In all but one of the eucalypt species investigated here, lyophilization increased extractability of condensed tannins and total phenolics by 2-10% (Table 4), which, depending on the reason for analysis, could be an error to be wary of. In one case (EPAU) lyophilization increased extractability from 73 % to 90% (Table 4). Hagerman (1988) also concluded that lyophilization can affect extractability of tannins from foliage and that the effect can vary seasonally. The different effects of lyophilization and fresh analysis can be partly, but not fully, compensated for by analysis of residues as well as extracts to give an estimate of extractability and total tannin (Table 4). Although the present study has concerned members of only one genus of plants, the phenolic chemistry of this genus is diverse (Hillis, 1966) and the species sampled were each from a separate infrageneric series (Chippendale, 1988). Therefore, the potential problems and pitfalls identified are likely to apply to many other plants. Inappropriate extraction conditions, especially lack of attention to the effects of light and temperature and the efficiency of the solvent in use, can make comparisons between plant species dubious and interpretation of physiological interactions between plants and animals very difficult. Therefore, we urge chemical ecologists analyzing plants for tannins and other phenolics to consider preparation and extraction conditions carefully, and we hope that this paper will assist.

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133

Acknowledgments--We thank Ms. Mandy Yialeloglou for skilled technical assistance in the performance and development of the techniques used here. We also thank Dr. A. Hagennan and Dr. C. McArthur of Miami University, Ohio, U.S.A., for helpful advice on analytical techniques. However, we are solely responsible for the final choice and implementation of techniques. Financial support for this work was provided by a grant (8889/06) from the Australian National Parks and Wildlife Service.

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Methods and pitfalls of extracting condensed tannins and other phenolics from plants: Insights from investigations onEucalyptus leaves.

Optimal conditions for extraction of tannins and other phenolics from tree foliage and their subsequent storage rarely have been investigated. We inve...
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