Evaluation of a semi-quantitative method for rapid screening of plant phenolic content

A rapid method for screening phenolic content in leaves is described and evaluated . The melhod is based on a treated paper strip that is squeezed around a leaf with pliars . Weaknesses of the method are some sub· jectivlty, lack of precision , and apparent insensitivity to phenolics present as glycosides. Strengths include its speed and independence of laboratory facilities. lmme· diate testing o{ leaves following detachment appears to be essential . The ct ude extraction in the field compares favorably with a more conventional extraction techn:que. The method is recommended for use in prel;minary screening of plant material when a large number of species must te examined within a short time and when laboratory facílities are not avaílable at the fie ld si te .


lNTRODUCTION
Plant phenolics constitute a large and varied chem ical class that includes pigments, metabolic compounds, and so-called secondary compounds.The last group is of special interest to ecologists and contains compounds that have been implicated as protective agents against herbivores and pathogens [cf.Levin, 1971).allelopathic substances [cf.Rice, 1974).and decomposition inhibitors (.cf. Handley, 1961;Benoit & Starkey, 1968a, 1968b).The chemistry and functions of pheno lics have been discussed at length (e.g., Levin, 1971;Walker, 1975;Swain, 1977).
The method chosen for phenol ic determination depends on one 's objectives.For chemical taxonomy, chromatographic techniques are often appropriate [Hathway, 1969).In the tanning industry, gelatin and hide powder precipitation are the basis of a complex and empírica!determination (White, 1958).Ecologists may be more interested in a measure of total tannin content.For this purpose colorime-Anthony B. Anderson (1) Theodore V. St. John (2) tric determinations of a water extract has often been employed (AIIen et a/., 1974).Feeny & Bostock (1968) used a more sophisticated gravimetric procedure on an acetone-water extraction.
Burns (1968) reviewed phenolic methods for use in assessing forage quality.Among his suggestions was a screening test based on paper strips impregnated with ferrir.ammonium citrate.Each strip is squeezed around a leaf to produce a dark spot proportional in intensity to the pheno lic concentration in the leaf .This method could have considerable appeal to tropica l ecologists.lt is independent of laboratory facilities, easily used in remote sites, and sufficiently rapid to allow evaluation of a large number of samples within a short time.Probably no alternative would permit examination of the great number of species characteristic of some tropical environments.For these reasons we undertook a criticai evaluation of the reproducibility and utility of Burns's method.

FIELD SITES
Leaf samples were obtained from two distinctive vegetation types in the vicinity of Manaus, Amazonas State, Brazil: (1) Amazon caatinga, a scrub vegetation on white sand (entisol)

LEAF COLLECTION
Leaf samples were obtained from a total of 210 species.Foliage was taken with a shotgun or by climbing the trees except at the white sand site, where they could be picked off the shrubs or low trees at ground levei.Ten leaves per species were collected for testing, usually from 2-3 separate branches.Leaves were usually obtained from the lower portion or center of the crowns.To test for variability within individual plants, a subsample of six leaves from various portions of the crown was obtained from one individual of each of ten white sand species.Whenever possible, sampling was limited to mature leaves believed to be of the current yeor's crop.Ali samples were collected during the rainy season, from January to April, 1977.Because of difficulties in collecting from tall trees on the oxisol site, leaf samples in both sites were taken from a single individual of each species.To test for variability among individual plants of a given species, subsamples were obtained from five individuais of ten white sand species.

FIELD SCREENING METHOD
The method for screening leaf phenolics was adapted from Burns (1968).A strip of filter paper previously impregnated with 0.5% (wt./vol.)ferric ammonium citrate was folded around the base of each leaf near the petiole (but not across the midrib) and squeezed with pliars.A concentration of 0.5% was used instead of the 2% recommended by Burns.A preliminary survey of 32 species showed that intensity of test spots is virtually independent of solution concentration over the range tested, and the lower concentration produced less discoloration of the test paper.We elimmated Burns's use of a second strip of untreated filter paper inside the test strip.The second strip was found to be impractical because many of the leaves were sclerophyllous and contained insufficient fluids to dampen the treated strip.Testing was usually carried ou tin the fi eld immediately after obtaining the leaves.The effect of a 5-hour lag time was determined in a subsample of six white sand species.

802-
Leaf phenolics react with ferric on thc test strips, producing a stained spol that vancs in intensity.On the basis of spot intensity.leaves can be assigned ot categories of leaf phenolic concentration.Although Burns recommended the use of ten categories, we were able to clearly distinguish only six.

COMPARISON WITH STANDARDS
The six categories of spot intensities produced by our samples were compared with three types of standards developed by dipping test strips into known concentrations of tannic acid, quebracho tann in, and wattle tannin o To test the permanence of the spot, the original standards were compared one year later with test strips (taken from the same batch as the origina l standards) di pped into newly prepared tannic acid solutionso

COMPARISON WITH SOLVENT EXTRACTION, ACID HYDROLYSIS, AND FOLIN-DENNIS TEST
The phenolic screening test was carried out on fresh leaves of eleven wh ite sand species and compared with the same test following (a) extraction in an acetone-water solution and (b) acid hydrolysis (Figo 1)0 Finally, the phenolic screening method was carried out on fifteen white sand species; separate leaf samples of these species were oven dried and analyzed by the Folin-Dennis methodo

VARIABILITY WITHIN PLANTS
Leaves of ten species were used to determine whether samples from different portions of the same plant differ detectably o In two of these species, one of the six tests differed from the others by a single category; the other eight species had no variation within the planto

VARIABILITY AMONG PLANTS OF A GIVEN SPECIES
Leaves of five i ndividuais in each of ten species were examined to determine whether detectable variability exists among plants of a The spot intensities that resulted from dipping test papers into the acetone-water extract were compared in an ordinal sense with the same series of eleven species tested in the field .The sequences of species were identical .This indicates that similar phenolic fractions are detected when testing fresh leaves in the field and when testing after extraction.

COMPARlSON AFTER ACID HYDROLYSIS
In the test for effects of acid hydrolysis, three of the eleven species showed increases in relative spot intensity after acid hydrolysis.The increases probably indicate that the aglycones liberated by acid are more reactive In this test than the glycosides of the original extract.

COMPARISON WITH FOLIN-DENNlS METHOD
Finally, oven-dried samples o f fifteen species were analyzed by the Folin-Dennis method .Again , we could not compare the two methods directly, but we could rank the two sets of values and compare the sequences.There was no apparent relationship between the two sequences.The difference may result from different sensitivities of the methods.A second source of error may be changes in detectable phenolic content following detachment, drying, and storage of leaves.The large shortterm changes reported above lead us to suspect that the latter effect is the primary source of error.

DlSCUSSION
The phenolic screening method described in this paper has severa I weaknesses.Among these is the subjectivity involved in categorizing spot intensity .Although the spot was usually a shade o f gray.many leaves produced reddish or brown spots .The colar had to be disregarded and only the intensity considered in classifying the spots.With practice, the two authors found good agreement when evaluatmg 804-spots independently .A second weakness of the phenolic screening methoó is lack of precision: phenollc levei is nominal rather than quantitativa .Third , the method appears to be insensitive to phenolics present as glycosides.The lack of a solvent extraction does not appear to constitute a weakness , as we obtained similar results with an extraction.
The strengths of the phenolic screening method are its speed and ease of appli ~ation .These attributes make it ideal for: (a) analysis of large sample sizes in a short time or (b) preliminary screening of samples before using more quantitativa or specific methods.One of the greatest advantages of this method may be its immediate application following leaf detachment, before potential alteration of phenolic compounds.
In conclusion.we offer the following suggestions for use of the phenolic screening method: (1) A single batch of test strips larger than the expected sample size should be prepared .We found variable results when using batches prepared on different occasions.
(2) Standards should be prepared at the outset, with a range of tannic acid concentrations .Spot categories should then be distinguished and assigned a relative value.
(3) The subjective element in evaluating spot intenslties may be reduced by having a slngle person assign categorias.This person should evaluate a preliminary sample to become accustomed to classifying test strips.
(4) Severa!(ca.ten) replicates of each sample should be obtained, and assignment to categories should be based on the most common SJ?Ot intensity.
(5) Before evaluation, the test strips should be allowed to dry .Evaluation should be made on the same day, and test strips should not be regarded as permanent.
(6) Depending on objectives, collection procedures should be standardized so as to minimize sampling variabliity within leaf, within plant, between plants, and/or between seasons.

APPENDIX -Relative phenolic content of 211 central
Amazonian native species.Species wi"hout an as• terisk were collected on the oxisol site; species with one asterisk were collected on the with sand site; species with two asterisks were collected on both sites .Phenolic content was evaluated on a scale of 1 to 6, with 1 having the lowest content.In cases where two values appear, the first refers to the white sand site and the second to the oxisol site.Evaluatlon ...     Mr. James Van May for providing commercial quebracho and wattle tannin preparations for use as a standard.

Species
Ms. Sandra Knapp and Dr. Philip W. Rundel for determ ining tanni n concentration by the Folin-Dennis method on fifteen species.
Field work was supported in part by a grant from t he Brazilian National Research Council to Dr. Paulo de Tarso Alvim.Preparation of th e manuscript was supported by U .S .National Science Foundation Grant DEB 78-11 201 .
Evaluation o o o given species o One individual of one species differed by a single category from the other four indiv4duals o In al i other species, every individual tested was in the same categoryo