Phenolics, Fibre, Alkaloids, Saponins, and Cyanogenic Glycosides in A

Biochemical Systematics and Ecology 31 (2003) 1221–1246 www.elsevier.com/locate/biochemsyseco

Phenolics, ﬁbre, alkaloids, saponins, and cyanogenic glycosides in a seasonal cloud forest in India S. Mali a, R.M. Borges b,∗ a National Innovation Foundation, Vastrapur, Ahmedabad 380 015, India b Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, India

Received 18 June 2001; accepted 3 February 2003

Abstract We investigated secondary compounds in ephemeral and non-ephemeral parts of trees and lianas of a seasonal cloud forest in the Western Ghats of India. We measured astringency, phenolic content, condensed tannins, gallotannins, ellagitannins, and fibre, and also screened for alkaloids, saponins and cyanogenic glycosides in 271 plant parts across 33 tree and 10 liana species which constituted more than 90% of the tree and liana species of this species- poor forest. Cyanogenic glycosides occurred only in the young leaves of Bridelia retusa (Euphorbiaceae), i.e. in 2.3% of species examined. Alkaloids were absent from petioles, ripe fruit and mature seeds examined. Saponins were found in all types of plant parts. Condensed tannins occurred in almost all plant parts examined (93.6%), while hydrolysable tannins were less ubiquitous (gallotannins in 31.2% of samples, and ellagitannins in 18.9%). Astringency levels were significantly correlated with total phenolic, condensed tannin, and hydrolysable tannin contents. Condensed tannin and hydrolysable tannin contents were not related. Immature leaves, flowers, and petioles had high astringency while lower levels were found in fruit. Flowers and fruit had the lowest fibre levels. There was no relationship between relative dominance of a species in the forest and the fibre or phenolic contents of its mature leaves. In each plant part category, the frequency of species containing tannins together with alkaloids or saponins was significantly lower than the frequency of species containing tannins alone. There was, however, no segregation between alkaloids and saponins.  2003 Published by Elsevier Science Ltd. Keywords: Condensed tannins; Hydrolysable tannins; Plant defences; Plant secondary compounds; Ratufa indica; Western Ghats

∗ Corresponding author. Tel.: +91-80-3602972; fax: +91-80-3601428. E-mail address: [email protected] (R.M. Borges).

0305-1978/03/$ - see front matter  2003 Published by Elsevier Science Ltd. doi:10.1016/S0305-1978(03)00079-6 1222 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246

1. Introduction

Data on community-level distribution of secondary compounds (mainly phenolics and alkaloids) are available for only a few tropical forests in Africa and Asia (McKey et al., 1978; Gartlan et al., 1980; McKey et al., 1981; Davies et al., 1988; Waterman et al., 1988; Kool, 1992). Especially with regard to phenolics, most of these studies have focused on only a few estimation methods. For example, most studies have not examined hydrolysable tannins or the relationship between condensed and hydrolysable tannins in plant parts. In this paper, we report on a quantitative analysis of phenolics, condensed tannins, hydrolysable tannins (gallotannins and ellagitannins), bovine serum albumin (BSA) assays for tannin astringency, digestibility reducers such as acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) for 271 ephemeral and non-ephemeral parts of 33 tree and 10 liana species within a seasonal cloud forest community in the Western Ghats of India. We also provide qualitative data on saponins, alkaloids, and cyanogenic glycosides for these resources. The sampled tree and liana species constituted more than 90% of the species at the site, and the samples were collected as part of a larger study on the foraging strategy of the Malabar giant squirrel Ratufa indica. We restricted our analysis to those compounds that have been found to affect the foraging strategy of arboreal mammalian herbivores such as primates (e.g. Oates et al., 1980; McKey et al., 1981; Waterman and Choo, 1981; Waterman and Kool, 1994) and giant squirrels (Borges, 1989; Borges, 1992). Data on the macro- and micro-nutrients within these plant parts will be presented elsewhere. Because of the enormous structural diversity and lack of general techniques, we restricted our analysis to the qualitative analysis of toxins. Owing to the structural diversity of tannins and the procedural difficulties involved in their quantitative analysis (Martin and Martin, 1982; Mole and Water- man, 1987a,b; Mole et al., 1989; Waterman and Mole, 1989), we employed only standard and improved methods recommended by Waterman and Mole (1994).We also used a combination of chemical and protein-precipitating methods to determine the biological activity of tannins. Therefore, our results are comparable with studies of other forest communities done elsewhere. Despite the limitations of our data set, in terms of missing analyses for some metabolites in some plant parts, owing to factors such as lack of adequate sample, insignificance in the giant squirrel diet or other logistic constraints, we also attempt in this paper to examine the co-occurrence of metabolites such as tannins, alkaloids and saponins in various plant parts in order to examine predictions about the possible synergisms or negative interactions between these metabolites. For example, since alkaloids and tannins react to form insoluble alkaloid-tannates in herbivore guts preventing reactions between tannins and proteins (Freeland and Janzen, 1974), and because the surfactant properties of saponins negate the anti-digestibility effects of tannins (Martin and Martin, 1984; Freeland et al., 1985), tannins are expected not to co-occur with either alkaloids or saponins in plant parts. Since alkaloids and saponins may have synergistic effects (e.g. Kerharo and Adam, 1974), they may be expected to co-occur to enhance herbivore deterrence. In this paper we therefore primarily report on the distribution of secondary com- S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246 1223 pounds in various plant parts and secondarily attempt to test a few predictions relat- ing to the co-occurrence of tannins, alkaloids and saponins in these resources.

2. Materials and methods

2.1. Study site

The study area was within the Bhimashankar Wildlife Sanctuary in Maharashtra State, India (19°21Ј–19°11ЈN, 73°31Ј–73°37ЈE, altitude 900 m, annual precipitation 3000 mm). This is a species-poor forest where only eight tree species contribute to 85.4% of relative dominance values, and the three most common species (Mangifera indica [Anacardiaceae], Memecylon umbellatum [Melastomataceae] and Olea dioica [Oleaceae]) contribute to 64.1% of relative dominance (Table 1). The forest is highly fragmented; our study site was situated in the largest and best protected fragment constituting a temple sacred grove (see Borges, 1990, 1993 for further descriptions).

2.2. Sample collection and processing

Samples represented 25 families and 13 orders of plants (Table 1), and thus covered a wide spectrum of species within the cloud forest. All analysed species with only two exceptions (Terminalia bellerica and Terminalia chebula) were part of the natural evergreen community of the cloud forest. As these chemical analyses were performed as part of a larger study on the foraging ecology of the giant squirrel R. indica (Mali, 1998), our sampling and choice of chemical analyses were designed to understand food preference and food avoidance, and were also inﬂuenced by the availability of adequate sample and other logistic constraints. Samples of both ephemeral and non-ephemeral items were collected at the time of year when they featured most in the diet of the giant squirrel (to control for seasonal variation in phytochemistry, if any) and were dried at 40–50 °C in the ﬁeld in kerosene ovens.

2.3. Qualitative ﬁeld tests on fresh material for alkaloids, saponins and cyanogenic glycosides

We field-tested for alkaloids using Dragendorff’s and Mayer’s reagents, and later performed confirmatory tests on dried material (Gartlan et al., 1980). For saponin detection, we vigorously agitated small aqueous extracts with distilled water and took a substantial and long-lasting lather formation to indicate the presence of saponins (Trease and Evans, 1972). Some seeds and fruit pulp were difficult to hom- ogenise adequately for saponin extraction and remained untested. We used the picrate test to detect cyanogenic glycosides (Conn, 1979).

2.4. Quantitative phytochemical analysis

The results of the chemical analyses presented here are single values estimated from samples pooled across several plant individuals (Appendix A, Table A1). 1224 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246

Table 1 Orders, families and species of trees and lianas for which phytochemical data were obtained and relative dominance of trees in the study site

Order Family Species Relative dominance

Trees Celastrales Celastraceae Cassine paniculata (Wt. and 1.4 Arn.) Cassine sp. –a Maytenus rothiana (Walp.) – Lobreau-Cal. Ericales Ebenaceae Diospyros montana Roxb. – Diospyros sylvatica Roxb. 1.5 Sapotaceae Vangueira spinosa Roxb. – Xantolis tomentosa (Roxb.) Raf. 7.0 Symplocaceae Symplocos beddomei Clarke – Gentianales Rubiaceae Bridelia retusa (L.) Sprengel 0.2 Canthium dicoccum (Gaert.) T. – and B.b Randia dumetorum (Retz.) Poirb 0.04 Lamiales Oleaceae Olea dioica Roxb.b 14.3 Laurales Lauraceae Actinodaphne angustifolia 0.2 (Blume) Litsea stocksii Hook. 1.0 Malphigiales Clusiaceae Garcinia talbotii Raizada ex. 2.1 Sant. Euphorbiaceae Macaranga peltata (Roxb.). 0.1 Muell.-Arg. Mallotus philippensis (Lam.) 1.4 Muell.-Arg. Salicaceae Flacourtia indica (Burman) 0.05 Merrill Myrtales Combretaceae Terminalia bellerica (Gaert.) – Roxb. Terminalia chebula Retz. 0.04 Melastomataceae Memecylon umbellatum N. 15.5 Burman Myrtaceae Syzygium cumini (L.) Skeels 6.5 Syzygium gardneri Thw. 2.5 Rosales Moraceae Artocarpus heterophyllus Lam. 0.02 Ficus callosa Willd. 3.1 Ficus racemosa L. 0.5 Ficus religiosa L. 0.09 Ficus tsjahela Burman – Sapindales Anacardiaceae Mangifera indica L. 34.3 Meliaceae Amoora lawii (Wt.) Bedd. 1.8 Dysoxylum binectariferum 0.4 (Roxb.) Bedd. Rutaceae Atalantia racemosa Wt. and Arn. 0.4 Sapindaceae Lepisanthes tetraphylla (Vahl) 1.7 Radlk. Lianas Ericales Myrsinaceae Embelia ribes Burman (continued on next page) S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1225

Table 1 (continued)

Order Family Species Relative dominance

Fabales Fabaceae Acacia concinna (Willd.). DC. Acacia sp. Mezoneuron cucullatum (Roxb.) Wt. and Arn. Gentianales Rubiaceae Randia rugulosa (Thw.) Hk.b Gnetales Gnetaceae Gnetum ula Brongn. Oxalidales Connaraceae Rourea santaloides Dalz. and Gibs. Ranunculales Menispermaceae Diploclisia glaucescens (Blume) Diels. Rosales Elaeagnaceae Elaeagnus conferta Roxb. Rhamnaceae Ventilago bombaiensis Dalz.

a In the relative dominance column, – indicates that the species occurred in such small numbers in the study plot that dominance values were below 0.001. Dominance values were not obtained for lianas. b Species authority citation from Saldanha and Nicolson (1976); for the rest read as Saldanha (1984, 1986).

Owing to the large number of different plant resources involved, it was not possible to examine seasonal variation, if any, in the chemistry of the resources. We estimated total phenolic content by the Folin–Ciocalteu method (modiﬁed by Singleton and Rossi, 1965; detailed in Waterman and Mole, 1994) using extracts in 50% aqueous methanol (Martin and Martin, 1982) and tannic acid to construct the standard curve. We used the proanthocyanidin method to estimate condensed tannins (Porter et al., 1986; detailed in Waterman and Mole, 1994) using extracts in 50% aqueous methanol (Martin and Martin, 1982) and quebracho tannin (supplied by Anne Hagerman, Miami University) to construct the standard curve. For hydrolysable tannins, we prepared the sample extracts in 70% acetone. We used the rhodanine method for gallotannins (Inoue and Hagerman, 1988), and constructed the standard curve using gallic acid. For ellagitannins we used the method of Wilson and Hagerman (1990), and constructed the standard curve using ellagic acid. We estimated the astringency of tannins using the BSA assay (Hagerman and Butler, 1978; Asquith and Butler, 1985) with Remazol brilliant blue and used tannic acid to construct the standard curve. We determined ﬁbre content (ADF, NDF and ADL) according to Goering and van Soest (1970).

2.5. Independence of data points

In our analysis of patterns, we have treated each species as an independent data point and we have not conducted phylogenetically independent contrasts (PIC). This is because our data are from 42 angiosperm and one gymnosperm species within 25 families and 13 orders (Table 1; mean number of species per family = 1.75 ± 1.06 SD; mode = 1; mean number of genera per family = 1.36 ± 0.57 SD). Therefore, 1226 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 in almost all cases, we were examining only one species per genus, and one genus per family.

3. Results

3.1. Occurrence of alkaloids, saponins, cyanogenic glycosides and phenolics

Cyanogenic glycosides were not found in any of the plant tissues examined (N = 54 bark items, N = 108 reproductive parts, N = 109 leaf items) except for the ﬂush leaves of B. retusa (Euphorbiaceae). Therefore, only one out of 43 plant species (2.3%) exhibited cyanogenesis. Summaries of the species-wise occurrence of the different compounds in the various plant parts are given in Table 2. Alkaloids were not found in petioles, semi-ripe and ripe fruit, and mature seeds examined (Appendix A, Table A1). Saponins were found in all types of plant parts (Appendix A, Table A1). Condensed tannins were found in all immature leaves, as well as all petioles and ﬂowers examined. Hydrolysable tannins were found in 68% of species, and 38% of plant samples. Gallotannins were found in only 31% of the samples examined while ellagitannins were found in still fewer samples (only 19%). Fewer plant species contained condensed or hydrolysable tannins in their bark and stems than in other plant parts.

3.2. Relative concentrations of secondary compounds in plant parts

Immature leaves, flowers, and petioles had high astringency while lower levels were found in fruit (Table 3). Tree twigs had low levels of astringency, condensed and hydrolysable tannins but high levels of fibre (Table 3). Inner bark had astringency and condensed tannin levels comparable to that of mature leaves while fibre levels were lower than those found in twigs (Table 3). Flowers and fruit had low fibre levels (Table 3). After Bonferroni’s correction (P Ͻ 0.001), only immature leaves were found to have significantly higher astringency, gallotannin and ellagitannin content than tree twigs, flowers were found to have significantly higher gallotannin levels than immature leaves, and tree twigs were found to have significantly higher ADF levels than mature seeds.

3.3. The quantitative relationship between various measures of phenolics

Astringency levels were strongly positively correlated with total phenolic, condensed tannin, gallotannin and ellagitanin contents (Table 4). Condensed tannin values were not correlated with gallotannin or ellagitannin contents. Gallotannin and ellagitannin contents were strongly positively correlated with each other (Table 4). Of the fibre components, since ADF, NDF and ADL are all highly correlated with each other, we used only ADF values in the correlations. We found no significant relation between any measure of phenolics and fibre after Bonferroni’s correction, except for the negative relationship between gallotannins and ADF (Table 4). S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1227 category where they indicate total number of category where they indicate percent samples ’ ’ All pooled All pooled ‘ ‘ phenolics tannins tannins ed, values are pooled for trees and lianas. fi CategoryImmature Alkaloids leaves TreesTrees Saponins and lianasMature leaves 23.5 (17)Trees AstringencyLianas 15.4 53.3 (13)Trees (15) Total and lianasPetioles 15.1 (33) 100 54.5Flowers (17) (11) 15.4 (26)Semi-ripe 14.3 fruit (7) Condensed 28.0pulp (25) 100 (13) 0 0 30.0 (7)Ripe (11) (20) fruit 100 pulp Hydrolysable 7.1 (16) 94.2 20.0Mature (14) (17) (5) seeds 0Tree 91.7 (15) twigs (12) 100 Gallotannins (12) 100Inner 0 60.0 bark (16) (5) (21) 0 33.3 100 (1) (9) (5)All 100 pooled (17) 30.0 Ellagitannins 100 42.8 (10) 100 (12) (7) 100 (11) (9) 100 58.8 15.0 (14) (17) (20) 33.3 81.2 (9) 10.8 (16) 83.3 (148) (6) 11.1 100 (9) (5) 100 72.7 (14) (11) 61.5 43.7 (13) (16) 36.5 85.7 (96) (21) 100 56.2 100 (13) 90.0 (17) (10) 37.3 (10) (17) 100 82.3 (5) 54.5 83.3 (17) 93.6 (11) (6) (125) 100 (15) 100 38.5 100 (13) 88.9 (13) (10) (18) 100 66.7 41.8 (8) 43.7 (6) (17) 93.3 (16) 100 50.0 98.3 (15) (16) (6) (118) 72.2 36.4 (18) (11) 50.0 50.0 (12) (10) 41.7 86.3 (12) (117) 75.0 75.0 (8) (16) 20.0 33.3 (15) (6) 29.4 (17) 31.2 (16) 50.0 37.3 37.5 (6) (16) (112) 50.0 50.0 14.3 (12) (10) 0 (14) (5) 20.0 (15) 16.7 (6) 31.2 (16) 31.2 (112) 33.3 (6) 16.7 20.0 (12) (10) 14.3 (14) 6.7 (15) 0 18.9 12.5 (5) 16.7 (111) (16) (6) 7.1 (14) 0 (5) analysed across species. Values in parentheses are number of species examined except for the samples analysed across species. Unless speci Table 2 Percent species containing alkaloids, saponins and phenolics in various plant part categories Values are expressed as percent species examined that contained the compound except for the 1228 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 nin, ). N 14.07 (17) 20.67 (10) 11.90 (21) 14.30 (14) 12.39 (13) 2.35 (9) 10.12 (17) 15.96 (17) ± ± ± ± ± ± ± ± SD) ± 0.001 (8) 49.70 0.09 (16) 38.48 0.13 (17) 16.10 0.04 (16) 27.00 0.53 (13) 25.93 0.21 (10) 35.27 0.14 (20) 33.18 0.38 (16) 33.98 ± ± ± ± ± ± ± ± 0.001 (5) 0.001 0.19 (16) 0.03 1.58 (18) 0.05 0.14 (15) 0.01 5.34 (13) 0.22 0.16 (10) 0.09 0.65 (16) 0.05 1.00 (17) 0.24 ± ± ± ± ± ± ± ± 20.65 (15) 0.06 21.56 (13) 2.70 20.57 (10) 0.10 20.29 (16) 0.28 19.02 (16) 0.56 4.34 (8) 0.001 20.49 (18) 0.72 7.76 (15) 0.05 ± ± ± ± ± ± ± ± 0.76 (16) 16.61 0.23 (8) 3.18 1.25 (18) 8.78 1.58 (15) 6.54 2.04 (13) 18.82 4.19 (10) 20.37 0.74 (16) 13.14 3.59 (16) 16.85 ± ± ± ± ± ± ± ± 7.52 (9) 3.01 10.54 (17) 2.70 7.11 (17) 0.87 2.77 (10) 0.29 6.41 (21) 1.00 5.43 (14) 1.13 7.30 (14) 1.99 7.31 (17) 1.05 ± ± ± ± ± ± ± ± Inner bark 6.35 seeds Tree twigs 1.95 pulp Mature 4.13 Ripe fruit 4.57 Flowers 9.51 leaves Petioles 10.42 leaves Mature 7.56 Category AstringencyImmature 11.37 Total phenolics Condensed tannins Gallotannins Ellagitannins ADF gallotannins as percent gallic acid and ellagitannins as percent ellagic acid in terms of dry weight. Values in parentheses are number of species ( Table 3 Levels of astringency, total phenolics, condensed tannins, gallotannins, ellagitannins and ADF in plant parts (mean Values are pooled for trees and lianas. Astringency and total phenolics expressed as percent tannic acid, condensed tannins as percent quebracho tan S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1229

Table 4 Correlates of various measures of phenolic compounds and ﬁbre

Total Condensed Gallotannin Ellagitannin ADF phenolics tannin

Astringency 0.48 (119)∗∗∗ 0.37 (118)∗∗∗ 0.20 (116)∗∗ 0.22 (115)∗∗ 0.13 (129)∗ Total phenolics 0.28 (121)∗∗∗ 0.36 (116)∗∗∗ 0.17 (114)∗ Ϫ0.08 (119) Condensed tannin 0.14 (115) 0.02 (113) 0.13 (118)∗ Gallotannin 0.32 (113)∗∗∗ Ϫ0.2 (116)∗∗ Ellagitannin Ϫ0.02 (114)

Values are Kendall’s correlation coefﬁcients. Values in parentheses are sample sizes (N). ∗P Ͻ 0.05; ∗∗P Ͻ 0.01; ∗∗∗P Ͻ 0.001 (∗∗ is signiﬁcant after Bonferroni’s correction for multiple tests).

Although correlations were performed between these phenolic measures separately for each plant part type, e.g. mature leaves, the results were not signiﬁcant after Bonferroni’s correction for multiple tests, except for the positive correlations between astringency and total phenolics in mature leaves of trees (Kendall’s t = 0.4, N = 17, P Ͻ 0.01) and between astringency and total phenolics (Kendall’s t = 0.61 N = 16, P Ͻ 0.01), as well as total phenolics and condensed tannins (Kendall’s t = 0.58, N = 16, P Ͻ 0.01) in inner bark.

3.4. Co-occurrence of condensed and hydrolysable tannins

Since almost all plant parts contained condensed tannins (Table 2), we were unable to examine the segregation between hydrolysable and condensed tannins in plant parts. Within each plant part we, therefore, compared the frequency of species containing both hydrolysable and condensed tannins to those containing condensed tannins alone using binomial probabilities, and we found that only in ripe fruit pulp was there a signiﬁcantly higher frequency of samples that contained condensed tannins but also did not contain hydrolysable tannins (N = 15, P Ͻ 0.02). Since there were samples that did not contain gallotannins, we examined the independence of occurrence of gallotannins and ellagitannins using a 2 × 2 contingency test, with Yates’ correction, and found that there was no segregation between gallotannins and ellagitannins in any plant part.

3.5. Co-occurrence of alkaloids, saponins and phenolics

Since tannins occurred in almost all plant parts examined, we were unable to examine whether the occurrence of tannins and alkaloids or tannins and saponins were independent of each other. We, therefore, examined whether significantly fewer numbers of plant parts of the different species contained both tannins and alkaloids or both tannins and saponins than those that contained tannins alone. We did this for each plant part category by calculating exact probabilities of the binomial since 1230 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 our sample sizes for each plant part category were less than 25 (Sokal and Rohlf, 1981, 708 pp.). Since all samples that gave positive results for phenolics with the Folin–Ciocalteu reagent were also astringent, and since astringency was measured for a greater number of samples (astringency is biologically more relevant to squirrel foraging as it is a measure of protein precipitation by tannins), we used astringency as an indication of the presence of phenolics in the samples. Except for tree twigs (N = 10) for which non-significant results were obtained, there were significantly more species that contained only tannins compared to those that contained both alkaloids and tannins in their different parts. For immature leaves: N = 17 species, P Ͻ 0.02; mature leaves: N = 18, P Ͻ 0.001; petiole: N = 9, P Ͻ 0.002; flowers: N = 14, P Ͻ 0.001; ripe fruit pulp: N = 14, P Ͻ 0.0001; mature seeds: N = 21, P Ͻ 0.0001; inner bark: N = 17, P Ͻ 0.02. However, with the exception of mature leaves (N = 16, P Ͻ 0.002) and tree twigs (N = 9, P Ͻ 0.05), the number of species containing both saponins and phenolics in all other categories was not significantly different from those containing phenolics alone. Since there were samples that did not contain alkaloids, we used a 2 × 2 contingency test, with Yates’ correction, to examine the pattern of co-occurrence of alkaloids and saponins and found no significant pattern of segregation between them. Furthermore, many samples contained neither alkaloids nor saponins (Table 2; Appendix A, Table A1).

3.6. Tree dominance and secondary metabolites

We examined the relationship between the relative dominance of tree species and the fibre and phenolic contents of their mature leaves, since it may be expected that dominant trees may have higher values of these compounds owing to their higher apparency (sensu Rhoades and Cates, 1976). However we did not find any significant relationship (Kendall’s correlation coefficients, P Ͼ 0.05).

4. Discussion

4.1. Distribution and content of secondary compounds in plant parts

4.1.1. Phenolics and fibre Almost all plant species in each plant part category we examined contained condensed tannins, while hydrolysable tannins were present in as few as 0% (tree twigs) to 61% (immature leaves of trees) of the species in each category. Condensed tannins are phylogenetically ancient secondary compounds while hydrolysable tannins are largely restricted to the dicots and are of more recent origin (Kubitzki and Gottlieb, 1984; Gottlieb et al., 1995). The lack of a strong detrimental effect of condensed tannins on insect and mammalian herbivores (Waterman and Kool, 1994; Ayres et al., 1997) has led to the belief that condensed tannins have evolved primarily in defence against microbes and fungi owing to their anti-microbial and fungistatic effects (Azaizeh et al., 1990). In the seasonal cloud forest of Bhimashankar, dense cloud settles on the stunted forest canopies, without lifting, for four monsoon months S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1231 each year (June through September/October). The need for protection against fungi at this time appears to be high and this may explain the ubiquitous presence of tannins in leaves, which is further evidenced by the low leaf litter decay (R.M. Borges, personal observation). The poor, acidic, leached soils (data from Indian Bureau of Soil Sciences) and the high levels of insolation at this site may have further resulted in phenolics such as condensed tannins being laid down in leaves and even in other plant parts due to overflow (Haslam, 1985) or a pluralistic combination of the various resource-related defence hypotheses (Berenbaum, 1995). Owing to the dearth of work on hydrolysable tannins in tropical rainforests we are unable to compare our results with other studies but hope that our results will be useful for further comparisons. Despite their powerful free-radical scavenging activity and alleged anti-carcinogenic effects (e.g. Sawa et al., 1999), virtually no information is available on the effect of hydrolysable tannins on various types of herbivores in natural systems (but see Whitten and Whitten, 1987; Clifford and Scal- bert, 2000) although their effect on large arboreal herbivores like the giant squirrel R. indica has been investigated (Borges and Mali, in preparation). Since we have used the Folin–Ciocalteu method for estimating total phenolics, which is recommended by Waterman and Mole (1994) as being better than the earlier Folin–Denis assay, and since all the earlier studies on community-wide distribution of secondary compounds in tropical forests have used the Folin–Denis method (e.g. Gartlan et al., 1980), our levels of total phenolics cannot be compared with other studies. However, as we have used the widely applied proanthocyanidin method for condensed tannins, our condensed tannin levels can be compared (Table 5) and were found to be nearly identical with the values found for another evergreen forest at Kakachi in southern India (Oates et al., 1980) despite the complete non-overlap of species between Bhimashankar and Kakachi. Furthermore, these condensed tannin levels were also found to be close to the values found for the two African and the two south-east Asian forests that have been most extensively studied (Table 5). Interestingly, the fibre levels (ADF) of the mature trees at Bhimashankar were also found to be nearly the same as those measured at Kakachi (Table 5). Immature leaves, flowers, and petioles had high astringency while lower levels were found in fruit. It is possible that either immature leaves, flowers and petioles actually do have greater protection by biologically active tannins as measured by their astringency or that the extractability of phenolics is greater in these tissues. High gallotannin levels were also found by Ossipov et al. (1997) in immature leaves; these levels declined as the leaves matured. Low astringency may be present in fruit as it is known that astringency levels decrease as fruit ripen (Goldstein and Swain, 1963). We cannot compare astringency levels between various stages of the same fruit owing to lack of sample sizes. Tree twigs had the lowest levels of astringency, condensed and hydrolysable tannins but the highest levels of fibre. Tree twigs have rarely been analysed chemically (Waterman and Kool, 1994). This pattern of allo- cation of secondary compounds to tree twigs may be a general strategy as twigs are protected by high lignin levels and therefore do not require protection from other compounds. Inner bark had astringency levels and condensed tannin levels comparable to those of mature leaves while fibre levels were lower than those of twigs. As 1232 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 200 73.2) – Ͻ 30.5) – Gartlan et al., 200 Lowland 71.7) 46.1 (21.5 – Ͻ 37.0) 4.8 (0 – 200 Lowland 77.2) 58.3 (40.7 – Ͻ 17.0) 8.8 (0 – Cameroon Malaysia 67.8) 47.0 (20.8 – 39.6) 5.4 (0 – 55.1) 35.4 (10.3 – 22.0) 5.8 (0 – ). 47.37) 39.4 (24.0 – 23.40) 6.9 (0 – 35.19 (19.0 15 14 23 38 17 33 , number of species; NA, rainfall value unavailable for Douala-Edea from above-mentioned sources. Numbers in parentheses N bre; fi a bre) fi a Values for ADF and condensed tannin are mean percents on dry weight basis (values other than for Bhimashankar are obtained from a leaves Condensed tannin in mature 6.86 (0 ParameterAltitude (m) Bhimashankar, IndiaN Kakachi, India 910 Kibale, Uganda Douala-Edea, Sepilok, Borneo Kuala Lompat, 1325 1400 Lowland Rainfall (mm)Forest typeADF ( 3000 Semi-evergreen Evergreen 3080 Evergreen Evergreen 1485 Evergreen NA Evergreen 3000 2000 1980; Oates et al., 1980; Waterman et al., 1988 Table 5 Comparison of Bhimashankar with other tropical forests ADF, acid detergent indicate range of values. S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1233 indicated by Milton (1979) and Waterman (1984), we also found that the secondary chemistry of flowers is more comparable to foliage that any other plant part.

4.1.2. Cyanogenic glycosides, alkaloids, and saponins Cyanogenic glycosides were present in only 2.3% of species screened. This virtual absence of cyanogenesis was also recorded in a lowland rain forest in Costa Rica wherein only 25 out of 488 species (5.1%) of woody plants screened were cyanogenic (Thomsen and Brimer, 1997). Cyanogenesis appears to be limited only to certain families such as Leguminosae, Rosaceae, Euphorbiaceae and Passifloraceae (Conn, 1979), and cyanogenic glycosides appear to be very much less ubiquitous as defence chemicals than alkaloids, saponins and phenolics. Alkaloids were absent from semi- ripe and ripe fruit, which could reflect the fact that defences that are deterrent to potential seed dispersers need to be minimised (McKey, 1974). Saponins were found in all plant parts examined. The lowest occurrence of saponins was found in tree twigs. Much more work needs to be done on the distribution of saponins in plant tissues, especially given their possible interactions with both condensed and hydrolysable tannins in influencing the potency of these secondary compounds.

4.2. The condensed tanninÐhydrolysable tannin interaction

Only in ripe fruit pulp did we find a higher number of species that contained condensed tannins rather than hydrolysable tannins. The role of hydrolysable tannins in defence against herbivores has barely been investigated. The seminal and detailed studies of food selection in primates, especially colobines (e.g. Gartlan et al., 1980; McKey et al., 1981; Waterman et al., 1988; Kool, 1992) have neither quantified hydrolysable tannins nor investigated their role in food selection. However, the bark- eating tropical squirrel Sundasciurus lowii was found to select barks with low levels of hydrolysable tannins (Whitten and Whitten, 1987). Since condensed tannins are probably not effective deterrents against insect and mammalian herbivores (Waterman and Kool, 1994; Reed, 1995; Ayres et al., 1997) and probably function largely as anti-microbial or anti-fungal agents (Waterman, 1983), it is possible that hydrolysable tannins have a more potent action against herbivores than condensed tannins (Swain, 1977; Zucker, 1983; Reed, 1995). Zimmer (1997) found that ingested gallotannins increased the surface tension of gut fluid, indicating reduced concentrations of free surfactants, while Barbehenn et al. (1996) found that the gut peritrophic membrane in polyphagous grasshoppers was easily permeated by several gallotannins. It is, therefore, interesting that we found that significantly fewer species had ripe fruit containing hydrolysable tannins rather than condensed tannins as these might deter dispersal agents. However, there are conflicting claims for beneficial and toxic effects caused by hydrolysable tannins such as ellagitannins in various animal species including rodents and ruminants (Clifford and Scalbert, 2000). Similarly almost no information is available on the occurrence of gallotannins and ellagitannins relative to each other. Our study has shown that gallotannins and ellagitannins are not significantly segregated in any plant part. Furthermore, of the 31 species that were examined for gallotannins and ellagitannins, only 10 species contained both 1234 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 these compounds, five contained only gallotannins, six only ellagitannins and 10 contained neither compound. The biological significance of these findings is as yet unclear and may merely reflect phylogenetic constraints (Gottlieb et al., 1993).

4.3. The alkaloidÐtannin interaction

In tropical forests, although alkaloids and phenolics have been widely investigated, few have examined their patterns of co-occurrence (Gartlan et al., 1980; Lebreton, 1982; Janzen and Waterman, 1984). Gartlan et al. (1980) found a segregation between alkaloids and tannins in mature leaves of Douala-Edea and Kibale forests, while Janzen and Waterman (1984) found a negative correlation between alkaloid and tannin contents in a dry forest in Costa Rica. This is expected as alkaloids and tannins are believed to form insoluble alkaloid-tannates in herbivore guts, thus negat- ing the effects of each other (Freeland and Janzen, 1974). The negative association between alkaloids and tannins was also predicted by Feeny (1976) from apparency theory. Within plant families, after correcting for species relatedness, Silvertown and Dodd (1996) found a negative association between the proportion of species containing tannins and those containing alkaloids. Our results also show that across almost all plant parts, the number of species containing tannins alone was greater than the number of species containing both alkaloids and tannins.

4.4. The saponinÐtannin interaction

Saponins are widespread in plants and cause haemolysis, enzyme inhibition, and alteration of gut surface tension in herbivores (Applebaum and Kirk, 1979). Although the anti-nutritional effects of saponins in various forages on domesticated herbivores (Klita et al., 1996; Newbold et al., 1997), and the anti-feedant effects of the saponins of a few plantation tree species on leaf-cutting ants (Folgarait et al., 1996) have been demonstrated, there has been no investigation of either the community-wide presence of saponins in tropical forests or of the effects of saponins on tropical forest herbivores. Martin and Martin (1984) showed that detergency negated the anti-digestibility effects of tannins in the tobacco hornworm, suggesting that the surfactant properties of saponins could function similarly. Freeland et al. (1985) demonstrated that the simultaneous consumption of tannins and saponins reduced the deleterious effects caused by the consumption of either saponins or tannins alone. These ﬁndings lead to the prediction that saponins and tannins should not co-occur in plant parts, which is the result that we obtained in this study when we found that within each plant part category the number of species containing tannins alone was greater than the number containing both saponins and tannins. We also found saponins to occur in all plant part categories, as has been found in other studies (Applebaum and Kirk, 1979).

4.5. The alkaloidÐsaponin interaction

The alkaloid–saponin interaction in herbivore guts and its possible inﬂuence on herbivore food selection has scarcely been investigated. Alkaloids and saponins may S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1235 cause greater deterrence to herbivores when they co-occur than when they occur independently owing to a synergistic effect, as was found for the seeds of Erythrophleum guineense (Caesalpiniaceae) (Kerharo and Adam, 1974). If this is the general case, then alkaloids and saponins may be expected to co-occur, or at least there does not appear to be any biological reason to expect a negative association between these compounds. In our study we were unable to ﬁnd any clear pattern of segregation between alkaloids and saponins and also little evidence of positive association. Much more work is needed in this area.

5. Summary

In summary, we have presented data on the correspondence between protein-precipitating assays and chemical tests for tannin activity; we have also measured both condensed and hydrolysable tannins (gallotannins and ellagitannins) in a variety of plant parts, analysed ﬁbre contents and screened for three types of toxins in plants from a wide array of families and orders within a tropical seasonal cloud forest in India.

Acknowledgements

This research was funded by a grant to RMB from the United States Fish and Wildlife Service. We thank the Wildlife Institute of India for collaboration. We thank Hema Somanathan for helping with data collection and analysis. We are grateful to Doyle McKey for useful suggestions throughout the study, and for helpful comments on this manuscript. We thank Anne Hagerman for providing the quebracho tannin and the protocols for tannin analysis.

Appendix A 1236 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 ) – – – – – – – A A AA PA PA A AA P A AP A AP continued on next page ( ––– ––– 0 0 0 40.9 38.7 30.0 A A 0.5 0 26.2 32.7 11.1 A A – –– content tannin bre and presence of alkaloids and saponins in trees and lianas at Bhimashankar fi 0.3 1.6 57.2 0.3 0.1 27.6 43.4 7.5 A A – ––––––– 3.26.8 0.2 0.7 3.3 7.6 0 0 0.6 0 23.0 31.0 26.6 36.8 22.9 20.1 A A P 6.60.60.4 1.1 0.2 4.7 1.0 0.1 0 0.2 0 38.9 57.6 33.3 30.1 43.5 A 29.1 P A A – ––––––– – ––– 2.4– ––––––– 0.4 0 0 0 32.2 44.4 28.3 A A – ––––––– – ––––––– – ––––––– – ––––––– – ––––––– 1.8– ––––––– 0.62.0– ––––––– – ––– 1.5 1.8 0 1.2 0 0 38.1 0 42.1 33.8 31.2 P 45.2 22.0 A A P 13.0 2.1 12.0 13.0 1.1 66.0 0.1 0 23.5 28.0 22.0 A A 21.1 1.3 23.4 0 0 41.5 43.8 32.2 A A a a a a a sp. 0 0.2 0 0 0 20.3 27.3 15.1 A P glaucescens Diploclisia cucullatum Mezoneuron Diospyros sylvatica Cassine Diospyros montana Mangifera indica Memecylon umbellatum Mallotus philippensis Maytenus rothiana Bridelia retusa Canthium dicoccum Atalantia racemosa angustifolia Amoora lawii Randia rugulosa Plant part and species Astringency TotalMature phenolic leaf Actinodaphne Condensed Gallotannin Ellagitannin ADF NDF ADL AlkaloidsEmbelia ribes Saponin Olea dioica Randia dumetorum Garcinia talbotii Gnetum ula Flacourtia indica Ficus racemosa Ficus callosa Litsea stocksii Macaranga peltata Table A1 Levels of phenolic compounds and S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1237 – – – AA AP AP AA ––– 27.5 27.4 14.7 A A 0 – 3.8 0 23.8 28.8 11.0 A A –– content tannin – ––––––– – ––––––– 4.0 0.2 1.8 0 0 50.5 39.3 30.6 A P – ––––––– 6.4– ––– 0.5 1.2 2.5 00.80.50.4 2.5 28.20.60.7 3.0 35.3 0.8 0.4 17.9 0.2 7.0 A 15.8 0 22.8 A 1.3 0.3 0.2 0.2 0 0.3 9.1 23.9 28.7 70.6 28.1 3.1 29.2 11.6 P 19.3 P P P A A 1.8 3.0 3.2 0 0 31.2 34.3 19.7 P P 13.4 1.7 0 1.0 022.5 29.915.4 36.5 11.2 20.5 1.4 A 17.3 21.8 A 1.3 1.5 1.3 0.9 35.1 25.5 34.8 29.8 18.0 11.3 A A P 16.0 12.0 1.6 1.3 0.6 10.1 10.3 9.6 A 18.617.0 2.6 1.5 56.7 0.7 0 0 0 0.3 36.6 37.0 23.6 32.1 23.1 35.5 A A A P 20.0 2.639.013.0 20.0 1.1 0.9 0.1 11.0 0 59.4 0 0 32.112.8 41.6 0 0 30.1 0.7 A 40.7 33.0 36.0 26.2 37.1 40.2 15.6 0 A A 0 P P 22.4 32.0 16.6 A A 17.613.2 0.8 0.8 14.3 14.0 0 0 0.1 0 64.3 50.1 60.6 57.9 60.9 A 58.3 A A P 17.0 1.4 0.1 0 0.2 40.3 40.0 26.1 A A a a ) a a a a continued Syzygium gardneri Terminalia bellerica Terminalia chebula Ventilago bombaiensis Syzygium cumini Syzygium gardneri Terminalia chebula Ventilago bombaiensis Plant part and species Astringency TotalRourea phenolic santaloides Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponin Ficus callosa Garcinia talbotii Gnetum ula Diploclisia glaucescens Diospyros sylvatica Xantolis tomentosa Immature leaf Actinodaphne angustifolia Cassine paniculata Mangifera indica Syzygium cumini Litsea stocksii Memecylon umbellatum Symplocos beddomei Symplocos beddomei Rourea santaloides Olea dioica Table A1 ( 1238 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 – – – – – – – – – A P A ––– ––– 20.6 20.3 16.4 A – 0 ––– 0.1 1.0 0 0 content tannin 2.9 2.02.00.2 3.60.4 0.27.1 1.28.0 0 0.1 3.0 15.4 0.7 3.1 0 1.1 0 28.8 0 2.4 33.5 0 0 0 29.9 17.5 18.6 0 0 0 16.1 A 16.2 18.3 A 34.7 20.2 10.8 22.6 44.5 26.1 A 6.5 32.8 12.1 P A A A P A A – ––––––– 3.06.0 0.2 0.2 3.6 13.2 0.1 0 0.3 0 33.4 49.1 38.3 58.5 19.5 43.0 A A P P 6.4 2.7 8.2 0 0 25.0 31.8 15.3 A A 1.0 1.1 1.10.6 0.03 0 30.6 41.6 30.1 A – 22.310.0 14.0 1.2 32.0 64.412.8 0.116.8 0 0.6 0.9 8.0 0 37.0 6.0 16.2 34.7 19.0 28.8 0.03 10.0 A 36.1 1.3 0 19.8 A A 22.4 15.4 23.8 A 24.6 16.1 15.3 A A A P 12.217.8 2.3 3.6 34.6 46.0 0.3 0.5 0 0 36.4 25.6 34.3 27.4 30.7 7.7 A A 19.5 2.0 7.8 0 0 61.5 44.6 33.1 A A 13.2 1.315.4 11.2 5.2 0 57.4 0 0 42.3 0 52.0 34.013.6 29.7 A 32.9 12.9 1.7 A 31.6 P 2.5 0 22.5 27.8 19.5 A ) a a a a a continued sp. Flower Actinodaphne angustifolia Diospyros montana Mezoneuron cucullatum Syzygium cumini glaucescens Lepisanthes tetraphylla Mangifera indica Memecylon umbellatum Mangifera indica Memecylon umbellatum Olea dioica Diploclisia Garcinia talbotii Macaranga peltata Diploclisia glaucescens angustifolia Cassine Diospyros sylvatica Syzygium cumini Plant part and species Astringency TotalPetiole phenolicActinodaphne Condensed Gallotannin Ellagitannin ADFGarcinia NDF talbotii Gnetum ula ADL Alkaloids Saponin Elaeagnus conferta Olea dioica Symplocos beddomei Table A1 ( S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1239 – – – – – – – – – – – – – A 6.9 8.8 1.4 A ––– 8.5 12.7 0.5 A P 26.6 35.2 11.1 P 35.1 33.9 22.4 A 0 53.1 35.9 37.7 A 0 52.7 53.8 27.3 P A – –– –––– –––– ––– 0.4 5.8 0 0 –––– content tannin 0.6 0.1 2.8 5.6 0.2 26.0 0 00.4 0.2 31.8 27.9 31.7 A P 0.2 2.3 0.4 2.4 0 0 27.7 37.1 26.4 A 0.4 0.5 0 1.8 0 7.9 16.6 2.7 A – 1.4 0.4 3.9 0 0 24.2 30.4 16.7 A 0.4 1.04.0 1.0 0.20 00.4 7.5 0.4 0 0 0 2.2 00.2 0 6.0 0 32.5 0.4 46.2 49.5 0 26.5 59.2 0 A 46.5 52.2 A 46.9 A 0 33.9 A 0.1 P 25.9 32.0 4.6 A A 15.5 0.3 6.6 0 0.2 57.7 47.1 57.0 A 15.2 0.8 25.6 0 0 28.4 22.6 22.1 A A 15.0 5.6 0 10.0 1.3 8.2 7.1 7.9 A 13.0 1.4 59.4 0.1 0 34.4 31.1 31.8 A 22.8 2.8 0.4 5.0 1.6 17.4 20.4 14.8 A ) a a a a a continued Ficus tsjahela Randia rugulosa Gnetum ula Ficus religiosa Gnetum ula heterophyllus Ficus racemosa Artocarpus Symplocos beddomei Terminalia chebula Semi-mature fruit pulp Garcinia talbotii Ficus racemosa angustifolia Amoora lawii Mangifera indica Ficus callosa glaucescens Xantolis tomentosa Entire immature fruit Actinodaphne Plant part and species Astringency TotalTerminalia phenolic chebula Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponin Garcinia talbotii Entire mature fruit Embelia ribes Immature fruit pulp Diploclisia Table A1 ( 1240 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 – – – – – – – – – – – – – – – A AA ––– ––– 4.0 17.1 1.1 A ––––– 6.1 10.8 1.7 A A 11.1 12.1 7.3 A 0 –––– 0 2.4 0 0 0.6 0.6 0.5 0.2 27.6 32.2 23.2 A –––– –––– content tannin 6.6 0.5 13.3 0 0 14.3 16.4 10.2 A 0.6 0 0.1 1.5 0 0 28.7 22.5 27.4 A 0.6 0.6 6.0 0 0 33.3 31.3 23.9 A 0.4 0.1 0.5 0 0 43.8 30.2 25.6 A – 1.0 0.7 0.9 0 0 8.7 14.7 8.5 A 0.40.90.64.50.4 0.4 0.1 0.8 0.2 6.4 1.5 3.4 7.6 5.2 1.1 0 0 0 0.03 0 0 0 0 0 0 4.9 29.1 31.0 8.6 7.5 43.5 26.3 9.4 3.5 9.5 20.0 7.4 A A A A A P A P 1.9 2.6 5.0 0 0 33.4 40.7 14.8 A 6.1 0.7 14.0 0 0 25.6 32.4 24.0 A – 0.2 0.8 0 1.31.0 0 0.2 4.4 0 18.9 1.0 00.1 A 0 12.8 17.5 12.3 A – ––– 7.1 1.9 1.1 00.7 0 42.1 57.3 12.4 A 11.8 0.6 11.5 0 0 35.5 47.3 33.3 A P 14.6 1.9 19.6 0.3 0 22.8 37.1 9.3 A A 14.5 1.7 25.2 0 0 52.2 41.8 44.4 A ) a a a a continued a sp. Ripe fruit pulp Acacia Xantolis tomentosa glaucescens Immature seed Diploclisia glaucescens Syzygium cumini Diospyros sylvatica Diploclisia Vangueria spinosa Symplocos beddomei angustifolia Amoora lawii Symplocos beddomei Syzygium cumini Syzygium gardneri Terminalia chebula Memecylon umbellatum Olea dioica Gnetum ula Olea dioica Gnetum ula Actinodaphne Litsea stocksii Mangifera indica Plant part and species Astringency TotalMangifera phenolic indica Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponin Garcinia talbotii Garcinia talbotii Table A1 ( S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1241 – – – – – – – – – – – – – – – – 6.2 23.8 1.2 A 5.7 29.6 2.6 A 6.9 35.0 6.4 A 4.5 6.4 1.0 A 9.3 32.1 1.2 A 6.4 33.4 2.8 P A 23.2 35.5 19.9 A A 16.0 18.3 13.8 A A – – 0 0 6.3 21.8 3.2 A –––– –––– –––– –– –––– –––– –––– content tannin 2.2 0.3 10.0 0 0 43.1 54.0 25.0 A 0.20.2 1.7 0 84.0 1.2 1.3 0 0 0 41.1 64.5 7.9 12.9 45.6 A 3.6 A A 0 0.2 0 0 0 19.8 23.2 15.0 A 0.1 0.5 0.4 0.4 0.1 0 0 0 40.0 51.9 27.8 A P 0.4 0 0.2 4.0 0 0.3 0.2 1.2 0 0 11.9 27.2 5.6 A 0.2 0.2 00.7 0.1 0.7 0 8.0 0 0 0 0 16.9 4.3 40.9 7.8 11.6 2.8 A A P 0.4 1.00 0.1 0 0.7 0 0 0 0 0 22.4 29.7 17.9 20.6 20.3 A 14.4 A 11.3 2.1 0.8 0.2 1.2 40.110.0 38.6 19.6 1.3 A 0.7 0 12.8 1.5 3.4 6.3 0.3 6.4 28.3 6.1 A 13.8 4.8 32.9 1.0 0.5 11.0 31.6 6.0 A A 23.3 3.0 3.0 0 0 3.8 56.6 3.8 A ) a a a a continued a sp. Mature seed Acacia concinna glaucescens Symplocos beddomei Diploclisia heterophyllus Diospyros sylvatica Gnetum ula Olea dioica Artocarpus Gnetum ula Terminalia chebula Semi-mature seed Garcinia talbotii binectariferum Garcinia talbotii Macaranga peltata Mangifera indica Memecylon umbellatum Olea dioica Mangifera indica Acacia Actinodaphne angustifolia Amoora lawii Plant part and species Astringency TotalMangifera phenolic indica Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponin Dysoxylum Litsea stocksii Lepisanthes tetraphylla Table A1 ( 1242 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 – – – – – AP A AA 17.2 A A – ––– ––––– ––––– 42.2 58.9 31.5 A 51.9 62.3 17.4 A A 15.885.9 17.9 4.4 A 0 – 0.3 53.5 62.5 43.9 P P 0 68.7 68.0 28.2 A A 0 51.3 60.9 32.5 A A 0 0 – – – –– –––– –––– content tannin 0.32.5– ––––––– 0.7 0.2 8.2 1.3 0 0 0 0 42.6 35.0 36.7 44.0 39.0 18.3 P A A A – ––– – ––––––– 00.100 0.57.3 0.1 1.0 2.9 27.0 0 0 24.2 34.2 9.7 P A 6.27.9 0.7 0.3 13.3 3.4 0 0 45.1 49.3 15.4 P P 0.10.7 2.00.3 0.9 0.1 0.6 0.3 3.6 0 0.6 0 0 0 0 61.8 66.8 55.7 17.4 67.4 P 19.0 A A A 1.0 0.2 1.7 1.0– ––– 0.1 0 0 0 23.0 30.0 23.0 A A 3.8 0.4 0 0 0 7.4 15.2 4.4 A – ––– 8.10.4 0.8 1.4 16.8 1.4 0 2.5 0 0 40.6 6.4 61.9 39.9 27.4 2.4 A A P 19.2 2.5 68.6 0.1 0 26.3 22.2 16.3 P A 10.0 1.6 0.7 2.0 0 15.8 33.3 6.2 A A ) continued sp. 0 0.1 0 0 0 36.8 49.1 15.2 A A Ficus callosa Diospyros sylvatica Diospyros montana Cassine paniculata Bridelia retusa Atalantia racemosa angustifolia Amoora lawii Inner bark Actinodaphne Xantolis tomentosa Syzygium cumini Symplocos beddomei Ficus racemosa Mangifera indica heterophyllus Cassine Ficus callosa Mallotus philippensis Olea dioica Artocarpus Stem of leaf spraysAmoora (Twigs) lawii Xantolis tomentosa Terminalia chebula Syzygium gardneri Terminalia bellerica Plant part and species Astringency TotalSymplocos phenolic beddomei Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponin Syzygium cumini Table A1 ( S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246 1243 bre; fi ids and – – – ondensed AA 61.4 50.3 53.0 A A indicates values not estimated; ADF, acid detergent – – 0 0 23.5 28.1 6.3 A A 0.1 0 27.3 32.3 25.0 A P indicates absence and ’ A ‘ – indicates presence; ’ P ‘ –– content tannin 0 0.1 0 0 0 50.7 62.3 42.2 A A 6.43.40.7 0.3 0.6 14.7 7.6 0 0 0 0.3 37.0 41.6 44.4 55.6 32.9 40.5 A A P A 0.66.4 1.2 0.2 52.0 13.2 0 0 0 0 7.8 48.3 10.6 50.3 2.8 46.1 A A P P 0 0.2 0– ––––––– 0 0 54.4 63.5 45.1 A P 5.0 0.5 5.4 0 0 45.8 65.6 41.8 A 0.4 0.2 3.4 0 0 26.1 40.6 9.5 A A 10.1 1.6 0 0 0 26.7 32.1 20.0 A 20.016.0 1.5 1.612.418.0 23.012.2 30.2 1.3 1.6 0 1.1 0.8 0 8.4 0 29.6 0 0 50.0 0.1 54.3 0 0 0.2 24.4 A 42.9 43.6 51.9 50.8 56.0 64.0 37.8 40.2 45.7 P P A P A P 14.6 1.9 bre; ADL, acid detergent lignin. Cyanogenic glycosides were absent in all these items and hence not shown. fi a ) a a a a continued Indicates liana. a Rourea santaloides Olea dioica Mallotus philippensis Mangifera indica Litsea stocksii Plant part and species Astringency TotalFicus phenolic racemosa Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponin Randia rugulosa Ficus religiosa Garcinia talbotii Ventilago bombaiensis Mezoneuron cucullatum Syzygium gardneri Syzygium cumini Symplocos beddomei Entire bark Gnetum ula Terminalia chebula Xantolis tomentosa tannins as percentsaponins quebracho were tannin, qualitatively gallotannins detected, asNDF, wherein neutral percent detergent gallic acid, and ellagitannins as percent ellagic acid on dry weight basis. Only alkalo Table A1 ( For parameters other than alkaloids and saponins, values are expressed as follows: astringency and total phenolic contents as percent tannic acid, c 1244 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221Ð1246

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