Phenolics, alkaloids, saponins and cyanogenic glycosides.pdf

1226 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246in almost all cases, we were examining only one species per genus, and one genusper family.3. Results3.1. Occurrence of alkaloids, saponins, cyanogenic glycosides and phenolicsCyanogenic 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 theflush leaves of B. retusa (Euphorbiaceae). Therefore, only one out of 43 plant species(2.3%) exhibited cyanogenesis. Summaries of the species-wise occurrence of thedifferent compounds in the various plant parts are given in Table 2. Alkaloids werenot found in petioles, semi-ripe and ripe fruit, and mature seeds examined (AppendixA, Table A1). Saponins were found in all types of plant parts (Appendix A, TableA1). Condensed tannins were found in all immature leaves, as well as all petiolesand flowers examined. Hydrolysable tannins were found in 68% of species, and 38%of plant samples. Gallotannins were found in only 31% of the samples examinedwhile ellagitannins were found in still fewer samples (only 19%). Fewer plant speciescontained condensed or hydrolysable tannins in their bark and stems than in otherplant parts.3.2. Relative concentrations of secondary compounds in plant partsImmature leaves, flowers, and petioles had high astringency while lower levelswere found in fruit (Table 3). Tree twigs had low levels of astringency, condensedand hydrolysable tannins but high levels of fibre (Table 3). Inner bark had astringencyand condensed tannin levels comparable to that of mature leaves while fibre levelswere lower than those found in twigs (Table 3). Flowers and fruit had low fibrelevels (Table 3). After Bonferroni’s correction (P 0.001), only immature leaveswere found to have significantly higher astringency, gallotannin and ellagitannin contentthan tree twigs, flowers were found to have significantly higher gallotannin levelsthan immature leaves, and tree twigs were found to have significantly higher ADFlevels than mature seeds.3.3. The quantitative relationship between various measures of phenolicsAstringency levels were strongly positively correlated with total phenolic, condensedtannin, gallotannin and ellagitanin contents (Table 4). Condensed tanninvalues were not correlated with gallotannin or ellagitannin contents. Gallotannin andellagitannin contents were strongly positively correlated with each other (Table 4).Of the fibre components, since ADF, NDF and ADL are all highly correlated witheach other, we used only ADF values in the correlations. We found no significantrelation 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) 1221–12461227Table 2Percent species containing alkaloids, saponins and phenolics in various plant part categoriesCategory Alkaloids Saponins Astringency Total Condensed Hydrolysable Gallotannins Ellagitanninsphenolics tannins tanninsImmature leavesTrees 15.4 (13) 54.5 (11) 100 (13) 100 (12) 100 (12) 61.5 (13) 38.5 (13) 41.7 (12)Trees and lianas 23.5 (17) 53.3 (15) 100 (17) 100 (16) 100 (16) 58.8 (17) 37.3 (17) 43.7 (16)Mature leavesTrees 15.4 (26) 30.0 (20) 91.7 (12) 100 (11) 72.7 (11) 54.5 (11) 36.4 (11) 37.3 (16)Lianas 14.3 (7) 20.0 (5) 100 (5) 100 (5) 100 (5) 50.0 (6) 50.0 (6) 33.3 (6)Trees and lianas 15.1 (33) 28.0 (25) 94.2 (17) 100 (17) 81.2 (16) 56.2 (17) 41.8 (17) 29.4 (17)Petioles 0 (11) 60.0 (5) 100 (9) 100 (10) 100 (10) 50.0 (10) 50.0 (10) 20.0 (10)Flowers 7.1 (14) 33.3 (9) 100 (14) 100 (13) 100 (13) 50.0 (12) 50.0 (12) 16.7 (12)Semi-ripe fruit 0 (7) 0 (1) 83.3 (6) 83.3 (6) 66.7 (6) 33.3 (6) 16.7 (6) 16.7 (6)pulpRipe fruit pulp 0 (15) 42.8 (7) 100 (14) 100 (15) 93.3 (15) 20.0 (15) 20.0 (15) 6.7 (15)Mature seeds 0 (21) 33.3 (9) 85.7 (21) 88.9 (18) 72.2 (18) 31.2 (16) 31.2 (16) 12.5 (16)Tree twigs 30.0 (10) 11.1 (9) 90.0 (10) 100 (8) 75.0 (8) 0 (5) 0 (5) 0 (5)Inner bark 15.0 (20) 43.7 (16) 82.3 (17) 100 (16) 75.0 (16) 14.3 (14) 14.3 (14) 7.1 (14)All pooled 10.8 (148) 36.5 (96) 93.6 (125) 98.3 (118) 86.3 (117) 37.5 (112) 31.2 (112) 18.9 (111)Values are expressed as percent species examined that contained the compound except for the ‘All pooled’ category where they indicate percent samplesanalysed across species. Values in parentheses are number of species examined except for the ‘All pooled’ category where they indicate total number ofsamples analysed across species. Unless specified, values are pooled for trees and lianas.

1228 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246Table 3Levels of astringency, total phenolics, condensed tannins, gallotannins, ellagitannins and ADF in plant parts (mean ± SD)Category Astringency Total phenolics Condensed tannins Gallotannins Ellagitannins ADFImmature 11.37 ± 10.54 (17) 2.70 ± 3.59 (16) 16.85 ± 19.02 (16) 0.56 ± 1.00 (17) 0.24 ± 0.38 (16) 33.98 ± 15.96 (17)leavesMature 7.56 ± 7.31 (17) 1.05 ± 0.74 (16) 13.14 ± 20.29 (16) 0.28 ± 0.65 (16) 0.05 ± 0.14 (20) 33.18 ± 10.12 (17)leavesPetioles 10.42 ± 7.52 (9) 3.01 ± 4.19 (10) 20.37 ± 20.57 (10) 0.10 ± 0.16 (10) 0.09 ± 0.21 (10) 35.27 ± 2.35 (9)Flowers 9.51 ± 7.30 (14) 1.99 ± 2.04 (13) 18.82 ± 21.56 (13) 2.70 ± 5.34 (13) 0.22 ± 0.53 (13) 25.93 ± 12.39 (13)Ripe fruit 4.57 ± 5.43 (14) 1.13 ± 1.58 (15) 6.54 ± 7.76 (15) 0.05 ± 0.14 (15) 0.01 ± 0.04 (16) 27.00 ± 14.30 (14)pulpMature 4.13 ± 6.41 (21) 1.00 ± 1.25 (18) 8.78 ± 20.49 (18) 0.72 ± 1.58 (18) 0.05 ± 0.13 (17) 16.10 ± 11.90 (21)seedsTree twigs 1.95 ± 2.77 (10) 0.29 ± 0.23 (8) 3.18 ± 4.34 (8) 0.001 ± 0.001 (5) 0.001 ± 0.001 (8) 49.70 ± 20.67 (10)Inner bark 6.35 ± 7.11 (17) 0.87 ± 0.76 (16) 16.61 ± 20.65 (15) 0.06 ± 0.19 (16) 0.03 ± 0.09 (16) 38.48 ± 14.07 (17)Values are pooled for trees and lianas. Astringency and total phenolics expressed as percent tannic acid, condensed tannins as percent quebracho tannin,gallotannins as percent gallic acid and ellagitannins as percent ellagic acid in terms of dry weight. Values in parentheses are number of species (N).

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461229Table 4Correlates of various measures of phenolic compounds and fibreTotal Condensed Gallotannin Ellagitannin ADFphenolics tanninAstringency 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)∗ 0.08 (119)Condensed tannin 0.14 (115) 0.02 (113) 0.13 (118)∗Gallotannin 0.32 (113)∗∗∗ 0.2 (116)∗∗Ellagitannin 0.02 (114)Values are Kendall’s correlation coefficients. Values in parentheses are sample sizes (N). ∗P 0.05;∗∗P 0.01; ∗∗∗ P 0.001 (∗∗ is significant after Bonferroni’s correction for multiple tests).Although correlations were performed between these phenolic measures separatelyfor each plant part type, e.g. mature leaves, the results were not significant afterBonferroni’s correction for multiple tests, except for the positive correlations betweenastringency and total phenolics in mature leaves of trees (Kendall’s t = 0.4, N =17, P 0.01) and between astringency and total phenolics (Kendall’s t = 0.61 N= 16, P 0.01), as well as total phenolics and condensed tannins (Kendall’s t =0.58, N = 16, P 0.01) in inner bark.3.4. Co-occurrence of condensed and hydrolysable tanninsSince almost all plant parts contained condensed tannins (Table 2), we were unableto examine the segregation between hydrolysable and condensed tannins in plantparts. Within each plant part we, therefore, compared the frequency of species containingboth hydrolysable and condensed tannins to those containing condensed tanninsalone using binomial probabilities, and we found that only in ripe fruit pulpwas there a significantly higher frequency of samples that contained condensed tanninsbut also did not contain hydrolysable tannins (N = 15, P 0.02). Since therewere samples that did not contain gallotannins, we examined the independence ofoccurrence of gallotannins and ellagitannins using a 2 × 2 contingency test, withYates’ correction, and found that there was no segregation between gallotannins andellagitannins in any plant part.3.5. Co-occurrence of alkaloids, saponins and phenolicsSince tannins occurred in almost all plant parts examined, we were unable toexamine whether the occurrence of tannins and alkaloids or tannins and saponinswere independent of each other. We, therefore, examined whether significantly fewernumbers of plant parts of the different species contained both tannins and alkaloidsor both tannins and saponins than those that contained tannins alone. We did thisfor each plant part category by calculating exact probabilities of the binomial since

1230 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246our 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 theFolin–Ciocalteu reagent were also astringent, and since astringency was measuredfor a greater number of samples (astringency is biologically more relevant to squirrelforaging as it is a measure of protein precipitation by tannins), we used astringencyas 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 significantlymore species that contained only tannins compared to those that contained both alkaloidsand tannins in their different parts. For immature leaves: N = 17 species, P 0.02; mature leaves: N = 18, P 0.001; petiole: N = 9, P 0.002; flowers: N= 14, P 0.001; ripe fruit pulp: N = 14, P 0.0001; mature seeds: N = 21, P 0.0001; inner bark: N = 17, P 0.02. However, with the exception of matureleaves (N = 16, P 0.002) and tree twigs (N = 9, P 0.05), the number of speciescontaining both saponins and phenolics in all other categories was not significantlydifferent from those containing phenolics alone. Since there were samples that didnot contain alkaloids, we used a 2 × 2 contingency test, with Yates’ correction, toexamine the pattern of co-occurrence of alkaloids and saponins and found no significantpattern of segregation between them. Furthermore, many samples containedneither alkaloids nor saponins (Table 2; Appendix A, Table A1).3.6. Tree dominance and secondary metabolitesWe examined the relationship between the relative dominance of tree species andthe fibre and phenolic contents of their mature leaves, since it may be expected thatdominant trees may have higher values of these compounds owing to their higherapparency (sensu Rhoades and Cates, 1976). However we did not find any significantrelationship (Kendall’s correlation coefficients, P 0.05).4. Discussion4.1. Distribution and content of secondary compounds in plant parts4.1.1. Phenolics and fibreAlmost all plant species in each plant part category we examined contained condensedtannins, 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 tanninsare phylogenetically ancient secondary compounds while hydrolysable tannins arelargely 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 condensedtannins on insect and mammalian herbivores (Waterman and Kool, 1994; Ayres etal., 1997) has led to the belief that condensed tannins have evolved primarily indefence against microbes and fungi owing to their anti-microbial and fungistaticeffects (Azaizeh et al., 1990). In the seasonal cloud forest of Bhimashankar, densecloud settles on the stunted forest canopies, without lifting, for four monsoon months

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461231each year (June through September/October). The need for protection against fungiat this time appears to be high and this may explain the ubiquitous presence oftannins in leaves, which is further evidenced by the low leaf litter decay (R.M.Borges, personal observation). The poor, acidic, leached soils (data from IndianBureau of Soil Sciences) and the high levels of insolation at this site may havefurther resulted in phenolics such as condensed tannins being laid down in leavesand even in other plant parts due to overflow (Haslam, 1985) or a pluralistic combinationof the various resource-related defence hypotheses (Berenbaum, 1995).Owing to the dearth of work on hydrolysable tannins in tropical rainforests weare unable to compare our results with other studies but hope that our results willbe useful for further comparisons. Despite their powerful free-radical scavengingactivity and alleged anti-carcinogenic effects (e.g. Sawa et al., 1999), virtually noinformation is available on the effect of hydrolysable tannins on various types ofherbivores in natural systems (but see Whitten and Whitten, 1987; Clifford and Scalbert,2000) although their effect on large arboreal herbivores like the giant squirrelR. 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 earlierFolin–Denis assay, and since all the earlier studies on community-wide distributionof 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 otherstudies. However, as we have used the widely applied proanthocyanidin method forcondensed tannins, our condensed tannin levels can be compared (Table 5) and werefound to be nearly identical with the values found for another evergreen forest atKakachi in southern India (Oates et al., 1980) despite the complete non-overlap ofspecies between Bhimashankar and Kakachi. Furthermore, these condensed tanninlevels were also found to be close to the values found for the two African andthe 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 alsofound to be nearly the same as those measured at Kakachi (Table 5).Immature leaves, flowers, and petioles had high astringency while lower levelswere found in fruit. It is possible that either immature leaves, flowers and petiolesactually do have greater protection by biologically active tannins as measured bytheir 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 fruitas it is known that astringency levels decrease as fruit ripen (Goldstein and Swain,1963). We cannot compare astringency levels between various stages of the samefruit 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 haverarely been analysed chemically (Waterman and Kool, 1994). This pattern of allocationof secondary compounds to tree twigs may be a general strategy as twigs areprotected by high lignin levels and therefore do not require protection from othercompounds. Inner bark had astringency levels and condensed tannin levels comparableto 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) 1221–1246Table 5Comparison of Bhimashankar with other tropical forestsParameter Bhimashankar, India Kakachi, India Kibale, Uganda Douala-Edea, Sepilok, Borneo Kuala Lompat,Cameroon MalaysiaAltitude (m) 910 1325 1400 Lowland 200 Lowland 200 Lowland 200Rainfall (mm) 3000 3080 1485 NA 3000 2000Forest type Semi-evergreen Evergreen Evergreen Evergreen Evergreen EvergreenADF (fibre) a 35.19 (19.0–47.37) 39.4 (24.0–55.1) 35.4 (10.3–67.8) 47.0 (20.8–77.2) 58.3 (40.7–71.7) 46.1 (21.5–73.2)Condensed tannin in mature 6.86 (0–23.40) 6.9 (0–22.0) 5.8 (0–39.6) 5.4 (0–17.0) 8.8 (0–37.0) 4.8 (0–30.5)leaves aN 15 14 23 38 17 33ADF, acid detergent fibre; N, number of species; NA, rainfall value unavailable for Douala-Edea from above-mentioned sources. Numbers in parenthesesindicate range of values.aValues for ADF and condensed tannin are mean percents on dry weight basis (values other than for Bhimashankar are obtained from Gartlan et al.,1980; Oates et al., 1980; Waterman et al., 1988).

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461233indicated by Milton (1979) and Waterman (1984), we also found that the secondarychemistry of flowers is more comparable to foliage that any other plant part.4.1.2. Cyanogenic glycosides, alkaloids, and saponinsCyanogenic glycosides were present in only 2.3% of species screened. This virtualabsence of cyanogenesis was also recorded in a lowland rain forest in Costa Ricawherein 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 certainfamilies such as Leguminosae, Rosaceae, Euphorbiaceae and Passifloraceae (Conn,1979), and cyanogenic glycosides appear to be very much less ubiquitous as defencechemicals than alkaloids, saponins and phenolics. Alkaloids were absent from semiripeand ripe fruit, which could reflect the fact that defences that are deterrent topotential seed dispersers need to be minimised (McKey, 1974). Saponins were foundin all plant parts examined. The lowest occurrence of saponins was found in treetwigs. Much more work needs to be done on the distribution of saponins in planttissues, especially given their possible interactions with both condensed andhydrolysable tannins in influencing the potency of these secondary compounds.4.2. The condensed tannin–hydrolysable tannin interactionOnly in ripe fruit pulp did we find a higher number of species that containedcondensed tannins rather than hydrolysable tannins. The role of hydrolysable tanninsin defence against herbivores has barely been investigated. The seminal and detailedstudies 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 quantifiedhydrolysable tannins nor investigated their role in food selection. However, the barkeatingtropical squirrel Sundasciurus lowii was found to select barks with low levelsof hydrolysable tannins (Whitten and Whitten, 1987). Since condensed tannins areprobably not effective deterrents against insect and mammalian herbivores(Waterman and Kool, 1994; Reed, 1995; Ayres et al., 1997) and probably functionlargely as anti-microbial or anti-fungal agents (Waterman, 1983), it is possible thathydrolysable tannins have a more potent action against herbivores than condensedtannins (Swain, 1977; Zucker, 1983; Reed, 1995). Zimmer (1997) found that ingestedgallotannins increased the surface tension of gut fluid, indicating reduced concentrationsof free surfactants, while Barbehenn et al. (1996) found that the gut peritrophicmembrane in polyphagous grasshoppers was easily permeated by severalgallotannins. It is, therefore, interesting that we found that significantly fewer specieshad ripe fruit containing hydrolysable tannins rather than condensed tannins as thesemight deter dispersal agents. However, there are conflicting claims for beneficial andtoxic effects caused by hydrolysable tannins such as ellagitannins in various animalspecies including rodents and ruminants (Clifford and Scalbert, 2000). Similarlyalmost no information is available on the occurrence of gallotannins and ellagitanninsrelative to each other. Our study has shown that gallotannins and ellagitannins arenot significantly segregated in any plant part. Furthermore, of the 31 species thatwere examined for gallotannins and ellagitannins, only 10 species contained both

1234 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246these compounds, five contained only gallotannins, six only ellagitannins and 10contained neither compound. The biological significance of these findings is as yetunclear and may merely reflect phylogenetic constraints (Gottlieb et al., 1993).4.3. The alkaloid–tannin interactionIn 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 segregationbetween alkaloids and tannins in mature leaves of Douala-Edea and Kibale forests,while Janzen and Waterman (1984) found a negative correlation between alkaloidand tannin contents in a dry forest in Costa Rica. This is expected as alkaloids andtannins are believed to form insoluble alkaloid-tannates in herbivore guts, thus negatingthe effects of each other (Freeland and Janzen, 1974). The negative associationbetween alkaloids and tannins was also predicted by Feeny (1976) from apparencytheory. Within plant families, after correcting for species relatedness, Silvertown andDodd (1996) found a negative association between the proportion of species containingtannins and those containing alkaloids. Our results also show that across almostall plant parts, the number of species containing tannins alone was greater than thenumber of species containing both alkaloids and tannins.4.4. The saponin–tannin interactionSaponins are widespread in plants and cause haemolysis, enzyme inhibition, andalteration of gut surface tension in herbivores (Applebaum and Kirk, 1979). Althoughthe 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 saponinsof a few plantation tree species on leaf-cutting ants (Folgarait et al., 1996) have beendemonstrated, there has been no investigation of either the community-wide presenceof saponins in tropical forests or of the effects of saponins on tropical forest herbivores.Martin and Martin (1984) showed that detergency negated the anti-digestibilityeffects of tannins in the tobacco hornworm, suggesting that the surfactant propertiesof saponins could function similarly. Freeland et al. (1985) demonstrated that thesimultaneous consumption of tannins and saponins reduced the deleterious effectscaused by the consumption of either saponins or tannins alone. These findings leadto the prediction that saponins and tannins should not co-occur in plant parts, whichis the result that we obtained in this study when we found that within each plantpart category the number of species containing tannins alone was greater than thenumber containing both saponins and tannins. We also found saponins to occur in allplant part categories, as has been found in other studies (Applebaum and Kirk, 1979).4.5. The alkaloid–saponin interactionThe alkaloid–saponin interaction in herbivore guts and its possible influence onherbivore food selection has scarcely been investigated. Alkaloids and saponins may

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461235cause greater deterrence to herbivores when they co-occur than when they occurindependently owing to a synergistic effect, as was found for the seeds ofErythrophleum guineense (Caesalpiniaceae) (Kerharo and Adam, 1974). If this is thegeneral case, then alkaloids and saponins may be expected to co-occur, or at leastthere does not appear to be any biological reason to expect a negative associationbetween these compounds. In our study we were unable to find any clear patternof segregation between alkaloids and saponins and also little evidence of positiveassociation. Much more work is needed in this area.5. SummaryIn summary, we have presented data on the correspondence between protein-precipitatingassays and chemical tests for tannin activity; we have also measured bothcondensed and hydrolysable tannins (gallotannins and ellagitannins) in a variety ofplant parts, analysed fibre contents and screened for three types of toxins in plantsfrom a wide array of families and orders within a tropical seasonal cloud forestin India.AcknowledgementsThis research was funded by a grant to RMB from the United States Fish andWildlife Service. We thank the Wildlife Institute of India for collaboration. We thankHema Somanathan for helping with data collection and analysis. We are grateful toDoyle McKey for useful suggestions throughout the study, and for helpful commentson this manuscript. We thank Anne Hagerman for providing the quebracho tanninand the protocols for tannin analysis.Appendix A

1236 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246Table A1Levels of phenolic compounds and fibre and presence of alkaloids and saponins in trees and lianas at BhimashankarPlant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninMature leafActinodaphne – – – – – – – – A –angustifoliaAmoora lawii 2.4 0.4 0 0 0 32.2 44.4 28.3 A AAtalantia racemosa – – – – – – – – P ABridelia retusa – – – – – – – – P ACanthium dicoccum – – – – 0 – – – A –Cassine sp. 0 0.2 0 0 0 20.3 27.3 15.1 A PDiospyros montana 3.2 0.2 3.3 0 0.6 23.0 26.6 22.9 A PDiospyros sylvatica 6.8 0.7 7.6 0 0 31.0 36.8 20.1 A –Diploclisia 0.3 1.6 57.2 0.3 0.1 27.6 43.4 7.5 A Aglaucescens aEmbelia ribes a 1.8 0.6 1.5 0 0 38.1 42.1 33.8 P AFicus callosa 21.1 1.3 23.4 0 0 41.5 43.8 32.2 A AFicus racemosa – – – – – – – – A –Flacourtia indica – – – – – – – – A –Garcinia talbotii – – – – – – – – A PGnetum ula a 2.0 1.8 1.2 0 0 31.2 45.2 22.0 A PLitsea stocksii – – – – – – – – A PMacaranga peltata – – – – 0 – – – A AMallotus philippensis 6.6 1.1 4.7 0.1 0.2 38.9 57.6 30.1 A AMangifera indica 13.0 2.1 12.0 – 0 40.9 38.7 30.0 A AMaytenus rothiana 0.6 0.2 1.0 0 0 33.3 43.5 29.1 P AMemecylon umbellatum 0.4 – – 0.5 0 26.2 32.7 11.1 A AMezoneuron – – – – – – – – A –cucullatum aOlea dioica – – – – – – – – A ARandia dumetorum – – – – – – – – P –Randia rugulosa a 13.0 1.1 66.0 0.1 0 23.5 28.0 22.0 A A(continued on next page)

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461237Table A1 (continued)Plant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninRourea santaloides a – – – – – – – – A ASymplocos beddomei 6.4 0.5 1.2 2.5 0 28.2 35.3 17.9 A ASyzygium cumini – – – – 0 – – – A ASyzygium gardneri 13.4 1.7 0 1.0 0 29.9 36.5 20.5 A ATerminalia bellerica – – – – – – – – A PTerminalia chebula – – – – – – – – A PVentilago bombaiensis a 20.0 2.6 20.0 0.1 0 32.1 41.6 30.1 A –Xantolis tomentosa 17.6 0.8 14.3 0 0.1 64.3 50.1 57.9 A AImmature leafActinodaphne 13.2 0.8 14.0 0 0 60.6 60.9 58.3 A PangustifoliaCassine paniculata 4.0 0.2 1.8 0 0 50.5 39.3 30.6 A PDiospyros sylvatica 39.0 1.1 11.0 0 0 40.7 26.2 40.2 A PDiploclisia 13.0 0.9 59.4 0 0 33.0 37.1 15.6 A Pglaucescens aFicus callosa 18.6 2.6 56.7 0 0 36.6 23.6 23.1 A AGarcinia talbotii 17.0 1.5 0.7 0 0.3 37.0 32.1 35.5 A PGnetum ula a 0.8 2.5 0.4 0 0.2 9.1 28.7 3.1 P PLitsea stocksii 17.0 1.4 0.1 0 0.2 40.3 40.0 26.1 A AMangifera indica 0.5 3.0 7.0 1.3 0 23.9 28.1 11.6 P AMemecylon umbellatum 0.4 0.8 15.8 0.3 – 27.5 27.4 14.7 A AOlea dioica 1.8 3.0 3.2 0 0 31.2 34.3 19.7 P PRourea santaloides a 0.6 0.2 22.8 0.2 0.3 70.6 29.2 19.3 P ASymplocos beddomei 0.7 – – 3.8 0 23.8 28.8 11.0 A ASyzygium cumini 22.5 11.2 17.3 1.3 1.3 35.1 34.8 18.0 A PSyzygium gardneri 15.4 1.4 21.8 1.5 0.9 25.5 29.8 11.3 A –Terminalia chebula 16.0 12.0 1.6 1.3 0.6 10.1 10.3 9.6 A –Ventilago bombaiensis a 12.8 0.7 36.0 0 0 22.4 32.0 16.6 A A

1238 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246Table A1 (continued)Plant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninPetioleActinodaphne 13.2 1.3 11.2 0 0 42.3 52.0 34.0 A –angustifoliaCassine sp. – 0.1 1.0 0 0 – – – A –Diospyros sylvatica 3.0 0.2 3.6 0.1 0.3 33.4 38.3 19.5 A PDiploclisia 6.0 0.2 13.2 0 0 49.1 58.5 43.0 A Pglaucescens aGarcinia talbotii 15.4 5.2 57.4 0 0 29.7 32.9 12.9 A PGnetum ula a 1.0 1.1 1.1 0.03 0 30.6 41.6 30.1 A –Macaranga peltata – – – – – – – – A –Mangifera indica 12.2 2.3 34.6 0.3 0 36.4 34.3 30.7 A –Memecylon umbellatum 17.8 3.6 46.0 0.5 0 25.6 27.4 7.7 A –Olea dioica 2.9 2.0 3.6 0 0 33.5 29.9 18.6 A ASyzygium cumini 22.3 14.0 32.0 0.1 0.6 37.0 34.7 19.0 A AFlowerActinodaphne 10.0 1.2 64.4 0 0 28.8 36.1 19.8 A AangustifoliaDiospyros montana 2.0 0.2 3.0 0 – 20.6 20.3 16.4 A –Diploclisia 0.2 1.2 15.4 0 0 16.1 16.2 10.8 A Pglaucescens aElaeagnus conferta a 0.6 – – – 0 – – – P –Garcinia talbotii 0.4 0.1 1.1 0 0 18.3 22.6 6.5 A PLepisanthes tetraphylla 7.1 0.7 28.8 0 0 34.7 44.5 32.8 A AMangifera indica 8.0 3.1 2.4 17.5 0 20.2 26.1 12.1 A AMemecylon umbellatum 12.8 0.9 6.0 0.03 1.3 22.4 23.8 16.1 A AMezoneuron 16.8 8.0 16.2 10.0 0 15.4 24.6 15.3 A Pcucullatum aOlea dioica 6.4 2.7 8.2 0 0 25.0 31.8 15.3 A ASymplocos beddomei 13.6 1.7 31.6 2.5 0 22.5 27.8 19.5 A –Syzygium cumini 19.5 2.0 7.8 0 0 61.5 44.6 33.1 A A

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461239Table A1 (continued)Plant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninTerminalia chebula 22.8 2.8 0.4 5.0 1.6 17.4 20.4 14.8 A –Xantolis tomentosa 13.0 1.4 59.4 0.1 0 34.4 31.1 31.8 A –Entire immature fruitActinodaphne 1.4 0.4 3.9 0 0 24.2 30.4 16.7 A –angustifoliaAmoora lawii 2.3 0.4 2.4 0 0 27.7 37.1 26.4 A –Artocarpus 5.6 0.2 26.0 0 0 31.8 27.9 31.7 A PheterophyllusFicus racemosa 15.2 0.8 25.6 0 0 28.4 22.6 22.1 A AGnetum ula a 1.0 1.0 0 0 0 32.5 46.2 26.5 A AEntire mature fruitEmbelia ribes a 4.0 0.2 7.5 0 0 49.5 59.2 46.5 A –Ficus callosa – 0.4 5.8 0 0 – – – A –Ficus racemosa 0.2 – – – 0 52.7 53.8 27.3 P AFicus religiosa 0.6 0.1 2.8 – 0 53.1 35.9 37.7 A –Ficus tsjahela 15.5 0.3 6.6 0 0.2 57.7 47.1 57.0 A –Randia rugulosa a 0 0.4 2.2 0 0 52.2 46.9 33.9 A PImmature fruit pulpDiploclisia 0.4 0 6.0 – – 35.1 33.9 22.4 A –glaucescens aGarcinia talbotii 0.4 – – – – 8.5 12.7 0.5 A PMangifera indica 0.4 0.5 0 1.8 0 7.9 16.6 2.7 A –Symplocos beddomei 0.4 – – – – 26.6 35.2 11.1 P –Terminalia chebula 15.0 5.6 0 10.0 1.3 8.2 7.1 7.9 A –Semi-mature fruit pulpGarcinia talbotii 0.2 – – – – 6.9 8.8 1.4 A –Gnetum ula a 0.2 0.4 0 0 0.1 25.9 32.0 4.6 A A

1240 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246Table A1 (continued)Plant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninMangifera indica 0.2 0.8 0 1.3 0 4.4 18.9 1.0 A –Olea dioica 1.9 2.6 5.0 0 0 33.4 40.7 14.8 A –Symplocos beddomei – 0 2.4 0 0 – – – A –Syzygium cumini 0 0.1 1.5 0 0 28.7 22.5 27.4 A –Xantolis tomentosa 6.6 0.5 13.3 0 0 14.3 16.4 10.2 A –Ripe fruit pulpAcacia sp. a – – – – 0 – – – – –Actinodaphne 6.1 0.7 14.0 0 0 25.6 32.4 24.0 A –angustifoliaAmoora lawii 1.0 0.7 0.9 0 0 8.7 14.7 8.5 A –Diospyros sylvatica 11.8 0.6 11.5 0 0 35.5 47.3 33.3 A PDiploclisia 0.6 0.6 6.0 0 0 33.3 31.3 23.9 A –glaucescens aGarcinia talbotii 1.0 0.2 0 0 0 12.8 17.5 12.3 A –Gnetum ula a 7.1 1.9 1.1 0 0 42.1 57.3 12.4 A –Litsea stocksii 14.5 1.7 25.2 0 0 52.2 41.8 44.4 A –Mangifera indica – 0.6 0.6 0.5 0.2 27.6 32.2 23.2 A –Memecylon umbellatum 14.6 1.9 19.6 0.3 0 22.8 37.1 9.3 A AOlea dioica 0.4 0.4 1.5 0 0 4.9 7.5 3.5 A ASymplocos beddomei 0.9 0.1 3.4 0 0 29.1 43.5 9.5 A PSyzygium cumini 0.6 0.8 7.6 0 0 8.6 9.4 7.4 A PSyzygium gardneri 4.5 0.2 5.2 0.03 0 31.0 26.3 20.0 A ATerminalia chebula 0.4 6.4 1.1 0 0 – – – A AVangueria spinosa 0.4 0.1 0.5 0 0 43.8 30.2 25.6 A –Immature seedDiploclisia 0.6 – – – – 11.1 12.1 7.3 A –glaucescens aGarcinia talbotii 0.1 – – – – 4.0 17.1 1.1 A –Gnetum ula a 0.7 – – – – 6.1 10.8 1.7 A A

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461241Table A1 (continued)Plant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninMangifera indica 0.2 – – – – 4.5 6.4 1.0 A –Terminalia chebula 11.3 2.1 0.8 0.2 1.2 40.1 38.6 19.6 A –Semi-mature seedGarcinia talbotii 0.5 – – – – 5.7 29.6 2.6 A –Gnetum ula a 0.4 – – – – 6.4 33.4 2.8 P AMangifera indica 0.4 – – 0 0 6.3 21.8 3.2 A –Olea dioica 0 0.2 0 0 0 19.8 23.2 15.0 A –Symplocos beddomei 2.2 0.3 10.0 0 0 43.1 54.0 25.0 A –Mature seedAcacia concinna a 1.0 0.1 0.7 0 0 22.4 29.7 20.6 A –Acacia sp. a 0 0 0 0 0 17.9 20.3 14.4 A –Actinodaphne 0 0.2 4.0 0 – 16.0 18.3 13.8 A AangustifoliaAmoora lawii 0.3 0.2 1.2 0 0 11.9 27.2 5.6 A –Artocarpus 0.1 – – – – 6.2 23.8 1.2 A –heterophyllusDiospyros sylvatica 0.2 1.7 84.0 1.3 0 41.1 64.5 12.9 A ADiploclisia 0.2 0 1.2 0 0 7.9 45.6 3.6 A –glaucescens aDysoxylum 0.2 – – – – 9.3 32.1 1.2 A –binectariferumGarcinia talbotii 10.0 1.3 0.7 0 – 6.9 35.0 6.4 A –Gnetum ula a 23.3 3.0 3.0 0 0 3.8 56.6 3.8 A –Lepisanthes tetraphylla 0 0.1 0 0 0 16.9 40.9 11.6 A PLitsea stocksii 0.7 0.7 8.0 0 0 4.3 7.8 2.8 A –Macaranga peltata 0.4 – – – – 23.2 35.5 19.9 A AMangifera indica 12.8 1.5 3.4 6.3 0.3 6.4 28.3 6.1 A –Memecylon umbellatum 13.8 4.8 32.9 1.0 0.5 11.0 31.6 6.0 A AOlea dioica 0.4 0.1 0 0 0 40.0 51.9 27.8 A P

1242 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246Table A1 (continued)Plant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninSymplocos beddomei 8.1 0.8 16.8 0 0 40.6 61.9 27.4 A PSyzygium cumini 0.4 1.4 1.4 2.5 0 6.4 39.9 2.4 A –Syzygium gardneri 10.0 1.6 0.7 2.0 0 15.8 33.3 6.2 A ATerminalia bellerica – – – – 0 – – – – –Terminalia chebula 3.8 0.4 0 0 0 7.4 15.2 4.4 A –Xantolis tomentosa 1.0 0.1 0 0 0 23.0 30.0 23.0 A AStem of leaf sprays (Twigs)Amoora lawii – – – – 0 – – – – –Artocarpus 1.0 0.2 1.7 – – 51.9 62.3 17.4 A AheterophyllusCassine sp. 0 0.1 0 0 0 36.8 49.1 15.2 A AFicus callosa 0.1 0.1 3.6 0 0 61.8 66.8 17.4 P AFicus racemosa 0.7 – – – – 15.8 17.9 4.4 A –Mallotus philippensis 2.0 0.6 0 0 0 55.7 67.4 19.0 A AMangifera indica 0.3 – – – – 85.9 – 17.2 A AOlea dioica 0.9 0.3 0.6 – 0 51.3 60.9 32.5 A ASymplocos beddomei 6.2 0.7 13.3 0 0 45.1 49.3 15.4 P PSyzygium cumini 7.9 0.3 3.4 – 0 68.7 68.0 28.2 A AXantolis tomentosa 0.5 0.1 2.9 0 0 24.2 34.2 9.7 P AInner barkActinodaphne 7.3 1.0 27.0 – 0.3 53.5 62.5 43.9 P PangustifoliaAmoora lawii 0 0.1 0 0 – 42.2 58.9 31.5 A –Atalantia racemosa – – – – – – – – A ABridelia retusa – – – – 0 – – – A PCassine paniculata 0.3 0.7 8.2 0 0 42.6 36.7 39.0 P ADiospyros sylvatica 2.5 0.2 1.3 0 0 35.0 44.0 18.3 A ADiospyros montana – – – – – – – – A –Ficus callosa 19.2 2.5 68.6 0.1 0 26.3 22.2 16.3 P A

S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–12461243Table A1 (continued)Plant part and species Astringency Total phenolic Condensed Gallotannin Ellagitannin ADF NDF ADL Alkaloids Saponincontent tanninFicus racemosa 0.6 1.2 52.0 0 0 7.8 10.6 2.8 A PFicus religiosa 6.4 0.2 13.2 0 0 48.3 50.3 46.1 A PGarcinia talbotii 10.1 1.6 0 0 0 26.7 32.1 20.0 A –Litsea stocksii 6.4 0.3 14.7 0 0 37.0 44.4 32.9 A PMallotus philippensis 3.4 0.6 7.6 0 0.3 41.6 55.6 40.5 A AMangifera indica 0.7 – – 0 0 23.5 28.1 6.3 A AOlea dioica 0 0.1 0 0 0 50.7 62.3 42.2 A ARourea santaloides a 20.0 1.5 23.0 0 0 50.0 54.3 24.4 A –Symplocos beddomei 0 0.2 0 0 0 54.4 63.5 45.1 A PSyzygium cumini 16.0 1.6 30.2 0.8 – 61.4 50.3 53.0 A ATerminalia chebula 14.6 1.9 – 0.1 0 27.3 32.3 25.0 A PXantolis tomentosa 0.4 0.2 3.4 0 0 26.1 40.6 9.5 A AEntire barkGnetum ula a 12.4 1.3 0 0 0 42.9 50.8 37.8 P PMezoneuron 18.0 1.6 8.4 0 0 43.6 56.0 40.2 P Acucullatum aRandia rugulosa a 12.2 1.1 29.6 0.1 0.2 51.9 64.0 45.7 A PSyzygium gardneri – – – – – – – – A AVentilago bombaiensis a 5.0 0.5 5.4 0 0 45.8 65.6 41.8 A –For parameters other than alkaloids and saponins, values are expressed as follows: astringency and total phenolic contents as percent tannic acid, condensedtannins as percent quebracho tannin, gallotannins as percent gallic acid, and ellagitannins as percent ellagic acid on dry weight basis. Only alkaloids andsaponins were qualitatively detected, wherein ‘P’ indicates presence; ‘A’ indicates absence and – indicates values not estimated; ADF, acid detergent fibre;NDF, neutral detergent fibre; ADL, acid detergent lignin. Cyanogenic glycosides were absent in all these items and hence not shown.aIndicates liana.

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