Accepted Manuscript

Title: Anti-inammatory and phytochemical properties of twelve medicinal plants used for treating gastro-intestinal ailments in South Africa Authors: O.A. Fawole, A.R. Ndhlala, S.O. Amoo, J.F. Finnie, J. Van Staden PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0378-8741(09)00153-6 doi:10.1016/j.jep.2009.03.012 JEP 5470 Journal of Ethnopharmacology 23-12-2008 5-3-2009 11-3-2009

Please cite this article as: Fawole, O.A., Ndhlala, A.R., Amoo, S.O., Finnie, J.F., Van Staden, J., Anti-inammatory and phytochemical properties of twelve medicinal plants used for treating gastro-intestinal ailments in South Africa, Journal of Ethnopharmacology (2008), doi:10.1016/j.jep.2009.03.012 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

* Manuscript

1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Anti-inflammatory and phytochemical properties of twelve medicinal plants used for treating gastro-intestinal ailments in South Africa

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

O.A. Fawole, A.R. Ndhlala, S.O. Amoo, J.F. Finnie, J. Van Staden*

Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences,

* Corresponding author, e-mail address: rcpgd@ukzn.ac.za (J. Van Staden)

Tel: +27 33 2605130; Fax: +27 33 2605897

Received..

Ethnopharmacological relevance: The investigated medicinal plants are commonly used for the treatment of pains and cramps related to gastro-intestinal tract infections in South African traditional medicine.

cyclooxygenase enzymes. Phytochemical analysis was also carried out in the quest to determine some plant metabolites that may be responsible for the observed anti-inflammatory activity. Materials and methods: The cyclooxygenase assay was used to test for the anti-inflammatory activity of the plant extracts using cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes. Total phenolic compounds including condensed tannins, gallotannins and flavonoids were

Ac

Aims of the study: This study aimed to evaluate the ability of the plant extracts to inhibit

ce p

te

Abstract

an
Page 1 of 29

us

cr

University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

ip t

26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

quantitatively determined using spectrophotometric methods. Qualitative tests for alkaloids and saponins were also carried out. Results: Most of the plant extracts evaluated showed dose dependent activity against COX-1 and/or COX-2 enzymes. Agapanthus campanulatus root dichloromethane extract showed the highest COX-2 inhibitory activity (83.7%) at 62.5 g/ml. The presence and/or amounts of phenolics, condensed tannins, gallotannins, flavonoids, alkaloids and saponins varied with plant parts and species.

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Conclusion: The results support the use of the investigated plant in treating pain and cramp related to gastro-intestinal tract infections. The observed anti-inflammatory activity could to some extent be attributed to the various plant secondary metabolites detected in the plant materials.

Keywords: Anti-inflammatory; Cyclooxygenase; Gastro-intestinal ailments; Secondary

1. Introduction

resulting in abdominal pains and cramps of varying degree (Barbara, 1998). Naik and Sketh (1976) defined inflammation as a complex, vascular lymphatic and local tissue reaction elicited in animals by the presence of viable and non-viable irritants. The intestine is vulnerable to muscle spasm in patients suffering from gastro-intestinal infections, and most patients suffering from such conditions often complain of abdominal cramps and pains (Sleisenger and Fordtrand, 1993). Escherichia coli and other gastro-intestinal pathogens

Ac

Gastro-intestinal ailments are associated with inflammation of the gastro-intestinal tract

ce p

te
2
Page 2 of 29

metabolites

an

us

cr

ip t

51
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

associated with food poisoning produce enterotoxins that induce watery diarrhoea and abdominal tissue damage through plasmid-encoded invasion factors, resulting in acute or chronic abdominal pains and cramps (Naik and Sketh, 1976; Sleisenger and Fordtrand, 1993). Non-steroidal anti-inflammatory drugs (NSAIDs) typically relieve inflammation and associated pain by inhibiting cyclooxygenase enzymes involved in the production of prostaglandins. These enzymes exist in two isoforms (COX-1 and COX-2) coded by distinct genes on different chromosomes (Polya, 2003). The two isoforms show about 50% homology and have similar catalytic activity, but are physiologically distinct (Pasinetti, 2001). Compounds that inhibit COX enzymes could therefore be considered to be potential antiinflammatory drugs. However, many of the commonly used anti-inflammatory agents are becoming less acceptable due to serious adverse reactions such as gastric intolerance, bone marrow depression and water and salt retention, resulting from prolonged use (Xiao et al., 2005). This necessitates the continued search for potent anti-inflammatory agents with

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

al., 1999; Khafagi and Dewedar, 2000). Some plant secondary metabolites such as alkaloids, phenols, tannins, glycosides, terpenoids, saponins, flavonoids and steroids have been implicated in their ability to inhibit the formation of pro-inflammatory signalling molecules

medicinal plants used traditionally in the treatment of pain associated with gastro-intestinal infections. The phytochemical components of these plants such as flavonoids, gallotannins, , condensed tannins, other phenolic compounds, alkaloids and saponins were also evaluated. The antimicrobial and genotoxicity evaluation of the same plant materials have earlier been reported (Fawole et al., 2009).

Ac

such as prostaglandin or leukotrienes (Polya, 2003). In the present study, we evaluated twelve

ce p

te

proved to be a more efficient method of drug discovery than random plant screening (Slish et

reduced or no side-effects. Studies based on the ethnobotanical use of plants have often

M
3

an

us

cr

ip t

Page 3 of 29

76
1 2 77 3 4 5 78 6 7 79 8 9 10 80 11 12 81 13 14 82 15 16 17 83 18 19 84 20 21 22 85 23 24 86 25 26 27 87 28 29 88 30 31 89 32 33 34 90 35 36 91 37 38 39 92 40 41 93 42 43 44 94 45 46 95 47 48 49 96 50 51 97 52 53 98 54 55 56 99 57 58 100 59 60 61 62 63 64 65

2. Material and methods

2.1. Plant material

Twelve traditional medicinal plants that are commonly used for the treatment of gastrointestinal ailments (Table 1) were collected between November (2007) and February (2008) from Mt. Gilboa (29 16.766' S, 30 17.627' E), Midmar (29 29.703' S, 30 12.417' E), University of KwaZulu-Natal Botanical Garden and Pietermaritzburg National Botanical Garden in KwaZulu-Natal Province, South Africa. Due to availability and consideration of potential sustainable harvesting of medicinal plants, the leaves of some plant species were substituted for their roots. Voucher specimens were identified by and lodged in the University of KwaZulu-Natal Herbarium, Pietermaritzburg. Plant materials were oven-dried at 50 C,

2.2. Anti-inflammatory activity

2.2.1. Preparation of extracts

(PE), dichloromethane (DCM) and 70% ethanol (EtOH) in a sonication bath (Julabo GMBH, West Germany) at room temperature for 1 h each. The extracts were then filtered under vacuum through Whatman No.1 filter paper. Water extracts were prepared non-sequentially and freeze-dried while organic extracts were concentrated in vacuo using a rotary evaporator at 30 C. The resultant extracts were air-dried at room temperature.

Ac

Ground plant materials (5 g) were sequentially extracted with 100 ml of petroleum ether

ce p

te
4
Page 4 of 29

ground into powders and stored in airtight containers at room temperature in the dark.

an

us

cr

ip t

101
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

2.2.2. Cyclooxygenase assays The cyclooxygenase assays (COX-1 and COX-2), as described by Eldeen and Van Staden (2008), were used to evaluate the anti-inflammatory activity of the extracts. Crude extracts were screened at a concentration of 250 g/ml for organic extracts and 2 mg/ml for aqueous extract. Extracts showing good ( 50%) COX-2 inhibitory activity were then further evaluated at concentrations of 125 g/ml and 62.5 g/ml in both COX-1 and COX-2 assays. Indomethacin (Sigma) (5 M for COX-1, 200 M for COX-2) was used as a positive control, while background in which the enzyme was inactivated with HCl before adding [14C] arachidonic acid, and solvent blank were used as negative controls. Percentage inhibition by the extracts was calculated by comparing the amount of radioactivity present in the sample to that in the solvent blank using the equation below:

102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

COX Inhibition (%) =

DPMbackground = Disintegrations per minute in which the enzyme was inactivated DPMblank = Disintegrations per minute for solvent used in dissolving plant extracts

experiment in duplicate.

2.3. Phytochemical analysis

2.3.1. Preparation of extracts

Ac

Results are expressed as means standard errors of two independent experiments, each

ce p

te

where DPMsample = Disintegrations per minute for plant extract

M
5
Page 5 of 29

an

us

cr

ip t

125
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Phenolic compounds were extracted from plant materials as described by Makkar (1999). Dried plant samples (2 g) were extracted with 10 ml of 50% aqueous methanol by sonication in cold water for 20 min. The extracts were then filtered under vacuum through Whatman No.1 filter paper.

126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149

2.3.2. Determination of total phenolics

The amount of total phenolics in plant samples was determined using the Folin Ciocalteu (Folin C.) assay for total phenolics as described by Makkar (1999). Fifty microlitres of each extract from the plant samples were transferred into test tubes and 950 l distilled water were added to make up to 1 ml, followed by 1 N Folin C. reagent (500 l) and 2% sodium carbonate (2.5 ml). A blank that contained aqueous methanol instead of plant extracts was also prepared. The test mixtures were incubated for 40 min at room temperature and the absorbance read at 725 nm using a UV- visible spectrophotometer (Varian). Each extract had

2.3.3. The butanol-HCl assay for condensed tannins Three millilitres of butanol-HCl reagent (95:5 v/v) were added to 500 l of each extract,

combination was vortexed and placed in a boiling water bath for 60 min. The absorbance was then read at 550 nm using a UV- visible spectrophotometer (Varian) against a blank prepared by mixing extract (500 l) with butanol-HCl reagent (3 ml) and ferric reagent (100 l), but without heating. Each extract had three replicates. Condensed tannin (% per dry matter) was calculated as equivalent amount of leucocyanidins using the formula developed by Porter et al. (1986):

Ac

followed by 100 l ferric reagent (2% ferric ammonium sulphate in 2 N HCl). The test

ce p

te
6
Page 6 of 29

(GAE).

three replicates and total phenolic concentrations were expressed as gallic acid equivalents

an

us

cr

ip t

150
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174

Condensed tannin = (A550 nm 78.26 dilution factor) (% dry matter)

2.3.4. Rhodanine assay for gallotannins Plant extracts (50 l) were made up to 1 ml with distilled water. One hundred microlitres of 0.4 N sulphuric acid and 600 l rhodanine were added to the diluted extracts. After 5 min, 200 l of 0.5 N potassium hydroxide were added and 4 ml distilled water after 2.5 min. The mixtures were left for a further 15 min at room temperature, after which the absorbance at 520 nm was read using a UV- visible spectrophotometer against a blank test that contained methanol instead of sample. Each extract was evaluated in replicates and gallotannin concentrations were expressed as gallic acid equivalents (GAE) (Makkar, 1999).

2.3.5. Vanillin assay for flavonoids

ml). Similar preparations of a blank that contained methanol instead of plant extracts were made. After 20 min at room temperature, absorbance at 500 nm was read using a UV- visible spectrophotometer (Varian Cary 50) against the blank. The flavonoids in the plant extracts

2.3.6. Thin layer chromatography for alkaloid detection Ten microlitres each of PE, DCM, EtOH and water extracts (50 mg/ml) (prepared as described in Section 2.2.1.) were spotted on thin layer chromatographic (TLC) plates (Silica gel 60 F254, Merck, Germany). The plates were developed using hexane:benzene:ethyl acetate (5:2:3) for PE and DCM extracts, while ethyl acetate:methanol:water (100:16.5:13.5) was

Ac

were expressed as catechin equivalents (Makkar, 1999).

ce p

te

tubes before adding 2.5 ml methanolic-HCl (95:5 v/v) and 2.5 ml vanillin reagent (1 g/100

Plant extracts (50 l) (in triplicate), were made up to 1 ml with distilled water in test

M
7
Page 7 of 29

an

us

cr

ip t

175
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

used for EtOH and water extracts. After development, the plates were dried, viewed under UV (254,366 nm) and the fluorescence noted. The presence of alkaloids was indicated by red coloration when the plates were sprayed with Dragendorff reagent (Robert, 1962; Wilfred and Ralph, 2006).

176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199

2.3.7. Froth test for saponins

Distilled water (10 ml) was added to 0.1 g of ground plant sample in a test tube. The test tube was corked and vigorously shaken for 2 min. The appearance of stable foam on the liquid surface for 45 min indicated the presence of saponins (Makkar, 1999). To confirm the presence of saponins, ten drops of olive oil were added to the aqueous extract (2 ml) in a test tube. The test tube was then corked and vigorously shaken. The formation of an emulsion confirmed the presence of saponins (Tadhani and Subhash, 2006).

3.1. Anti-inflammatory activity

presented in Table 2. Plant extracts showing a minimum inhibition of 50% are considered to have good activity (Eldeen and Van Staden, 2008). All the PE and DCM extracts of the plant material (with the exception of Antidesma venosum leaf and Protea simplex bark) showed good activity against both COX-1 and COX-2 (inhibition of prostaglandin synthesis ranging from 58.8 to 103%). Generally, all the ethanol (except Diospyros lycioides leaf and Watsonia tabularis corm) and water extracts showed weak or no activity (inhibition < 50%) against

Ac

The percentage inhibition of COX-1 and COX-2 by all the extracts at 250 g/ml is

ce p

te
8
Page 8 of 29

3. Results and discussion

an

us

cr

ip t

200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

COX-2. However, water extracts of Agapanthus campanulatus leaf, Becium obovatum root, Cyperus textilis root, Protea simplex leaf and bark, and Watsonia tabularis corm exhibited good activity mainly against COX-1 enzyme with inhibition ranging from 50.3 to 90.5%. Most of the plant extracts evaluated showed dose dependent activity against COX-1 and/or COX-2 enzymes (Figs 1 and 2 respectively). Interestingly, at 62.5 g/ml concentration, 16 out of 28 PE and DCM extracts evaluated still showed good activity (> 50%) against the COX-2 enzyme. Generally, most of the extracts showed higher percentage inhibition for COX-1 than for COX-2 at the highest screening concentration (250 g/ml). Although COX-2 specific inhibitors have been suggested to be potential classical non-steroid anti-inflammatory drugs due to their reduced or no side effects, some authors have reported that they also have a non-negligible risk of gastro-intestinal toxicity in some patients (MacAulay and Blackburn, 2002; Bertin, 2004; Warner and Mitchell, 2008). However, the observed activity in many of the extracts supports their uses in South African traditional medicine. In the case of extracts

201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224

active at other sites in the inflammatory pathways and/or contain compounds showing better activity in vivo as they undergo metabolic transformation (McGaw et al., 1997; Garcia et al., 2003). In the human inflammatory process, for example, anti-inflammatory activity of

mediated signalling pathway in immune cells that lead to the production of inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines and inducible cyclooxygenase (iCOX) (Polya, 2003). Moreover, the presence of comparable activities in the leaves and root or stem of Agapanthus campanulatus, Cyperus textilis, Diospyros lycioides, and Protea simplex supports the idea of plant part substitution for sustainable use of many highly threatened plants (Zschocke and Van Staden, 2000).

Ac

medicinal plants could be manifested in the inhibition of nuclear transcriptase factor (NFB)

ce p

te

traditional medicine which may result in COX inhibition. Some of these extracts might be

showing weak or no activity in these assays, high dosages of extract are often used in

M
9

an

us

cr

ip t

Page 9 of 29

225
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249

3.2. Phytochemical analysis

3.2.1. Total phenolics composition Figure 3 shows the total phenolic compounds concentrations of the investigated plant species. All the plant species evaluated contained some phenolic compounds. The highest concentration of total phenolics was detected in C. textilis leaf (84.5 mgGAE/g). In addition, B. obovatum root, P. simplex leaf and bark, and C. textilis root contained total phenolic concentrations 50 mgGAE/g. Phenolic compounds are of important pharmacological value, some having anti-inflammatory properties (Bruneton, 1995). Different types of phenolic compounds such as flavonoids, condensed tannins, and gallotannins are known to inhibit some molecular targets of pro-inflammatory mediators in inflammatory responses (Sharma et al., 1994; Iwalewa et al., 2007). Specific types of phenolic compounds present in these

3.2.2. Condensed tannins contents

The amounts of condensed tannins expressed as percentage per dry matter are shown in Fig. 4. Highest amounts of condensed tannins were detected in C. textilis leaves and roots

B. obovatum root, A. campanulatus root and Vernonia natalensis leaf. Condensed tannins (proanthocyanidins) are essentially derived from (+) gallocatechin, () epicatechin, (+) catechin and epigallocatechin, and their derivatives via carbon to carbon (C-C) links. These compounds are antagonists of particular hormone receptors or inhibitors of particular enzymes such as cyclooxygenase enzymes (Polya, 2003).

Ac

while low levels were detected in Gladiolus dalenii. No condensed tannins were detected in

ce p

te
10
Page 10 of 29

species were therefore investigated.

an

us

cr

ip t

250
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

3.2.3. Rhodanine assay for gallotannins Figure 5 presents the amounts of gallotannins present in the investigated plants. With the exceptions of A. campanulatus root and G. dalenii corm, gallotannins are detected in all the investigated species. The highest amount of gallotannin was detected in P. simplex leaf (13.5 g/g dry matter). Gallotannins exert various biological effects ranging from antiinflammatory to anticancer and antiviral properties (Erde`lyi et al., 2005). The mechanisms underlying the anti-inflammatory effect of tannins include the scavenging of radicals and inhibition of the expression of inflammatory mediators, such as some cytokines, inducible nitric-oxide synthase, and cyclooxygenase-2 (Polya, 2003; Erde`lyi et al., 2005). The high amounts of gallotannins present in some of the evaluated plant materials could in part be responsible for the observed high anti-inflammatory activity.

251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274

3.2.4. Vanillin assay for flavonoids

and Haworthia limifolia leaves respectively. According to Talhouk et al. (2007), flavonoids are known to act on the inflammatory response via many routes and block molecules like COX, iNOS, cytokines, nuclear factor-B and matrix metalloproteinases. Some flavonoids

induced mouse paw oedema model (Pelzer et al., 1998).

3.2.5. Alkaloids detection The presence or absence of alkaloids in the investigated plant extracts are summarized in Table 3. Twelve out of 48 extracts evaluated showed the presence of alkaloids. Previous researchers have reported the presence of alkaloids in A. venosum, Vernonia sp., and D.

Ac

have been reported to be effective against acute inflammation in vivo using a carrageenin-

ce p

te

Fig. 6. The highest (7.4 mg/g) and the lowest (0.24 mg/g) amounts were detected in C. textilis

The flavonoid concentrations present in the investigated plant materials are shown in

M
11
Page 11 of 29

an

us

cr

ip t

275
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

lycioides (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996; Ndukwe et al., 2004). Some alkaloids such as isoquinoline, indole and diterpene are known to have good antiinflammatory activity (Barbosa-Filho et al., 2006).

276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299

3.2.6. Saponins detection All the evaluated plant materials except H. limifolia, P. simplex, A. venosum and D. princeps leaves tested positive for saponins. The anti-inflammatory activities of some saponin derivatives such as triterpenoids saponins have been reported (Sahu and Mahato, 1994). According to Sparg et al. (2004), many saponins extracted from plant sources produce an inhibition of inflammation in the mouse carrageenan-induced oedema assay.

4. Conclusions

of many of the investigated plant species have not been reported, yet they are extensively used in traditional medicine. To a large extent, the results in this study validate the traditional medicinal use of the evaluated plant species in treating stomach pains and cramps associated

considered sufficient for further studies aimed at isolating and identifying the active principle(s) as well as potential combination effects (if any) of the isolated compounds as some of these plants are frequently included in multiple decoctions.

Acknowledgements

Ac

with gastro-intestinal infections. The study of their phytochemical constituents might be

ce p

te

As far as we can ascertain, the anti-inflammatory activity and phytochemical properties

M
12
Page 12 of 29

an

us

cr

ip t

300
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324

Mrs Alison Young of the University of KwaZulu-Natal Botanical Garden and Gary Stafford of the Research Centre for Plant Growth and Development, UKZN are thanked for assistance in plant collection. The National Research Foundation and the University of KwaZulu-Natal are gratefully acknowledged for financial assistance.

References

Barbara, B.B., 1998. Gastroenteritis. Medical Update for Psychiatrists 4, 95-98. Barbosa-Filho, J.M., Piuvezam, M.R., Moura, M.D., Silva, M.S., Lima, K.V.B., Leito daCunha, E.V., Fechine, I.M., Takemura, O.S., 2006. Anti-inflammatory activity of alkaloids: a twenty-century review. Revista Brasileira de Farmacognosia 6, 1.

Bruneton, J., 1995. Pharmacognosy, Phytochemistry, Medicinal Plants. Intercept Ltd., Hampshire.

Eldeen, I.M.S., Van Staden, J., 2008. Cyclooxygenase inhibition and antimycobacterial

224-229.

Erde`lyi, K., Kiss, A., Bakondi, E., Bai, P., Szabo, C., Gergely, P., Erdo di, F., Vira g, L., 2005. Gallotannin inhibits the expression of chemokines and inflammatory cytokines in A549 Cells. Molecular Pharmacology 68, 895-904. Fawole, O.A., Finnie, J.F., Van Staden, J., 2009. Antimicrobial activity and mutagenic effects of twelve traditional medicinal plants used to treat ailments related to the gastro-

Ac

effects of extracts from Sudanese medicinal plants. South African Journal of Botany 74,

ce p

te

worthy of scrutiny. Joint Bone Spine 71, 454-456.

Bertin, P., 2004. Should gastroprotective agents be given with COX-2 inhibitors? A question

M
13

an

us

cr
Page 13 of 29

ip t

325
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

intestinal

tract

in

South

Africa.

South

African

Journal

of

Botany

326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348

doi:10.1016/j.sajb.2008.11.002. Garcia, D., Leiro, J., Delgado, R., Sanmartin, M.L., Ubeira, F.M., 2003. Mangifera indica L. extract (Vimang) and mangiferin modulate mouse humoral immune responses. Phytotherapy Research 17, 1182-1187. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A., 1996. Zulu Medicinal Plants. An Inventory. University of Natal Press, Pietermaritzburg.

Iwalewa, E.O., McGaw, L.J., Naidoo, V., Eloff, J.N., 2007. Inflammation: the foundation of diseases and disorders. A review of phytomedicines of South African origin used to treat pain and inflammatory conditions. African Journal of Biotechnology 6, 2868-2885. Khafagi, I.K., Dewedar, A., 2000. The efficiency of random versus ethno-directed research in the evaluation of Sinai medicinal plants for bioactive compounds. Journal of Ethnopharmacology 71, 365-376.

Journal 135, 30-34.

Makkar, H.P.S., 1999. Quantification of Tannins in Tree Foliage: A laboratory manual for the FAO/IAEA co-ordinated research project on use of nuclear and related techniques to

feeding ruminants on the tanniferous tree foliage. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria. Margaret, R., 1990. Indigenous Healing Plants. Southern Book Publisher, South Africa. McGaw, L.J., Jger, A.K., Van Staden, J., 1997. Prostaglandin synthesis inhibitory activity in Zulu, Xhosa and Sotho medicinal plants. Phytotherapy Research 11, 113-117.

Ac

develop simple tannins assays for predicting and improving the safety and efficiency of

ce p

te

they safer than traditional nonsteroidal anti-inflammatory drugs? Canadian Pharmacists

MacAulay, S., Blackburn, D., 2002. Selective COX-2 inhibitors for patients with arthritis: are

M
14
Page 14 of 29

an

us

cr

ip t

349
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Naik, S.R., Sketh, U.K., 1976. Inflammatory process and screening methods for antiinflammatory agents- A review. Journal of Postgraduate Medicine 22, 5-21. Ndukwe, K.C., Lamikanra, A., Okeke, I.N., 2004. Antibacterial activity in plants used as chewing sticks in Africa. Drugs of the Future 29, 1221. Pasinetti, G.M., 2001. Cyclooxygenase and Alzheimers disease: implication for preventive initiatives to slow the progression of clinical dementia. Archives of Gerontology and Geriatrics 33, 13-28.

350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372

Pelzer, L.E., Guardia, T., Osvaldo Juarez, A., Guerreiro, E., 1998. Acute and chronic antiinflammatory effects of plant flavonoids. Farmaco 53, 421-424.

Polya, G.M., 2003. Biochemical targets of plant bioactive compounds. A pharmacological reference guide to sites of action and biological effects. CRC Press, Florida. Pooley, E., 1998. A Field Guide to Wild Flowers in KwaZulu-Natal and the Eastern Region. Natal Flora Publication Trust, Durban.

Robert, F.R., 1962. Simple test for alkaloid-containing plants. Economic Botany 16, 171-172. Sahu, N.P., Mahato, S.B., 1994. Anti-inflammatory triterpene saponins of Pithecellobium dulce: characterization of an echinocystic acid bisdesmoside. Phytochemistry 37, 1425-

Sharma, S., Stutzman, J.D., Kellof, G.J., Steele, V.E., 1994. Screening of potential chemopreventive agents using biochemical markers of carcinogenesis. Cancer Research 54, 5848-5855. Sleisenger, M.H., Fordtrand, J.S., 1993. Gastrointestinal Disease: Pathophysiology, Diagnosis, Management. Saunders, Philadelphia.

Ac

1427.

ce p

te

prodelphinidins to cyanidin and delphinidin. Phytochemistry 25, 223-230.

Porter, L.J., Hrstich, L.N., Chan, B.G., 1986. The conversion of procyanidins and

M
15
Page 15 of 29

an

us

cr

ip t

373
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Slish, D.F., Ueda, H., Arvigo, R., Balick, M.J., 1999. Ethnobotany in the search for vasoactive herbal medicines. Journal of Ethnopharmacology 66, 159-165. Sparg, S.G., Light, M.E., Van Staden, J., 2004. Biological activities and distribution of plant saponins. Journal of Ethnopharmacology 94, 219-243. Tadhani, M., Subhash, R., 2006. Preliminary studies on Stevia rebaudiana leaves: proximal composition, mineral analysis and phytochemical screening. Journal of Medical Science 6, 321-326.

374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

Talhouk, R.S., Karam, C., Fostok, S., El-Jouni, W., Barbour, E.K., 2007. Anti-inflammatory bioactivities in plant extracts. Journal of Medicinal Food 10, 1-10.

Warner, T.D., Mitchell, J.A., 2008. COX-2 selectivity alone does not define the cardiovascular risks associated with non-steroidal anti-inflammatory drugs. Lancet 371, 270273.

Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern

Xiao, J., Jiang, X., Chen, X., 2005. Antibacterial, anti-inflammatory and diuretic effect of flavonoids from Marchantia convoluta. African Journal of Traditional, Complementary and Alternative Medicines 2, 244-252.

bullata? A pharmacological investigation in terms of cycloxygenase-1 and -2 inhibition. Journal of Ethnopharmacology 71, 473-478.

Ac

Zschocke, S., Van Staden, J., 2000. Cryptocarya species - substitute plants for Ocotea

ce p

te

Wilfred, V., Ralph, N., 2006. Phenolic Compound Biochemistry. Springer, Netherlands.

and Eastern Africa, 2nd ed. Livingstone, London.

M
16

an

us

cr

ip t

Page 16 of 29

1 2 3 4 5 6 395 7 8 396 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Table 1 Medicinal plants used against gastrointestinal problems in South Africa. Family Agapanthaceae Asphodelaceae Asteraceae Species Agapanthus campanulatus Leighton Haworthia limifolia Marloth Vernonia natalensis Sch. Bip. ex Walp. Cucurbitaceae Cyperaceae Ebenaceae

Voucher number Traditional uses

an
FAW 3 NU FAW 6 NU FAW 2 NU FAW 9 NU FAW 10 NU FAW 7 NU FAW 12 NU 17

FAW 4 NU

Cucumis hirsutus Sond

ep te

Cyperus textiles Thunb.

Ac c

Diospyros lycioides Desf. Antidesma venosum E. Mey.ex Gladiolus dalenii van Geel

Euphorbiaceae Iridaceae

Tul.

us

cr
Root decoctions are taken orally or as enemas for stomach problems in children (Watt and Breyer- Brandwijk, 1962). Decoction made from the leaves is used for stomach trouble (Hutchings et al., 1996). Decoctions from leaves and stems are used for stomach cramps, nervous spasms of the stomach and other stomach ailments (Hutchings et al., 1996). Leaf and root decoctions are used for abdominal pain as well as diarrhoea (Hutchings et al., 1996). Root infusions are used as enemas for children with various stomach pains and cramps (Hutchings et al., 1996). Bark and root decoctions are taken for bloody faeces and dysentery (Hutchings et al., 1996). Leaf decoctions are used for abdominal cramps and dysentery. (Hutchings et al., 1996). Corm ground down to a fine meal and taken this mixed with warm water in small quantities to relieve dysentery, diarrhoea and stomach cramps (Margaret, 1990).

ip t

Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 397 25 26 398 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Watsonia tabularis Bak Lamiaceae Becium obovatum E. Mey. Ex Benth. Melastomataceae Proteaceae Dissotis princeps (Kunth) Triana Protea simplex E. Phillips

FAW 5 NU FAW 8 NU

an
FAW 1 NU 18

FAW 11 NU

d Ac c ep te

us

cr
1996).

Corms are used for diarrhoea in humans and calves (Hutchings et al., 1996). Warm water infusions of pounded roots and leaf are administered as enemas to treat children with stomach ailments as well as abdominal pain (Pooley, 1998).

Leaf infusions are administered as enemas for dysentery and diarrhoea (Hutchings et al., 1996). Decorticated root and bark infusions are used for dysentery, diarrhoea and stomach pains in humans (Hutchings et al.,

ip t

Page 18 of 29

1 2 3 4 5 6 399 7 8 400 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Table 2

Percentage inhibition Plant Plant species Agapanthus campanulatus Antidesma venosum Becium obovatum Cucumis hirsutus Cyperus textilis Diospyros lycioides Dissotis princeps Gladiolus dalenii Protea simplex L R L R L R L L S L L L B 92.6 1.1 97.7 1.4 103.0 0.8 78.5 4.0 91.7 2.1 part COX-1 PE DCM

us
Water

Inhibitory activity (COX-1 and COX-2) of different plant extracts evaluated at 250 g/ml

cr
COX-2 PE DCM EtOH Water 74.2 5.9 33.4 3.3 36.2 6.1 85.3 1.9 26.0 9.4 61.3 0.69 0.0 37.1 0.2 13.1 5.7 4.8 2.8 34.4 0.1 30.7 10.5 57.8 14.4 90.5 1.2 72.3 9.1 78.0 4.4 46.6 12.3 76.6 0.8 80.3 3.5 75.6 9.8 75.0 1.8 91.6 1.9 67.7 0.3 60.6 7.7 68.4 5.7 82.4 2.3 72.4 1.1 41.0 7.4 68.1 3.8 97.0 1.2 40.9 9.9 62.6 9.3 81.5 4.1 73.5 2.4 83.0 0.1 84.8 1.3 65.9 1.7 67.2 9.5 100.6 3.5 72.3 2.8 68.4 3.7 35.8 2.4 16.9 9.5 9.1 21.0 40.9 10.5 0.0 0.0 47.9 24.3 32.8 1.0 72.0 2.3 37.9 10.1 22.1 1.1 35.6 5.7 0.0 0.0 20.0 2.1

78.4 7.2

an
EtOH 12.8 0.8 48.1 9.3 84.3 7.0 4.1 3.0 29.1 5.0 81.4 8.9 79.8 8.9 90.4 4.3 70.6 1.2 87.1 4.0 53.1 4.4 1.3 4.1 23.7 6.1 89.2 7.8 19

ip t

47.5 3.7 28.8 4.0 0.0 1.2 17.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20.9 6.9 16.7 0.3

Ac c
C

Haworthia limifolia

ep te
91.7 5.0 86.3 0.7 92.8 0.9 73.8 3.6 58.8 1.2 88.3 1.5 88.3 3.8 100.1 0.8 94.2 3.7

98.4 1.0 72.8 4.3 86.4 2.6 101.8 1.1 88.5 4.9 88.4 0.5 94.0 5.7 81.0 2.1 82.7 2.3 101.8 0.3 83.5 1.9 80.6 8.7 86.1 7.1

Page 19 of 29

1 2 3 4 5 6 7 8 9 401 10 11 12 402 13 14 403 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Vernonia natalensis Watsonia tabularis

L C

88.5 3.2 97.6 5.3

77.5 3.4 73.9 5.1

51.2 12.4 38.7 3.3

cr

ip t
86.7 0.9 91.5 0.9 63.4 0.5 80.5 8.7 0.0 51.1 8.8

0.0 0.0

Percentage inhibition of prostaglandin synthesis by indomethacin was 70 3.3 for COX-1 and 68.9 2.5 for COX-2.

Ac c

ep te
20
Page 20 of 29

an

B- bark, C-corm, L-leaf, R-root, S-stem

us

90.3 3.4

50.3 1.8

404
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Table 3 Detection of alkaloids in twelve medicinal plants traditionally used for treating gastrointestinal ailments in South Africa. Plant name Plant part Extracts PE Agapanthus campanulatus Roots Antidesma venosum Becium obovatum Cucumis hirsutus Cyperus textiles Leaves Roots Leaves Roots Leaves Diospyros lycioides Leaves Stems Dissotis princeps Gladiolus dalenii Leaves Corms + + Leaves DCM EtOH

405 406

us
+ -

an
+ + -

M
21

d
+ + -

te

ce p

Haworthia limifolia Protea simplex

Leaves Leaves Bark Leaves Corms

Ac

Vernonia natalensis Watsonia tabularis - = absence + = presence

407 408

cr
+ + + + Page 21 of 29

ip t
+

409
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure Legends

410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433

Fig. 1. Dose-dependent COX-1 percentage inhibition by different plant extracts. (A) Petroleum ether extracts (B) Dichloromethane extracts, and (C) Ethanol extracts. Percentage inhibition of prostaglandin synthesis by Indomethacin was 70 3.3. A.c = Agapanthus campanulatus; B.o = Becium obovatum; C.h = Cucumis hirsutus; C.t = Cyperus textilis; D.l = Diospyros lycioides; D.p = Dissotis princeps; G.d = Gladiolus dalenii; H.l = Haworthia limifolia; P.s = Protea simplex; V.n = Vernonia natalensis; W.t = Watsonia tabularis.

Fig. 2. Dose-dependent COX-2 percentage inhibition by different plant extracts. (A) Petroleum ether extracts (B) Dichloromethane extracts, and (C) Ethanol extracts. Percentage inhibition of prostaglandin synthesis by Indomethacin was 68.9 2.5.

H.l = Haworthia limifolia; P.s = Protea simplex; V.n = Vernonia natalensis; W.t = Watsonia tabularis.

traditionally used for treating gastro-intestinal ailments. 1-Haworthia limifolia (leaf), 2-Cucumis hirsutus (leaf), 3-Becium obovatum (root), 4-Protea simplex (leaf), 5-Protea simplex (bark), 6-Agapanthus campanulatus (root), 7-Cyperus textilis (root), 8-Cyperus textilis (leaf), 9-Vernonia natalensis (leaf), 10-Watsonia tabularis (corm), 11-Antidesma venosum (leaf), 12- Diospyros lycioides (leaf), 13-Diospyros lycioides (stem), 14-Dissotis princeps (leaf), 15-Gladiolus dalenii (corm).

Ac

Fig. 3. Total phenolic compounds per dry matter (DM) of twelve medicinal plants

ce p

te

Cyperus textilis; D.l = Diospyros lycioides; D.p = Dissotis princeps; G.d = Gladiolus dalenii;

A.c = Agapanthus campanulatus; B.o = Becium obovatum; C.h = Cucumis hirsutus; C.t =

M
22

an

us
Page 22 of 29

cr

ip t

434
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459

Fig. 4. Percentage condensed tannins as leucocyanidins equivalents of twelve medicinal plants traditionally used for treating gastro-intestinal ailments. 1-Haworthia limifolia (leaf), 2-Cucumis hirsutus (leaf), 3-Becium obovatum (root), 4-Protea simplex (leaf), 5-Protea simplex (bark), 6-Agapanthus campanulatus (root), 7-Cyperus textilis (root), 8-Cyperus textilis (leaf), 9-Vernonia natalensis (leaf), 10-Watsonia tabularis (corm), 11-Antidesma venosum (leaf), 12- Diospyros lycioides (leaf), 13-Diospyros lycioides (stem), 14-Dissotis princeps (leaf), 15-Gladiolus dalenii (corm).

Fig. 5. Gallotannin concentrations per dry matter (DM) of twelve medicinal plants traditionally used for treating gastro-intestinal ailments.

1-Haworthia limifolia (leaf), 2-Cucumis hirsutus (leaf), 3-Becium obovatum (root), 4-Protea simplex (leaf), 5-Protea simplex (bark), 6-Agapanthus campanulatus (root), 7-Cyperus textilis

14-Dissotis princeps (leaf), 15-Gladiolus dalenii (corm).

Fig. 6. Flavonoid concentration as catechin equivalent of twelve medicinal plants traditionally

1-Haworthia limifolia (leaf), 2-Cucumis hirsutus (leaf), 3-Becium obovatum (root), 4-Protea simplex (leaf), 5-Protea simplex (bark), 6-Agapanthus campanulatus (root), 7-Cyperus textilis (root), 8-Cyperus textilis (leaf), 9-Vernonia natalensis (leaf), 10-Watsonia tabularis (corm), 11-Antidesma venosum (leaf), 12- Diospyros lycioides (leaf), 13-Diospyros lycioides (stem), 14-Dissotis princeps (leaf), 15-Gladiolus dalenii (corm).

Ac

used for treating gastro-intestinal ailments.

ce p

te

11-Antidesma venosum (leaf), 12- Diospyros lycioides (leaf), 13-Diospyros lycioides (stem),

(root), 8-Cyperus textilis (leaf), 9-Vernonia natalensis (leaf), 10-Watsonia tabularis (corm),

M
23

an

us

cr
Page 23 of 29

ip t

460
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

120

A
100

Leaf

Root

Corm

Stem

Percentage Inhibition

62.5 g/ml 125 g/ml 250 g/ml

80

60

40

20

120

B
100

Leaf

Root

Corm

Percentage inhibition

80

60

40

20

0 100

te

A.c C.h C.t

D.l D.p H.l Leaf

P.s V.n A.c B.o C.t G.d W.t Corm

M
D.l

C
80

Percentage inhibition

60

40

20

Ac

ce p

D.l

W.t

461 462 463 464

Plant species

Figure 1

24
Page 24 of 29

an

us
Stem

A.c C.h C.t

D.l D.p H.l

P.s V.n A.c B.o C.t G.d W.t

D.l

cr

ip t

465
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

100

Leaf

Root

Corm

Stem 62.5 g/ml 125 g/ml 250 g/ml

80

Percentage inhibition

60

40

0 120

B
100

Leaf

Root

Corm

Percentage inhibition

80

60

40

20

0 80

M
C
Leaf

te

A.c C.h C.t D.l

D.p H.l

P.s V.n A.c B.o C.t G.d W.t

Corm

Percentage inhibition

60

40

20

Ac

ce p

D.l

466 467 468 469

Plant species

Figure 2

25
Page 25 of 29

an
D.l W.t

us
Stem

A.c C.h C.t D.l

D.p H.l

P.s V.n A.c B.o C.t G.d W.t

D.l

cr

20

ip t

470
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Total phenolic concentration (mg GAE/g DM)

100 75 50 25 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sample
471 472 473 474 475 476 477 478 479 480 481 482 483 484 485

Ac

ce p

te
26
Page 26 of 29

an

Figure 3

us

cr

ip t

486
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sample
487 488 489 490 491 492 493 494 495 496 497 498 499 500 501

Ac

ce p

te
27
Page 27 of 29

an

Figure 4

us

cr

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

Condensed tannins (% per dry matter)

ip t

502
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sample
503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520

Figure 5

Ac

ce p

te
28
Page 28 of 29

an

us

cr

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Gallotannin concentration g GAE/g DM

ip t

521
1 2 522 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 523 24 25 524 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Flavonoid concentration (mg Catechin equivalent/g DM)

8 7 6 4 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 5

Sample
Figure 6

Ac

ce p

te
29
Page 29 of 29

an

us

cr

ip t