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Proanthocyanidin diversity in the EU ‘HealthyHay’ sainfoin (Onobrychis viciifolia) germplasm collection
Phytochemistry 77 (2012) 197–208
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Proanthocyanidin diversity in the EU ‘HealthyHay’ sainfoin (Onobrychis viciifolia) germplasm collection Elisabetta Stringano a,⇑, Christine Hayot Carbonero b,1, Lydia M.J. Smith b, Ronald H. Brown a, Irene Mueller-Harvey a a Chemistry and Biochemistry Laboratory, Food Production and Quality Division, School of Agriculture, Policy and Development, University of Reading, P.O. Box 236, 1 Earley Gate, Reading RG6 6AT, UK b National Institute for Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
a r t i c l e
i n f o
Article history: Received 16 November 2011 Received in revised form 3 January 2012 Available online 6 February 2012 Keywords: Sainfoin Tannins Proanthocyanidins Thiolytic degradation Germplasm screening
a b s t r a c t This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80 g/100 g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P < 0.001; R2 = 0.49). A negative correlation existed between PA content (thiolysis) and mDP (P < 0.05; R2 = 0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Plant breeding is facing a paradigm shift and needs to develop new cultivars that can deliver lower environmental impact from agricultural production (Pilippot and Hallin, 2011). Recent evaluations of rice germplasm have already identified 500% differences in methane emissions, whilst a survey of wheat and other germplasm material discovered an enormous potential for plants with much higher biological nitrification inhibition activity. Sainfoin (Onobrychis viciifolia) is receiving renewed interest because of its many positive attributes (Hayot Carbonero et al., 2011; Website). Consumption of sainfoin shows promise for reducing environmental emissions of nitrogen and methane from ruminant animals (Hatew et al., 2011; Theodoridou et al., 2010) and unlike many
Abbreviations: BM, benzyl mercaptan; C, catechin; Cis, cis-flavanols (epicatechin or epigallocatechin); EC, epicatechin; EGC, epigallocatechin; G, gallocatechin; HPLC, high performance liquid chromatography; mDP, mean degree of polymerisation; PA, proanthocyanidin; PC, procyanidins; PD, prodelphinidins; trans, trans-flavanols (catechin or gallocatechin). ⇑ Corresponding author. Present address: CNR (National Research Council), Istituto di Ricerca Sulle Acque (Water Research Institute), 70132 Bari, Italy. Tel.: +44 (0)118 378 6619; fax: +44 (0)118 935 2421. E-mail address: [email protected] (E. Stringano). 1 Present address: University of Illinois at Urbana-Champaign, 1102 South Good win Avenue, Turner Hall, Urbana, IL 61801, USA. 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2012.01.013
other forage legumes, sainfoin generates high voluntary intakes without causing bloat (Mueller-Harvey, 2009). In addition, its flowers are a valuable source of pollen and nectar for bees (Clement et al., 2006; Kells, 2001; McGregor, 1976) and could thus contribute to alleviating problems associated with pollinator decline. Sainfoin has also been shown to control parasitic worms, which are posing a serious threat to animal welfare and farming (Hoste et al., 2006; Novobilsky´ et al., 2011). Content and composition of tannins, also called proanthocyanidins (PAs), are heritable traits that are amenable to plant breeding (Orians et al., 2000; Scioneaux et al., 2011). PAs are also the active ingredients in sainfoin and their variation is, therefore, worth exploring amongst currently available accessions. Although several studies have reported better absorption of nitrogen and essential amino acids by ruminants from tannin-containing sainfoin than from iso-nitrogenous but tannin-free forages, contradictory findings also exist (Hayot Carbonero et al., 2011). Here, we set out to investigate whether the contradictory reports could be due to variation in tannins among different sainfoin lines as indicated previously (Marais et al., 2000). It was for these reasons that the EU ‘HealthyHay’ project established a 300+ sainfoin germplasm collection (Website), which was evaluated for nutritional, anti-parasitic, chemical, biochemical, genetic and agronomic characteristics. The present study focussed on variation in PA content and composition within a subset of 37 diverse sainfoin accessions.
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E. Stringano et al. / Phytochemistry 77 (2012) 197–208
Fig. 1. General structure of proanthocyanidins (R@OH: prodelphinidins; R@H: procyanidins).
Fig. 2. General structure of anthocyanidins (R@OH or H).
Many analytical methods have been used to quantify and characterise PAs in plants (Fig. 1). The widely used HCl–butanol method is an acid-catalysed oxidative depolymerisation of the PA interflavanyl linkages and yields anthocyanidins (Fig. 2), which can be determined spectrophotometrically (Porter et al., 1985). However, PA measurements based on anthocyanidin yields have several limitations: (a) water in the reaction medium can suppress the anthocyanidin yield (Waterman, 1994); (b) the number of hydroxy groups in the A and B rings affect the wavelength of the absorbance maximum of the anthocyanidin products (Fig. 2) (Hemingway, 1989); (c) colour yield is not always linearly correlated with tannin concentration (Waterman, 1994); (d) the ratio of reagent to sample, the temperature and the length of the reaction time affect the colour yield (Scalbert, 1992; Waterman, 1994); (e) the lack of appropriate standards remains a problem for quantifying complex mixtures of PAs (Giner-Chavez et al., 1997; Hagerman and Butler, 1994); and (f) the acid–butanol assay is generally used to quantify soluble PAs, however, some PAs are insoluble in common solvents and this can lead to an underestimation (Makkar et al., 1999; Reed, 1986). Although the HCl–butanol reaction has also been applied directly to plant samples (Kayani et al., 2007; Makkar et al., 1999; Yu and Dahlgren, 2000) the above limitations still apply and, in addition, the presence of chlorophyll interferes with anthocyanidin measurements (Watterson and Butler, 1983). Taken together these limitations mean that the HCl–butanol method is not suitable for quantitative PA analysis in extracts or whole plants. Therefore, Gea et al. (2011) recently developed a thiolysis method for the in situ analysis of complex PA mixtures in sainfoin ( O. viciifolia). This method has now been used to screen
Table 1 Samples collected from the EU ‘HealthyHay’ sainfoin germplasm collection (na = not available). Accession number
Species
1005 1007 1012r1a 1012r2a 1013 1017r1 1017r2 1019 1026r1 1026r2 1028 1041 1043 1071 1077 1103 1104 1110 1113 1123 1127 1156 1157r1 1157r2 1163 1165r1 1165r2 1169 1179 1197 1199 1200 1210 1213
Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis
viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia
Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop.
Variety
Country of origin
Status
Perly na Ambra Ambra Somborne Teruel Teruel Taja Buciansky Buciansky Simpro Camaras Bivolari Hampshire Common Nova Korunga na CPI 63750 CPI 63753 CPI 63763 CPI 63767 Dukorastushchii Miatiletka Miatiletka Giant Rees ‘‘A’’ Rees ‘‘A’’ CPI 63810 CPI 63820 CPI 63838 CPI 63840 CPI 63841 Premier CPI 63854
Switzerland China Italy Italy UK Spain Spain Poland Slovakia Slovakia France Romania Romania UK Canada Turkey Turkey Turkey Spain Turkey USA, Washington Former Soviet Union Former Soviet Union Former Soviet Union UK UK UK Lithuania Spain Norway Former Soviet Union Germany Switzerland Switzerland
Cultivar na Cultivar Cultivar Cultivated Cultivated Cultivated Cultivar Cultivar Cultivar Cultivar na na Cultivated Cultivar na na na na Wild Cultivated Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar na na na na na Cultivar Cultivated
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E. Stringano et al. / Phytochemistry 77 (2012) 197–208 Table 1 (continued)
a
Accession number
Species
1220 1230 1253r1 1253r2 1256 1260 1261r1 1261r2 1262r1 1262r2 1264r1 1264r2
Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis
viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. antasiatica antasiatica
Variety
Country of origin
Status
247 Visnovsky Tu86-43-03 Tu86-43-04 Wkt 10 X93234 Line 107 Line 108 Cotswold Common Cotswold Common Sisiani Local Sisiani Local
Morocco Czech Republic Turkey, Hakkari Turkey, Hakkari Turkey, Afyon China, Xinjiang Armenia Armenia UK UK Armenia Armenia
na Cultivar Cultivated Cultivated Wild Wild Cultivated Cultivated Cultivated Cultivated Cultivated Cultivated
r1 and r2 refer to two replicate samples that were collected from the experimental site.
Table 2 Proanthocyanidin (PA) monomer composition and content in sainfoin accessions (g/100 g freeze-dried plant; SD in brackets); C = catechin, EC = epicatechin, GC = gallocatechin, EGC = epigallocatechin. Accession number
C
EC
GC
EGC
Starter units 1005 1007 1012r1
a
1012r2
a
1013 1017 r1 1017 r2 1019 1026r1 1026r2 1028 1041 1043 1071 1077 1103 1104 1110 1113 1123 1127 1156 1157r1 1157r2 1163 1165r1
0.020 (0.0024) 0.013 (0.0016) 0.024 (0.0054) 0.007 (0.0019) 0.021 (0.0036) 0.013 (0.0027) 0.017 (0.0023) 0.024 (0.0037) 0.014 (0.0047) 0.020 (0.0063) 0.012 (0.0007) 0.017 (0.0019) 0.020 (0.0024) 0.028 (0.0005) 0.006 (0.0008) 0.017 (0.0011) 0.008 (0.0014) 0.009 (0.0020) 0.010 (0.0013) 0.012 (0.0010) 0.007 (0.0017) 0.015 (0.0017) 0.013 (0.0024) 0.012 (0.0010) 0.021 (0.0032) 0.035 (0.0028)
C
EC
GC
EGC
Total PA content
0.290 (0.0438) 0.167 (0.0232) 0.286 (0.0414) 0.139 (0.0028) 0.349 (0.0591) 0.194 (0.0181) 0.312 (0.0202) 0.257 (0.0325) 0.161 (0.0436) 0.214 (0.0059) 0.173 (0.0203) 0.205 (0.0189) 0.165 (0.0469) 0.260 (0.0238) 0.128 (0.0016) 0.152 (0.0035) 0.108 (0.0265) 0.112 (0.0325) 0.138 (0.0115) 0.123 (0.0046) 0.113 (0.0154) 0.129 (0.0171) 0.164 (0.0172) 0.167 (0.0031) 0.354 (0.0316) 0.415 (0.0391)
0.230 (0.1038) 0.209 (0.0257) 0.166 (0.0466) 0.179 (0.0367) 0.328 (0.0612) 0.163 (0.0114) 0.167 (0.0153) 0.249 (0.0291) 0.187 (0.0474) 0.283 (0.0762) 0.132 (0.0183) 0.141 (0.0330) 0.208 (0.0117) 0.131 (0.0104) 0.067 (0.0056) 0.136 (0.0422) 0.179 (0.0144) 0.197 (0.0321) 0.175 (0.0172) 0.203 (0.0140) 0.229 (0.0460) 0.221 (0.0431) 0.201 (0.0297) 0.271 (0.0162) 0.206 (0.0596) 0.240 (0.0480)
0.630 (0.0926) 0.707 (0.0673) 0.884 (0.1200) 0.499 (0.0115) 0.745 (0.1503) 0.484 (0.0914) 0.740 (0.0537) 0.851 (0.0621) 0.499 (0.1183) 0.781 (0.0324) 0.434 (0.0577) 0.771 (0.0748) 0.840 (0.2198) 0.542 (0.0147) 0.495 (0.0855) 0.554 (0.0201) 0.373 (0.0757) 0.414 (0.0868) 0.473 (0.0350) 0.487 (0.0133) 0.517 (0.1068) 0.427 (0.1091) 0.533 (0.0623) 0.650) (0.0174) 0.582 (0.0752) 0.763 (0.1339)
1.250 (0.2689) 1.140 (0.1145) 1.422 (0.2222) 0.870 (0.0513) 1.529 (0.2845) 0.906 (0.1170) 1.292 (0.0896) 1.459 (0.1088) 0.933 (0.2231) 1.362 (0.1158) 0.786 (0.0861) 1.159 (0.1209) 1.290 (0.2756) 1.053 (0.0257) 0.711 (0.0837) 0.905 (0.0591) 0.700 (0.0963) 0.781 (0.1569) 0.824 (0.0731) 0.883 (0.0030) 0.899 (0.1755) 0.851 (0.1815) 0.962 (0.0998) 1.156 (0.0363) 1.239 (0.1832) 1.564 (0.2164)
Extender units 0.010 (0.0031) 0.003 (0.0002) 0.005 (0.0014) 0.009 (0.0021) 0.017 (0.0042) 0.011 (0.0012) 0.012 (0.0033) 0.008 (0.0023) 0.007 (0.0012) 0.003 (0.0032) 0.012 (0.0027) 0.009 (0.0060) 0.003 (0.0006) 0.021 (0.0014) 0.004 (0.0006) 0.010 (0.0003) 0.007 (0.0013) 0.011 (0.0008)
0.020 (0.0077) 0.016 (0.0019) 0.023 (0.0022) 0.012 (0.0011) 0.014 (0.0038) 0.009 (0.0032) 0.015 (0.0034) 0.036 (0.0016) 0.019 (0.0010) 0.027 (0.0045)
0.009 (0.0016) 0.004 (0.0012) 0.007 (0.0026) 0.007 (0.0021) 0.009 (0.0008) 0.007 (0.0016) 0.018 (0.0010)
0.022 (0.0026)
0.004 (0.0076) 0.037 (0.0045) 0.026 (0.0019)
0.023 (0.0014)
0.017 (0.0042)
0.022 (0.0031) 0.017 (0.0016) 0.014 (0.0017) 0.017 (0.0037) 0.027 (0.0069)
*
0.014 (0.0124)
0.050 (0.0178) 0.024 (0.0015) 0.034 (0.0052) 0.025 (0.0049) 0.055 (0.0079) 0.032 (0.0019) 0.028 (0.0030) 0.034 (0.0023) 0.046 (0.0083) 0.034 (0.0146) 0.023 (0.0039) 0.011 (0.0092) 0.017 (0.0053) 0.031 (0.0138) 0.012 (0.0006) 0.013 (0.0001) 0.026 (0.0019) 0.020 (0.0023) 0.029 (0.0115) 0.027 (0.0023) 0.030 (0.0064) 0.030 (0.0058) 0.028 (0.0062) 0.033 (0.0025) 0.053 (0.0137) 0.064 (0.0064)
(continued on next page)
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E. Stringano et al. / Phytochemistry 77 (2012) 197–208
Table 2 (continued) Accession number
C
EC
GC
EGC
Starter units 1165r2 1169 1179 1197 1199 1200 1210 1213 1220 1230 1253r1 1253r2 1256 1260 1261r1 1261r2 1262r1 1262r2 1264r1 1264r2
EC
GC
EGC
Total PA content
0.025 (0.0036) 0.008 (0.0012) 0.005 (0.0010) 0.006 (0.0025) 0.007 (0.0005) 0.003 (0.0003) 0.010 (0.0047) 0.007 (0.0013) 0.001 (0.0022) 0.007 (0.0012) 0.005 (0.0017) 0.005 (0.0005) 0.003 (0.0008) 0.003 (0.0005) 0.007 (0.0007) 0.005 (0.0006) 0.013 (0.0029) 0.015 (0.0007) 0.005 (0.0007) 0.003 (0.0013)
0.025 (0.0084) 0.035 (0.0031) 0.003 (0.0048) 0.012 (0.0031) 0.013 (0.0005) 0.020 (0.0060) 0.018 (0.0018) 0.031 (0.0007) 0.014 (0.0024) 0.043 (0.0014) 0.050 (0.0038) 0.049 (0.0023) 0.122 (0.0104) 0.017 (0.0015)
0.007 (0.0121)
0.018 (0.0035)
0.003 (0.0059)
0.011 (0.0104)
0.046 (0.0024) 0.041 (0.0074) 0.035 (0.0057) 0.017 (0.0037) 0.021 (0.0072) 0.021 (0.0017) 0.033 (0.0097) 0.030 (0.0066) 0.014 (0.0027) 0.021 (0.0073) 0.016 (0.0007) 0.015 (0.0019) 0.022 (0.0102) 0.016 (0.0025) 0.019 (0.0037) 0.024 (0.0009) 0.053 (0.0150) 0.080 (0.0045) 0.025 (0.0038) 0.014 (0.0049)
0.542 (0.0310) 0.369 (0.0108) 0.426 (0.0648) 0.144 (0.0240) 0.157 (0.0083) 0.195 (0.0183) 0.254 (0.0355) 0.281 (0.0168) 0.083 (0.0109) 0.158 (0.0211) 0.117 (0.0114) 0.108 (0.0094) 0.104 (0.0124) 0.100 (0.0048) 0.163 (0.0088) 0.143 (0.0011) 0.296 (0.0692) 0.399 (0.0264) 0.174 (0.0122) 0.086 (0.0323)
0.293 (0.1308) 0.277 (0.0372) 0.092 (0.0083) 0.177 (0.0552) 0.131 (0.0165) 0.103 (0.0141) 0.176 (0.0789) 0.242 (0.0131) 0.137 (0.0276) 0.184 (0.0459) 0.146 (0.0065) 0.141 (0.0141) 0.725 (0.0308) 0.168 (0.0289) 0.151 (0.0139) 0.131 (0.0307) 0.157 (0.0342) 0.209 (0.0651) 0.178 (0.0455) 0.101 (0.0226)
1.032 (0.0651) 1.256 (0.0414) 0.482 (0.0591) 0.537 (0.0896) 0.552 (0.0500) 0.537 (0.0639) 0.655 (0.1108) 1.117 (0.0539) 0.382 (0.0490) 0.637 (0.0938) 0.655 (0.0116) 0.594 (0.0408) 1.791 (0.0411) 0.484 (0.0181) 0.616 (0.0415) 0.398 (0.0024) 0.643 (0.1539) 0.837 (0.0840) 0.601 (0.0357) 0.356 (0.1233)
2.006 (0.2165) 2.011 (0.0967) 1.096 (0.1393) 0.901 (0.1339) 0.893 (0.0778) 0.906 (0.1048) 1.166 (0.2200) 1.735 (0.0778) 0.641 (0.0864) 1.068 (0.1585) 0.998 (0.0237) 0.921 (0.0566) 2.802 (0.0606) 0.798 (0.0498) 0.971 (0.0509) 0.712 (0.0271) 1.196 (0.2777) 1.566 (0.1625) 0.996 (0.0934) 0.567 (0.1846)
Empty cells signify that flavanol subunit was not detected. r1 and r2 refer to two replicate samples that were collected from the experimental site.
a
3.0
Proanthocyanidin content
2.5
2.0
1.5
1.0
0.5
0.0
1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
PAs %
*
0.036 (0.0031) 0.026 (0.0034) 0.053 (0.0080) 0.010 (0.0010) 0.013 (0.0013) 0.026 (0.0023) 0.019 (0.0039) 0.026 (0.0040) 0.011 (0.0014) 0.018 (0.0018) 0.009 (0.0012) 0.009 (0.0007) 0.016 (0.0010) 0.010 (0.0003) 0.012 (0.0022) 0.011 (0.0001) 0.022 (0.0047) 0.026 (0.0011) 0.012 (0.0012) 0.007 (0.0024)
C Extender units
Fig. 3. Proanthocyanidin contents of sainfoin accessions (g/100 g freeze-dried plant).
1
0
1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
trans %
0
50 1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
mDP 90
1
100
1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
PD %
E. Stringano et al. / Phytochemistry 77 (2012) 197–208
Fig. 6. Proportion of trans-flavan-3-ols in sainfoin accessions.
201
Mean degree of polymerisation
80 70
60
50
40
30
20
10
0
Fig. 4. Mean degree of polymerisation of proanthocyanidins in sainfoin accessions.
Prodelphinidins
95
90
80 85
75
65 70
60
55
Fig. 5. Proportion of prodelphinidins in sainfoin accessions.
35
trans- flavan-3-ols
30
25
20
15
10
5
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Table 3 Pairwise correlations between PA content, mean degree of polymerisation (mDP), % prodelphinidins (PD), % trans flavan-3-ols and absorbance at 550 nm (HCl–butanol method; A550) in sainfoin accessions from the EU ‘HealthyHay’ germplasm collection. PA content mDP PD trans A550 (HCl–butanol) * ** ***
mDP
PD
trans
to explore any linkages between PA composition, sainfoin accessions and geographical origin. 2. Results and discussion
*
0.30 0.02 0.07 0.49***
0.14 0.08 0.21
2.1. Proanthocyanidin analysis 0.39** 0.34*
0.27
p < 0.05. p < 0.01. p < 0.001.
representative samples from the EU ‘HealthyHay’ sainfoin germplasm collection at NIAB (Cambridge, UK) in order to: to characterise the extent of PA diversity in this sainfoin collection to investigate correlations amongst PA structural traits within this collection to identify chemovars with contrasting PA composition for subsequent investigations into their biological effects and for supporting a future sainfoin breeding programme, and
Table 1 lists the O. viciifolia accessions that were tested. These sainfoin lines were selected from the 300+ accessions in the EU ‘HealthyHay’ collection as the most diverse accessions based on morphology, vigour, disease resistance and also flower colour (Hayot Carbonero, 2011), as the latter suggested differences in flavonoid metabolism. PAs in sainfoin samples were analysed directly by thiolytic degradation (Gea et al., 2011). This yielded information on PA content and composition (Tables 2 and S1). PA contents showed 4.9-fold differences, which ranged from 0.57 to 2.80 g PA per 100 g of freeze-dried sainfoin (NIAB accession number 1264 vs. NIAB 1256; Fig. 3). This variation is much larger than previously reported (Kelman et al., 1997) for a Lotus pedunculatus Cav. or L. corniculatus L. collection, where PAs ranged from 2.96 to 8.73 and from 1.56 to 4.22 g/100 g, respectively. A recent survey (Gruber et al., 2008) found 4-fold differences in soluble leaf PA among 14% of Lotus accessions, but 86% of accessions had no extractable leaf PA. Considerable variation has also been described for Lathyrus
Representative Countries Height Spain, Turkey
China, Slovakia, Former Soviet Union (Eastern Europe, Asia) Germany, UK (Western Europe) Slovakia, Former Soviet Union, Turkey, Czech Rep, Romania, Poland (Eastern Europe)
Cluster tree based on PA content, mDP, % PD and % trans-flavan-3-ols
Switzerland, UK, France, Spain, Italy (Western Europe)
Canada, USA, Armenia (Rest of World)
Fig. 7. Clustering of sainfoin accessions based on all thiolysis parameters (proanthocyanidin content, mean degree of polymerisation, % prodelphinidins and % trans-flavanol units).
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Representative Countries Height
Armenia, Turkey, Canada
Cluster tree based on PA content (thiolysis)
Turkey, Armenia, China, Former Soviet Union (Asia)
Spain, Switzerland, UK, Romania, Czech Rep, Slovakia, Poland, Italy (West & East Europe)
Fig. 8. Clustering of sainfoin accessions based on proanthocyanidin content (a) thiolysis method (g PA/100 g plant) and (b) HCl–butanol method (A550
sativus L. genotypes, where PA contents ranged from 0.0 to 4.38 g/ kg (Deshpande and Campbell, 1992). Of particular note was the large variation in the mean degree of polymerisation (mDP). This varied 7-fold from 12 to 84 (NIAB 1071 vs. NIAB 1127; Fig. 4), which corresponds to average polymer sizes of ca. 3600–25200 Da. Such high mDP values agree with two previous reports (Bate-Smith, 1973; Jones et al., 1976) and indicate that the currently available sainfoin germplasm does indeed contain extremely polydisperse PAs (Foo et al., 1982). Whilst the thiolysis method used here (Gea et al., 2011) measured both extractable and non-extractable PAs, leaves from three Lotus accessions showed only a 3-fold variation in the largest, extractable PA fraction (1260– 3802 Da) (Gruber et al., 2008). Considerable differences were also observed amongst the proportion of prodelphinidins (PD), which varied 1.8-fold from 53 to 95 (NIAB 1179, vs. NIAB 1256; Fig. 5) and the proportion of trans-flavanol units varied 2.8-fold from 12 to 34 (NIAB 1077 vs. NIAB 1156; Fig. 6). These results are interesting because PA content and composition were shown to be inherited in F1 hybrids of Salix and Populus (Orians et al., 2000; Scioneaux et al., 2011) and therefore this variation offers opportunities for optimising the anti-parasitic, nutritional and environmental effects of sainfoin (Hatew et al., 2011; Manolaraki et al., 2011). 2.2. Correlations amongst sainfoin PA traits PA contents were determined by the thiolysis and HCl–butanol assays and were positively correlated (P < 0.001), but this
nm).
correlation was quite low (R2 = 0.49) (Table 3). This suggests that the two assays do not reflect the same PA properties. We also noticed that across the accessions, PA contents (HCl–butanol) were negatively correlated to % PD (P < 0.05), although the biochemical significance of this observation is unclear. Within this germplasm collection, several other low, but significant, correlations could be seen among the tannin traits. A negative correlation existed between PA content and mDP (P < 0.05). This suggests that accessions with high PA contents do not necessarily have large PA polymers. Instead, lower tannin contents are more likely to reflect higher mDP values. The positive correlation (P < 0.01) between % trans-flavan-3-ols (i.e. catechin or gallocatechin units) and % PD (i.e. gallocatechin or epigallocatechin units) may simply reflect the presence of gallocatechin in both parameters. Alternatively, it may indicate that accessions differ especially in the biosynthesis of gallocatechin subunits, as epigallocatechin accounts for most of the PA subunits and therefore the PD percentage (Table 2). 2.3. Cluster analysis of sainfoin accessions based on PA composition The ‘‘complete linkage’’ clustering method, which is also called the farthest neighbour method, defines the distance between two clusters to be the maximum distance between their individual components. In other words, the distance between two clusters is given by the value of the longest link between the clusters, which is shown as ‘height’ on the y-axis. Figs. 7, 8b, 9 and 11 depict
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Representative Countries Height
Switzerland, Spain, UK, Italy, Germany (Western Europe)
Cluster tree based on PA content (HCl-butanol method)
Armenia, Former Soviet Union, Turkey, Romania, Slovakia (Asia, Eastern Europe)
UK, Italy, France, Switzerland (Western Europe)
Former Soviet Union, Turkey, China (Asia)
Fig. 8 (continued)
clusters, which contain several groups that differ in height. Thus, the resulting dendrogrammes consist of many U-shaped brackets, which connect these sub-groups via a hierarchical tree. The height of each U represents the distance between two sub-groups. However in Figs. 8a and 10 there are hardly any height differences. Only accessions 1256, 1169, 1165r2 and 1213 show distinct height differences compared to the other accessions, which had much lower and relatively similar PA contents (Fig. 8a). The dendrogramme in Fig. 7 shows the cluster tree that was derived from all thiolysis parameters, i.e. PA content, mDP, percentage of PD and trans-flavan-3-ols. Two Western European, two Eastern European and one Rest-of-World cluster can be seen. Figs. 8–11 show the cluster trees derived from individual thiolysis parameters. It is worth noting that clustering based just on PD contents reveals some similarities to the overall data clustering (Fig. 10 vs. Fig. 7): the two accessions from Spain and Turkey (1179 and 1256) clustered outside the two main groups, but accessions from Asia and Eastern Europe (China, Former Soviet Union, Armenia, Turkey, Romania, Slovakia, Czech Republic, Poland and Lithuania) clustered together, as did accessions from Western Europe (Spain, France, Germany, Switzerland and Italy).
The cluster tree based on PA contents (thiolysis method) showed that accessions from Asia (Turkey, Armenia, China, Former Soviet Union) had a tendency to group together (Fig. 8a). Interestingly, the PA content as measured by the HCl–butanol method generated more regional clusters than the thiolysis method: two Western European, one Asian/Eastern European and one Asian cluster (Fig. 8b). These observations coincide with reports that PA contents in Lotus were also affected by country of origin (Gruber et al., 2008; Kelman et al., 1997). Using the average PA polymer size or mDP-values (Fig. 9), several accession clusters could be seen: (i) ‘Eastern’ countries tended to group together (China, Turkey, Czech Republic and Romania, Poland and Turkey); (ii) so did Canada, USA and Armenia and (iii) Western Europe (UK, Germany, Spain, Switzerland and Italy). In contrast, a lineage map based on PD percentages (Fig. 10) generated one UK cluster, two Turkish sub-groups, one Eastern European/Asian cluster (China, Slovakia, Former Soviet Union, Czech Republic, Armenia, Turkey, Lithuania, Poland and Romania) and one Western European cluster (Spain, France, Germany, Switzerland, Italy). Clustering based on the percentage of transflavan-3-ols (Fig. 11) also revealed Turkish and UK clusters, a Southern European cluster (Spain and Italy), a Western European
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Representative Countries Height Canada, USA, Armenia
China, Turkey, Czech Republic
Cluster tree based on mDP
Romania, Poland, Turkey
UK, Germany, Spain, Switzerland, Italy (Western Europe)
Turkey, Armenia
Fig. 9. Clustering of sainfoin accessions based on the mean degree of polymerisation.
cluster (UK, Norway, Spain, Switzerland) and an a cluster from Armenian, Romanian and Turkish accessions. This type of linkage information can be useful to identify relationships between PA composition and accession origin. To summarise, most accessions clustered into two main groups in terms of PA composition: Western Europe (UK, France, Switzerland, Spain, Italy) and Eastern Europe/Asia (China, Former Soviet Union, Turkey, Slovakia, Romania, Poland). However, several accessions from Armenia, Canada and USA tended to cluster into separate groups (Fig. 7). 2.4. Relationship between proanthocyanidins and phylogenetic characteristics of sainfoin The above clusters coincided with the strong links that were observed between geographical origin and agronomic performance within the ‘HealthyHay’ germplasm (Hayot Carbonero, 2011; Website): (i) morphological traits differed between accessions from Western Europe and from the rest of the world; (ii) the best agronomical accessions originated mainly from Eastern Europe and (iii) accessions from Western Europe and from the rest of the world could be classified as two different genetic subspecies (Hayot Carbonero et al., in press). There was a clear division among the morphological traits (more than 50% differences) between Western European accessions, which had more leaflets (25 vs. 21 per leaf) and thick stems
(29 vs. 21), and Eastern European accessions, which had more inflorescences (8 vs. 7 per stem). Accessions from Western Europe tended to flower earlier (140 vs. 146 days). Accessions from the rest of the world had longer organs and more leaves and inflorescences compared to Western European accessions. As a result there was good agreement between accession clusters derived from morphological traits and flowering date (Hayot Carbonero, 2011; Website) and tannin traits (Fig. 7). Western European accessions tended to cluster together when all tannin traits (PA content, mDP, % PD and % trans) were combined. These observations can be explained by the fact that sainfoin leaves contain more PAs than stems and that PAs in leaves tend to have higher mDP values and higher PD and trans-flavanol proportions than stems (Häring et al., 2007; Theodoridou et al., 2010). However, in contrast to Lotus (Kelman et al., 1997), no direct relationship could be found between erect or prostrate sainfoin accessions in terms of PA compositions. Results from the present study coincide instead with another report on Lotus (Gruber et al., 2008), where also no relationship could be found between leaf PA contents and morphological traits. 3. Conclusions Screening of contrasting accessions from this EU ‘HealthyHay’ sainfoin germplasm collection discovered a large variation in PA contents and composition. It is possible that such PA differences may have contributed to previous, contradictory reports about the anthelmintic
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Representative Countries Height Spain, Turkey
UK
Cluster tree based on % PD
China, Slovakia, Former Soviet Union, Czech Rep, Armenia, Turkey, Lithuania, Poland, Romania (Eastern Europe/
Turkey
Spain, France, Germany, Switzerland, Italy (Western Europe)
Fig. 10. Clustering of sainfoin accessions based on proportion of prodelphinidins.
or nutritional value of sainfoin (Hayot Carbonero, 2011; Website). The present study also identified distinct sainfoin chemovars, which is an important finding because PA content is negatively related to in vitro digestibility (Kelman, 2006) and some PA compositional traits have been linked to useful anti-parasitic, nutritional and environmental effects (Hatew et al., 2011; Manolaraki et al., 2011). The use of contrasting chemovars will be valuable for establishing robust PA structure–activity relationships and thus enable development of new sainfoin cultivars with optimum bioactive PA composition. Such an approach should facilitate the delivery of consistent anthelmintic, nutritional and environmental benefits. It is envisaged that optimising PA contents and composition will contribute towards developing more sustainable ruminant feeding systems for the future.
4. Experimental 4.1. General experimental procedures The HPLC equipment and analysis conditions used in this study were the same as used previously (Gea et al., 2011). Hydrochloric acid (36%), acetone (analytical reagent grade), dichloromethane
(HPLC grade), methanol (HPLC grade) and ascorbic acid were obtained from ThermoFisher Scientific (Loughborough, UK). (±)-Dihydroquercetin (98%) was obtained from Apin Chemicals (Abingdon, UK). Benzyl mercaptan (BM) (98%), (+)-catechin (C), ( )-epicatechin (EC), ( )-gallocatechin (GC) and ( )-epigallocatechin (EGC) were obtained from Sigma–Aldrich (Poole, UK).
4.2. Plant material Sainfoin samples were collected in June and July 2008 at NIAB (Cambridge, UK). Sourcing of seeds and growing conditions were described previously (Hayot Carbonero, 2011). Sainfoin accessions were harvested when about 50% of stems showed open flowers on the lowest half of the flower stem. Plant material was then weighed, packed in special bags (Nalgene low density polyethylene bags; 22.9 45.7 cm) and frozen at 20 °C. The frozen samples were subsequently transported to the freeze drying facility at the Archaeological Trust in York. The freeze dried material was then transported back to NIAB, ground to <8 mm at the University of Reading and transferred to Wageningen University (The Netherlands). Here the samples were first ground through a 1 mm screen using a Wiley mill. Subsequently, the ground material was passed
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Representative Countries Height
Turkey
Spain, Italy (Southern Europe) Cluster tree based on % trans-flavan-3-ols
UK, Norway, Spain, Switzerland (Western Europe)
Armenia, Romania, Turkey UK
UK Fig. 11. Clustering of sainfoin accessions based on the proportion of trans-flavanols.
through a sample divider, which is a rotating divider that distributes the original material into sub-samples with the same composition as the original bulk sample. 4.3. Analysis of sainfoin proanthocyanidins Proanthocyanidins were analysed by the in situ thiolysis method as described (Gea et al., 2011). 4.4. Cluster analysis of sainfoin accessions based on PA composition Cluster analysis was performed with The R Project for Statistical Computing (version 2.12.1) software for data manipulation, calculation and graphical display (CRAN, 2002). The ‘‘euclidean’’ method was applied to compute the distance matrix. The ‘‘complete linkage’’ method was then used for the hierarchical clustering. Acknowledgement This investigation was supported by the European Commission (Marie Curie Research Training Network, ‘HealthyHay’, MRTN-CT2006-035805). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2012.01.013.
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Proanthocyanidin diversity in the EU ‘HealthyHay’ sainfoin (Onobrychis viciifolia) germplasm collection Elisabetta Stringano a,⇑, Christine Hayot Carbonero b,1, Lydia M.J. Smith b, Ronald H. Brown a, Irene Mueller-Harvey a a Chemistry and Biochemistry Laboratory, Food Production and Quality Division, School of Agriculture, Policy and Development, University of Reading, P.O. Box 236, 1 Earley Gate, Reading RG6 6AT, UK b National Institute for Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
a r t i c l e
i n f o
Article history: Received 16 November 2011 Received in revised form 3 January 2012 Available online 6 February 2012 Keywords: Sainfoin Tannins Proanthocyanidins Thiolytic degradation Germplasm screening
a b s t r a c t This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80 g/100 g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P < 0.001; R2 = 0.49). A negative correlation existed between PA content (thiolysis) and mDP (P < 0.05; R2 = 0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Plant breeding is facing a paradigm shift and needs to develop new cultivars that can deliver lower environmental impact from agricultural production (Pilippot and Hallin, 2011). Recent evaluations of rice germplasm have already identified 500% differences in methane emissions, whilst a survey of wheat and other germplasm material discovered an enormous potential for plants with much higher biological nitrification inhibition activity. Sainfoin (Onobrychis viciifolia) is receiving renewed interest because of its many positive attributes (Hayot Carbonero et al., 2011; Website). Consumption of sainfoin shows promise for reducing environmental emissions of nitrogen and methane from ruminant animals (Hatew et al., 2011; Theodoridou et al., 2010) and unlike many
Abbreviations: BM, benzyl mercaptan; C, catechin; Cis, cis-flavanols (epicatechin or epigallocatechin); EC, epicatechin; EGC, epigallocatechin; G, gallocatechin; HPLC, high performance liquid chromatography; mDP, mean degree of polymerisation; PA, proanthocyanidin; PC, procyanidins; PD, prodelphinidins; trans, trans-flavanols (catechin or gallocatechin). ⇑ Corresponding author. Present address: CNR (National Research Council), Istituto di Ricerca Sulle Acque (Water Research Institute), 70132 Bari, Italy. Tel.: +44 (0)118 378 6619; fax: +44 (0)118 935 2421. E-mail address: [email protected] (E. Stringano). 1 Present address: University of Illinois at Urbana-Champaign, 1102 South Good win Avenue, Turner Hall, Urbana, IL 61801, USA. 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2012.01.013
other forage legumes, sainfoin generates high voluntary intakes without causing bloat (Mueller-Harvey, 2009). In addition, its flowers are a valuable source of pollen and nectar for bees (Clement et al., 2006; Kells, 2001; McGregor, 1976) and could thus contribute to alleviating problems associated with pollinator decline. Sainfoin has also been shown to control parasitic worms, which are posing a serious threat to animal welfare and farming (Hoste et al., 2006; Novobilsky´ et al., 2011). Content and composition of tannins, also called proanthocyanidins (PAs), are heritable traits that are amenable to plant breeding (Orians et al., 2000; Scioneaux et al., 2011). PAs are also the active ingredients in sainfoin and their variation is, therefore, worth exploring amongst currently available accessions. Although several studies have reported better absorption of nitrogen and essential amino acids by ruminants from tannin-containing sainfoin than from iso-nitrogenous but tannin-free forages, contradictory findings also exist (Hayot Carbonero et al., 2011). Here, we set out to investigate whether the contradictory reports could be due to variation in tannins among different sainfoin lines as indicated previously (Marais et al., 2000). It was for these reasons that the EU ‘HealthyHay’ project established a 300+ sainfoin germplasm collection (Website), which was evaluated for nutritional, anti-parasitic, chemical, biochemical, genetic and agronomic characteristics. The present study focussed on variation in PA content and composition within a subset of 37 diverse sainfoin accessions.
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Fig. 1. General structure of proanthocyanidins (R@OH: prodelphinidins; R@H: procyanidins).
Fig. 2. General structure of anthocyanidins (R@OH or H).
Many analytical methods have been used to quantify and characterise PAs in plants (Fig. 1). The widely used HCl–butanol method is an acid-catalysed oxidative depolymerisation of the PA interflavanyl linkages and yields anthocyanidins (Fig. 2), which can be determined spectrophotometrically (Porter et al., 1985). However, PA measurements based on anthocyanidin yields have several limitations: (a) water in the reaction medium can suppress the anthocyanidin yield (Waterman, 1994); (b) the number of hydroxy groups in the A and B rings affect the wavelength of the absorbance maximum of the anthocyanidin products (Fig. 2) (Hemingway, 1989); (c) colour yield is not always linearly correlated with tannin concentration (Waterman, 1994); (d) the ratio of reagent to sample, the temperature and the length of the reaction time affect the colour yield (Scalbert, 1992; Waterman, 1994); (e) the lack of appropriate standards remains a problem for quantifying complex mixtures of PAs (Giner-Chavez et al., 1997; Hagerman and Butler, 1994); and (f) the acid–butanol assay is generally used to quantify soluble PAs, however, some PAs are insoluble in common solvents and this can lead to an underestimation (Makkar et al., 1999; Reed, 1986). Although the HCl–butanol reaction has also been applied directly to plant samples (Kayani et al., 2007; Makkar et al., 1999; Yu and Dahlgren, 2000) the above limitations still apply and, in addition, the presence of chlorophyll interferes with anthocyanidin measurements (Watterson and Butler, 1983). Taken together these limitations mean that the HCl–butanol method is not suitable for quantitative PA analysis in extracts or whole plants. Therefore, Gea et al. (2011) recently developed a thiolysis method for the in situ analysis of complex PA mixtures in sainfoin ( O. viciifolia). This method has now been used to screen
Table 1 Samples collected from the EU ‘HealthyHay’ sainfoin germplasm collection (na = not available). Accession number
Species
1005 1007 1012r1a 1012r2a 1013 1017r1 1017r2 1019 1026r1 1026r2 1028 1041 1043 1071 1077 1103 1104 1110 1113 1123 1127 1156 1157r1 1157r2 1163 1165r1 1165r2 1169 1179 1197 1199 1200 1210 1213
Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis
viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia viciifolia
Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop. Scop.
Variety
Country of origin
Status
Perly na Ambra Ambra Somborne Teruel Teruel Taja Buciansky Buciansky Simpro Camaras Bivolari Hampshire Common Nova Korunga na CPI 63750 CPI 63753 CPI 63763 CPI 63767 Dukorastushchii Miatiletka Miatiletka Giant Rees ‘‘A’’ Rees ‘‘A’’ CPI 63810 CPI 63820 CPI 63838 CPI 63840 CPI 63841 Premier CPI 63854
Switzerland China Italy Italy UK Spain Spain Poland Slovakia Slovakia France Romania Romania UK Canada Turkey Turkey Turkey Spain Turkey USA, Washington Former Soviet Union Former Soviet Union Former Soviet Union UK UK UK Lithuania Spain Norway Former Soviet Union Germany Switzerland Switzerland
Cultivar na Cultivar Cultivar Cultivated Cultivated Cultivated Cultivar Cultivar Cultivar Cultivar na na Cultivated Cultivar na na na na Wild Cultivated Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar na na na na na Cultivar Cultivated
199
E. Stringano et al. / Phytochemistry 77 (2012) 197–208 Table 1 (continued)
a
Accession number
Species
1220 1230 1253r1 1253r2 1256 1260 1261r1 1261r2 1262r1 1262r2 1264r1 1264r2
Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis Onobrychis
viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. viciifolia Scop. antasiatica antasiatica
Variety
Country of origin
Status
247 Visnovsky Tu86-43-03 Tu86-43-04 Wkt 10 X93234 Line 107 Line 108 Cotswold Common Cotswold Common Sisiani Local Sisiani Local
Morocco Czech Republic Turkey, Hakkari Turkey, Hakkari Turkey, Afyon China, Xinjiang Armenia Armenia UK UK Armenia Armenia
na Cultivar Cultivated Cultivated Wild Wild Cultivated Cultivated Cultivated Cultivated Cultivated Cultivated
r1 and r2 refer to two replicate samples that were collected from the experimental site.
Table 2 Proanthocyanidin (PA) monomer composition and content in sainfoin accessions (g/100 g freeze-dried plant; SD in brackets); C = catechin, EC = epicatechin, GC = gallocatechin, EGC = epigallocatechin. Accession number
C
EC
GC
EGC
Starter units 1005 1007 1012r1
a
1012r2
a
1013 1017 r1 1017 r2 1019 1026r1 1026r2 1028 1041 1043 1071 1077 1103 1104 1110 1113 1123 1127 1156 1157r1 1157r2 1163 1165r1
0.020 (0.0024) 0.013 (0.0016) 0.024 (0.0054) 0.007 (0.0019) 0.021 (0.0036) 0.013 (0.0027) 0.017 (0.0023) 0.024 (0.0037) 0.014 (0.0047) 0.020 (0.0063) 0.012 (0.0007) 0.017 (0.0019) 0.020 (0.0024) 0.028 (0.0005) 0.006 (0.0008) 0.017 (0.0011) 0.008 (0.0014) 0.009 (0.0020) 0.010 (0.0013) 0.012 (0.0010) 0.007 (0.0017) 0.015 (0.0017) 0.013 (0.0024) 0.012 (0.0010) 0.021 (0.0032) 0.035 (0.0028)
C
EC
GC
EGC
Total PA content
0.290 (0.0438) 0.167 (0.0232) 0.286 (0.0414) 0.139 (0.0028) 0.349 (0.0591) 0.194 (0.0181) 0.312 (0.0202) 0.257 (0.0325) 0.161 (0.0436) 0.214 (0.0059) 0.173 (0.0203) 0.205 (0.0189) 0.165 (0.0469) 0.260 (0.0238) 0.128 (0.0016) 0.152 (0.0035) 0.108 (0.0265) 0.112 (0.0325) 0.138 (0.0115) 0.123 (0.0046) 0.113 (0.0154) 0.129 (0.0171) 0.164 (0.0172) 0.167 (0.0031) 0.354 (0.0316) 0.415 (0.0391)
0.230 (0.1038) 0.209 (0.0257) 0.166 (0.0466) 0.179 (0.0367) 0.328 (0.0612) 0.163 (0.0114) 0.167 (0.0153) 0.249 (0.0291) 0.187 (0.0474) 0.283 (0.0762) 0.132 (0.0183) 0.141 (0.0330) 0.208 (0.0117) 0.131 (0.0104) 0.067 (0.0056) 0.136 (0.0422) 0.179 (0.0144) 0.197 (0.0321) 0.175 (0.0172) 0.203 (0.0140) 0.229 (0.0460) 0.221 (0.0431) 0.201 (0.0297) 0.271 (0.0162) 0.206 (0.0596) 0.240 (0.0480)
0.630 (0.0926) 0.707 (0.0673) 0.884 (0.1200) 0.499 (0.0115) 0.745 (0.1503) 0.484 (0.0914) 0.740 (0.0537) 0.851 (0.0621) 0.499 (0.1183) 0.781 (0.0324) 0.434 (0.0577) 0.771 (0.0748) 0.840 (0.2198) 0.542 (0.0147) 0.495 (0.0855) 0.554 (0.0201) 0.373 (0.0757) 0.414 (0.0868) 0.473 (0.0350) 0.487 (0.0133) 0.517 (0.1068) 0.427 (0.1091) 0.533 (0.0623) 0.650) (0.0174) 0.582 (0.0752) 0.763 (0.1339)
1.250 (0.2689) 1.140 (0.1145) 1.422 (0.2222) 0.870 (0.0513) 1.529 (0.2845) 0.906 (0.1170) 1.292 (0.0896) 1.459 (0.1088) 0.933 (0.2231) 1.362 (0.1158) 0.786 (0.0861) 1.159 (0.1209) 1.290 (0.2756) 1.053 (0.0257) 0.711 (0.0837) 0.905 (0.0591) 0.700 (0.0963) 0.781 (0.1569) 0.824 (0.0731) 0.883 (0.0030) 0.899 (0.1755) 0.851 (0.1815) 0.962 (0.0998) 1.156 (0.0363) 1.239 (0.1832) 1.564 (0.2164)
Extender units 0.010 (0.0031) 0.003 (0.0002) 0.005 (0.0014) 0.009 (0.0021) 0.017 (0.0042) 0.011 (0.0012) 0.012 (0.0033) 0.008 (0.0023) 0.007 (0.0012) 0.003 (0.0032) 0.012 (0.0027) 0.009 (0.0060) 0.003 (0.0006) 0.021 (0.0014) 0.004 (0.0006) 0.010 (0.0003) 0.007 (0.0013) 0.011 (0.0008)
0.020 (0.0077) 0.016 (0.0019) 0.023 (0.0022) 0.012 (0.0011) 0.014 (0.0038) 0.009 (0.0032) 0.015 (0.0034) 0.036 (0.0016) 0.019 (0.0010) 0.027 (0.0045)
0.009 (0.0016) 0.004 (0.0012) 0.007 (0.0026) 0.007 (0.0021) 0.009 (0.0008) 0.007 (0.0016) 0.018 (0.0010)
0.022 (0.0026)
0.004 (0.0076) 0.037 (0.0045) 0.026 (0.0019)
0.023 (0.0014)
0.017 (0.0042)
0.022 (0.0031) 0.017 (0.0016) 0.014 (0.0017) 0.017 (0.0037) 0.027 (0.0069)
*
0.014 (0.0124)
0.050 (0.0178) 0.024 (0.0015) 0.034 (0.0052) 0.025 (0.0049) 0.055 (0.0079) 0.032 (0.0019) 0.028 (0.0030) 0.034 (0.0023) 0.046 (0.0083) 0.034 (0.0146) 0.023 (0.0039) 0.011 (0.0092) 0.017 (0.0053) 0.031 (0.0138) 0.012 (0.0006) 0.013 (0.0001) 0.026 (0.0019) 0.020 (0.0023) 0.029 (0.0115) 0.027 (0.0023) 0.030 (0.0064) 0.030 (0.0058) 0.028 (0.0062) 0.033 (0.0025) 0.053 (0.0137) 0.064 (0.0064)
(continued on next page)
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E. Stringano et al. / Phytochemistry 77 (2012) 197–208
Table 2 (continued) Accession number
C
EC
GC
EGC
Starter units 1165r2 1169 1179 1197 1199 1200 1210 1213 1220 1230 1253r1 1253r2 1256 1260 1261r1 1261r2 1262r1 1262r2 1264r1 1264r2
EC
GC
EGC
Total PA content
0.025 (0.0036) 0.008 (0.0012) 0.005 (0.0010) 0.006 (0.0025) 0.007 (0.0005) 0.003 (0.0003) 0.010 (0.0047) 0.007 (0.0013) 0.001 (0.0022) 0.007 (0.0012) 0.005 (0.0017) 0.005 (0.0005) 0.003 (0.0008) 0.003 (0.0005) 0.007 (0.0007) 0.005 (0.0006) 0.013 (0.0029) 0.015 (0.0007) 0.005 (0.0007) 0.003 (0.0013)
0.025 (0.0084) 0.035 (0.0031) 0.003 (0.0048) 0.012 (0.0031) 0.013 (0.0005) 0.020 (0.0060) 0.018 (0.0018) 0.031 (0.0007) 0.014 (0.0024) 0.043 (0.0014) 0.050 (0.0038) 0.049 (0.0023) 0.122 (0.0104) 0.017 (0.0015)
0.007 (0.0121)
0.018 (0.0035)
0.003 (0.0059)
0.011 (0.0104)
0.046 (0.0024) 0.041 (0.0074) 0.035 (0.0057) 0.017 (0.0037) 0.021 (0.0072) 0.021 (0.0017) 0.033 (0.0097) 0.030 (0.0066) 0.014 (0.0027) 0.021 (0.0073) 0.016 (0.0007) 0.015 (0.0019) 0.022 (0.0102) 0.016 (0.0025) 0.019 (0.0037) 0.024 (0.0009) 0.053 (0.0150) 0.080 (0.0045) 0.025 (0.0038) 0.014 (0.0049)
0.542 (0.0310) 0.369 (0.0108) 0.426 (0.0648) 0.144 (0.0240) 0.157 (0.0083) 0.195 (0.0183) 0.254 (0.0355) 0.281 (0.0168) 0.083 (0.0109) 0.158 (0.0211) 0.117 (0.0114) 0.108 (0.0094) 0.104 (0.0124) 0.100 (0.0048) 0.163 (0.0088) 0.143 (0.0011) 0.296 (0.0692) 0.399 (0.0264) 0.174 (0.0122) 0.086 (0.0323)
0.293 (0.1308) 0.277 (0.0372) 0.092 (0.0083) 0.177 (0.0552) 0.131 (0.0165) 0.103 (0.0141) 0.176 (0.0789) 0.242 (0.0131) 0.137 (0.0276) 0.184 (0.0459) 0.146 (0.0065) 0.141 (0.0141) 0.725 (0.0308) 0.168 (0.0289) 0.151 (0.0139) 0.131 (0.0307) 0.157 (0.0342) 0.209 (0.0651) 0.178 (0.0455) 0.101 (0.0226)
1.032 (0.0651) 1.256 (0.0414) 0.482 (0.0591) 0.537 (0.0896) 0.552 (0.0500) 0.537 (0.0639) 0.655 (0.1108) 1.117 (0.0539) 0.382 (0.0490) 0.637 (0.0938) 0.655 (0.0116) 0.594 (0.0408) 1.791 (0.0411) 0.484 (0.0181) 0.616 (0.0415) 0.398 (0.0024) 0.643 (0.1539) 0.837 (0.0840) 0.601 (0.0357) 0.356 (0.1233)
2.006 (0.2165) 2.011 (0.0967) 1.096 (0.1393) 0.901 (0.1339) 0.893 (0.0778) 0.906 (0.1048) 1.166 (0.2200) 1.735 (0.0778) 0.641 (0.0864) 1.068 (0.1585) 0.998 (0.0237) 0.921 (0.0566) 2.802 (0.0606) 0.798 (0.0498) 0.971 (0.0509) 0.712 (0.0271) 1.196 (0.2777) 1.566 (0.1625) 0.996 (0.0934) 0.567 (0.1846)
Empty cells signify that flavanol subunit was not detected. r1 and r2 refer to two replicate samples that were collected from the experimental site.
a
3.0
Proanthocyanidin content
2.5
2.0
1.5
1.0
0.5
0.0
1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
PAs %
*
0.036 (0.0031) 0.026 (0.0034) 0.053 (0.0080) 0.010 (0.0010) 0.013 (0.0013) 0.026 (0.0023) 0.019 (0.0039) 0.026 (0.0040) 0.011 (0.0014) 0.018 (0.0018) 0.009 (0.0012) 0.009 (0.0007) 0.016 (0.0010) 0.010 (0.0003) 0.012 (0.0022) 0.011 (0.0001) 0.022 (0.0047) 0.026 (0.0011) 0.012 (0.0012) 0.007 (0.0024)
C Extender units
Fig. 3. Proanthocyanidin contents of sainfoin accessions (g/100 g freeze-dried plant).
1
0
1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
trans %
0
50 1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
mDP 90
1
100
1005_Perly 1007_China 1012rep1_Ambra 1012rep2_Ambra 1013_Somborne 1017rep1_Teruel 1017rep2_Teruel 1019_Taja 1026rep1_Buciansky 1026rep2_Buciansky 1028_Simpro 1041_Camaras 1043_Bivolari 1071_Hampshire Com 1077_Nova 1103_Korunga 1104_Turkey 1110_CPI 63750 1113_CPI 63753 1123_CPI 63763 1127_CPI 63767 1156_Dukorastushchii 1157rep1_Miatiletka 1157rep2_Miatiletka 1163_Giant 1165rep1_Rees "A" 1165rep2_Rees "A" 1169_CPI 63810 1179_CPI 63820 1197_CPI 63838 1199_CPI 63840 1200_CPI 63841 1210_Premier 1213_CPI 63854 1220_247 1230_Visnovsky 1253rep1_Tu86-43-03 1253rep2_Tu86-43-04 1256_Wkt 10 1260_X93234 1261rep1_Line 107 1261rep2_Line 108 1262rep1_Cotswold Com 1262rep2_Cotswold Com 1264rep1_Sisiani Local 1264rep2_Sisiani Local
PD %
E. Stringano et al. / Phytochemistry 77 (2012) 197–208
Fig. 6. Proportion of trans-flavan-3-ols in sainfoin accessions.
201
Mean degree of polymerisation
80 70
60
50
40
30
20
10
0
Fig. 4. Mean degree of polymerisation of proanthocyanidins in sainfoin accessions.
Prodelphinidins
95
90
80 85
75
65 70
60
55
Fig. 5. Proportion of prodelphinidins in sainfoin accessions.
35
trans- flavan-3-ols
30
25
20
15
10
5
202
E. Stringano et al. / Phytochemistry 77 (2012) 197–208
Table 3 Pairwise correlations between PA content, mean degree of polymerisation (mDP), % prodelphinidins (PD), % trans flavan-3-ols and absorbance at 550 nm (HCl–butanol method; A550) in sainfoin accessions from the EU ‘HealthyHay’ germplasm collection. PA content mDP PD trans A550 (HCl–butanol) * ** ***
mDP
PD
trans
to explore any linkages between PA composition, sainfoin accessions and geographical origin. 2. Results and discussion
*
0.30 0.02 0.07 0.49***
0.14 0.08 0.21
2.1. Proanthocyanidin analysis 0.39** 0.34*
0.27
p < 0.05. p < 0.01. p < 0.001.
representative samples from the EU ‘HealthyHay’ sainfoin germplasm collection at NIAB (Cambridge, UK) in order to: to characterise the extent of PA diversity in this sainfoin collection to investigate correlations amongst PA structural traits within this collection to identify chemovars with contrasting PA composition for subsequent investigations into their biological effects and for supporting a future sainfoin breeding programme, and
Table 1 lists the O. viciifolia accessions that were tested. These sainfoin lines were selected from the 300+ accessions in the EU ‘HealthyHay’ collection as the most diverse accessions based on morphology, vigour, disease resistance and also flower colour (Hayot Carbonero, 2011), as the latter suggested differences in flavonoid metabolism. PAs in sainfoin samples were analysed directly by thiolytic degradation (Gea et al., 2011). This yielded information on PA content and composition (Tables 2 and S1). PA contents showed 4.9-fold differences, which ranged from 0.57 to 2.80 g PA per 100 g of freeze-dried sainfoin (NIAB accession number 1264 vs. NIAB 1256; Fig. 3). This variation is much larger than previously reported (Kelman et al., 1997) for a Lotus pedunculatus Cav. or L. corniculatus L. collection, where PAs ranged from 2.96 to 8.73 and from 1.56 to 4.22 g/100 g, respectively. A recent survey (Gruber et al., 2008) found 4-fold differences in soluble leaf PA among 14% of Lotus accessions, but 86% of accessions had no extractable leaf PA. Considerable variation has also been described for Lathyrus
Representative Countries Height Spain, Turkey
China, Slovakia, Former Soviet Union (Eastern Europe, Asia) Germany, UK (Western Europe) Slovakia, Former Soviet Union, Turkey, Czech Rep, Romania, Poland (Eastern Europe)
Cluster tree based on PA content, mDP, % PD and % trans-flavan-3-ols
Switzerland, UK, France, Spain, Italy (Western Europe)
Canada, USA, Armenia (Rest of World)
Fig. 7. Clustering of sainfoin accessions based on all thiolysis parameters (proanthocyanidin content, mean degree of polymerisation, % prodelphinidins and % trans-flavanol units).
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E. Stringano et al. / Phytochemistry 77 (2012) 197–208
Representative Countries Height
Armenia, Turkey, Canada
Cluster tree based on PA content (thiolysis)
Turkey, Armenia, China, Former Soviet Union (Asia)
Spain, Switzerland, UK, Romania, Czech Rep, Slovakia, Poland, Italy (West & East Europe)
Fig. 8. Clustering of sainfoin accessions based on proanthocyanidin content (a) thiolysis method (g PA/100 g plant) and (b) HCl–butanol method (A550
sativus L. genotypes, where PA contents ranged from 0.0 to 4.38 g/ kg (Deshpande and Campbell, 1992). Of particular note was the large variation in the mean degree of polymerisation (mDP). This varied 7-fold from 12 to 84 (NIAB 1071 vs. NIAB 1127; Fig. 4), which corresponds to average polymer sizes of ca. 3600–25200 Da. Such high mDP values agree with two previous reports (Bate-Smith, 1973; Jones et al., 1976) and indicate that the currently available sainfoin germplasm does indeed contain extremely polydisperse PAs (Foo et al., 1982). Whilst the thiolysis method used here (Gea et al., 2011) measured both extractable and non-extractable PAs, leaves from three Lotus accessions showed only a 3-fold variation in the largest, extractable PA fraction (1260– 3802 Da) (Gruber et al., 2008). Considerable differences were also observed amongst the proportion of prodelphinidins (PD), which varied 1.8-fold from 53 to 95 (NIAB 1179, vs. NIAB 1256; Fig. 5) and the proportion of trans-flavanol units varied 2.8-fold from 12 to 34 (NIAB 1077 vs. NIAB 1156; Fig. 6). These results are interesting because PA content and composition were shown to be inherited in F1 hybrids of Salix and Populus (Orians et al., 2000; Scioneaux et al., 2011) and therefore this variation offers opportunities for optimising the anti-parasitic, nutritional and environmental effects of sainfoin (Hatew et al., 2011; Manolaraki et al., 2011). 2.2. Correlations amongst sainfoin PA traits PA contents were determined by the thiolysis and HCl–butanol assays and were positively correlated (P < 0.001), but this
nm).
correlation was quite low (R2 = 0.49) (Table 3). This suggests that the two assays do not reflect the same PA properties. We also noticed that across the accessions, PA contents (HCl–butanol) were negatively correlated to % PD (P < 0.05), although the biochemical significance of this observation is unclear. Within this germplasm collection, several other low, but significant, correlations could be seen among the tannin traits. A negative correlation existed between PA content and mDP (P < 0.05). This suggests that accessions with high PA contents do not necessarily have large PA polymers. Instead, lower tannin contents are more likely to reflect higher mDP values. The positive correlation (P < 0.01) between % trans-flavan-3-ols (i.e. catechin or gallocatechin units) and % PD (i.e. gallocatechin or epigallocatechin units) may simply reflect the presence of gallocatechin in both parameters. Alternatively, it may indicate that accessions differ especially in the biosynthesis of gallocatechin subunits, as epigallocatechin accounts for most of the PA subunits and therefore the PD percentage (Table 2). 2.3. Cluster analysis of sainfoin accessions based on PA composition The ‘‘complete linkage’’ clustering method, which is also called the farthest neighbour method, defines the distance between two clusters to be the maximum distance between their individual components. In other words, the distance between two clusters is given by the value of the longest link between the clusters, which is shown as ‘height’ on the y-axis. Figs. 7, 8b, 9 and 11 depict
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Representative Countries Height
Switzerland, Spain, UK, Italy, Germany (Western Europe)
Cluster tree based on PA content (HCl-butanol method)
Armenia, Former Soviet Union, Turkey, Romania, Slovakia (Asia, Eastern Europe)
UK, Italy, France, Switzerland (Western Europe)
Former Soviet Union, Turkey, China (Asia)
Fig. 8 (continued)
clusters, which contain several groups that differ in height. Thus, the resulting dendrogrammes consist of many U-shaped brackets, which connect these sub-groups via a hierarchical tree. The height of each U represents the distance between two sub-groups. However in Figs. 8a and 10 there are hardly any height differences. Only accessions 1256, 1169, 1165r2 and 1213 show distinct height differences compared to the other accessions, which had much lower and relatively similar PA contents (Fig. 8a). The dendrogramme in Fig. 7 shows the cluster tree that was derived from all thiolysis parameters, i.e. PA content, mDP, percentage of PD and trans-flavan-3-ols. Two Western European, two Eastern European and one Rest-of-World cluster can be seen. Figs. 8–11 show the cluster trees derived from individual thiolysis parameters. It is worth noting that clustering based just on PD contents reveals some similarities to the overall data clustering (Fig. 10 vs. Fig. 7): the two accessions from Spain and Turkey (1179 and 1256) clustered outside the two main groups, but accessions from Asia and Eastern Europe (China, Former Soviet Union, Armenia, Turkey, Romania, Slovakia, Czech Republic, Poland and Lithuania) clustered together, as did accessions from Western Europe (Spain, France, Germany, Switzerland and Italy).
The cluster tree based on PA contents (thiolysis method) showed that accessions from Asia (Turkey, Armenia, China, Former Soviet Union) had a tendency to group together (Fig. 8a). Interestingly, the PA content as measured by the HCl–butanol method generated more regional clusters than the thiolysis method: two Western European, one Asian/Eastern European and one Asian cluster (Fig. 8b). These observations coincide with reports that PA contents in Lotus were also affected by country of origin (Gruber et al., 2008; Kelman et al., 1997). Using the average PA polymer size or mDP-values (Fig. 9), several accession clusters could be seen: (i) ‘Eastern’ countries tended to group together (China, Turkey, Czech Republic and Romania, Poland and Turkey); (ii) so did Canada, USA and Armenia and (iii) Western Europe (UK, Germany, Spain, Switzerland and Italy). In contrast, a lineage map based on PD percentages (Fig. 10) generated one UK cluster, two Turkish sub-groups, one Eastern European/Asian cluster (China, Slovakia, Former Soviet Union, Czech Republic, Armenia, Turkey, Lithuania, Poland and Romania) and one Western European cluster (Spain, France, Germany, Switzerland, Italy). Clustering based on the percentage of transflavan-3-ols (Fig. 11) also revealed Turkish and UK clusters, a Southern European cluster (Spain and Italy), a Western European
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Representative Countries Height Canada, USA, Armenia
China, Turkey, Czech Republic
Cluster tree based on mDP
Romania, Poland, Turkey
UK, Germany, Spain, Switzerland, Italy (Western Europe)
Turkey, Armenia
Fig. 9. Clustering of sainfoin accessions based on the mean degree of polymerisation.
cluster (UK, Norway, Spain, Switzerland) and an a cluster from Armenian, Romanian and Turkish accessions. This type of linkage information can be useful to identify relationships between PA composition and accession origin. To summarise, most accessions clustered into two main groups in terms of PA composition: Western Europe (UK, France, Switzerland, Spain, Italy) and Eastern Europe/Asia (China, Former Soviet Union, Turkey, Slovakia, Romania, Poland). However, several accessions from Armenia, Canada and USA tended to cluster into separate groups (Fig. 7). 2.4. Relationship between proanthocyanidins and phylogenetic characteristics of sainfoin The above clusters coincided with the strong links that were observed between geographical origin and agronomic performance within the ‘HealthyHay’ germplasm (Hayot Carbonero, 2011; Website): (i) morphological traits differed between accessions from Western Europe and from the rest of the world; (ii) the best agronomical accessions originated mainly from Eastern Europe and (iii) accessions from Western Europe and from the rest of the world could be classified as two different genetic subspecies (Hayot Carbonero et al., in press). There was a clear division among the morphological traits (more than 50% differences) between Western European accessions, which had more leaflets (25 vs. 21 per leaf) and thick stems
(29 vs. 21), and Eastern European accessions, which had more inflorescences (8 vs. 7 per stem). Accessions from Western Europe tended to flower earlier (140 vs. 146 days). Accessions from the rest of the world had longer organs and more leaves and inflorescences compared to Western European accessions. As a result there was good agreement between accession clusters derived from morphological traits and flowering date (Hayot Carbonero, 2011; Website) and tannin traits (Fig. 7). Western European accessions tended to cluster together when all tannin traits (PA content, mDP, % PD and % trans) were combined. These observations can be explained by the fact that sainfoin leaves contain more PAs than stems and that PAs in leaves tend to have higher mDP values and higher PD and trans-flavanol proportions than stems (Häring et al., 2007; Theodoridou et al., 2010). However, in contrast to Lotus (Kelman et al., 1997), no direct relationship could be found between erect or prostrate sainfoin accessions in terms of PA compositions. Results from the present study coincide instead with another report on Lotus (Gruber et al., 2008), where also no relationship could be found between leaf PA contents and morphological traits. 3. Conclusions Screening of contrasting accessions from this EU ‘HealthyHay’ sainfoin germplasm collection discovered a large variation in PA contents and composition. It is possible that such PA differences may have contributed to previous, contradictory reports about the anthelmintic
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Representative Countries Height Spain, Turkey
UK
Cluster tree based on % PD
China, Slovakia, Former Soviet Union, Czech Rep, Armenia, Turkey, Lithuania, Poland, Romania (Eastern Europe/
Turkey
Spain, France, Germany, Switzerland, Italy (Western Europe)
Fig. 10. Clustering of sainfoin accessions based on proportion of prodelphinidins.
or nutritional value of sainfoin (Hayot Carbonero, 2011; Website). The present study also identified distinct sainfoin chemovars, which is an important finding because PA content is negatively related to in vitro digestibility (Kelman, 2006) and some PA compositional traits have been linked to useful anti-parasitic, nutritional and environmental effects (Hatew et al., 2011; Manolaraki et al., 2011). The use of contrasting chemovars will be valuable for establishing robust PA structure–activity relationships and thus enable development of new sainfoin cultivars with optimum bioactive PA composition. Such an approach should facilitate the delivery of consistent anthelmintic, nutritional and environmental benefits. It is envisaged that optimising PA contents and composition will contribute towards developing more sustainable ruminant feeding systems for the future.
4. Experimental 4.1. General experimental procedures The HPLC equipment and analysis conditions used in this study were the same as used previously (Gea et al., 2011). Hydrochloric acid (36%), acetone (analytical reagent grade), dichloromethane
(HPLC grade), methanol (HPLC grade) and ascorbic acid were obtained from ThermoFisher Scientific (Loughborough, UK). (±)-Dihydroquercetin (98%) was obtained from Apin Chemicals (Abingdon, UK). Benzyl mercaptan (BM) (98%), (+)-catechin (C), ( )-epicatechin (EC), ( )-gallocatechin (GC) and ( )-epigallocatechin (EGC) were obtained from Sigma–Aldrich (Poole, UK).
4.2. Plant material Sainfoin samples were collected in June and July 2008 at NIAB (Cambridge, UK). Sourcing of seeds and growing conditions were described previously (Hayot Carbonero, 2011). Sainfoin accessions were harvested when about 50% of stems showed open flowers on the lowest half of the flower stem. Plant material was then weighed, packed in special bags (Nalgene low density polyethylene bags; 22.9 45.7 cm) and frozen at 20 °C. The frozen samples were subsequently transported to the freeze drying facility at the Archaeological Trust in York. The freeze dried material was then transported back to NIAB, ground to <8 mm at the University of Reading and transferred to Wageningen University (The Netherlands). Here the samples were first ground through a 1 mm screen using a Wiley mill. Subsequently, the ground material was passed
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Representative Countries Height
Turkey
Spain, Italy (Southern Europe) Cluster tree based on % trans-flavan-3-ols
UK, Norway, Spain, Switzerland (Western Europe)
Armenia, Romania, Turkey UK
UK Fig. 11. Clustering of sainfoin accessions based on the proportion of trans-flavanols.
through a sample divider, which is a rotating divider that distributes the original material into sub-samples with the same composition as the original bulk sample. 4.3. Analysis of sainfoin proanthocyanidins Proanthocyanidins were analysed by the in situ thiolysis method as described (Gea et al., 2011). 4.4. Cluster analysis of sainfoin accessions based on PA composition Cluster analysis was performed with The R Project for Statistical Computing (version 2.12.1) software for data manipulation, calculation and graphical display (CRAN, 2002). The ‘‘euclidean’’ method was applied to compute the distance matrix. The ‘‘complete linkage’’ method was then used for the hierarchical clustering. Acknowledgement This investigation was supported by the European Commission (Marie Curie Research Training Network, ‘HealthyHay’, MRTN-CT2006-035805). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2012.01.013.
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