Bioactive compounds, aucubin and acteoside, in plantain (Plantago lanceolata L.) and their effect on in vitro rumen fermentation

Animal Feed Science and Technology 222 (2016) 158–167

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Bioactive compounds, aucubin and acteoside, in plantain (Plantago lanceolata L.) and their effect on in vitro rumen fermentation Soledad Navarrete a,∗ , Peter D. Kemp a , Sarah J. Pain b , Penny J. Back b a b

Institute of Agriculture and Environment, Private Bag 11222, Massey University, Palmerston North 4442, New Zealand Institute of Veterinary, Animal and Biomedical Sciences, Private Bag 11222, Massey University, Palmerston North 4442, New Zealand

a r t i c l e

i n f o

Article history: Received 4 May 2016 Received in revised form 7 October 2016 Accepted 17 October 2016 Keywords: Plantain Aucubin Acteoside In vitro fermentation Ammonia Volatile fatty acids

a b s t r a c t Plantain (Plantago lanceolata L.) contains bioactive compounds with antimicrobial activity that can potentially influence ruminal fermentation. This study aimed to identify the concentration of the bioactive compounds catalpol, aucubin, and acteoside in plantain cv. ‘Ceres Tonic’ through two consecutive growing seasons (2011–2012 and 2012–2013). Then the herbage with highest levels of bioactive compounds was used to evaluate their effect on rumen in vitro fermentation. Plantain cv. ‘Ceres Tonic’ had almost nil concentration of catalpol. Both aucubin and acteoside concentrations increased (P < 0.05) through the growing season. Aucubin increased from 1.78 to 3.80 mg/g DM in the first and from 0.44 to 6.87 mg/g DM in the second growing season; while, acteoside increased from 23.6 to 35.4 mg/g DM and from 0.5 to 41.7 mg/g DM, respectively. The in vitro experiment evaluated the effect of aucubin and acteoside on ammonia (NH3 ), volatile fatty acids (VFA) and gas production (GP) parameters. Aucubin and acteoside were added to chicory (Cichorium intybus L.) as negative and to plantain as positive controls. The treatments were (i) chicory (CH); (ii) chicory + 10 mg aucubin/g DM (C+au); (iii) chicory + 20 mg aucubin/g DM (C+2au); (iv) chicory + 40 mg acteoside/g DM (C+ac); (v) plantain naturally containing 7 mg/g DM of aucubin and 36 mg/g DM of acteoside (PL); (vi) plantain + extra 10 mg aucubin/g DM (P+au); and (vii) plantain + extra 36 mg acteoside/g DM (P+ac). Plantain with natural concentrations of bioactives produced 40% less NH3 than chicory over 24 h. The exogenous addition of both bioactive compounds reduced net NH3 production by CH and PL. The increase in potential GP from acteoside fermentation suggested its use as an energy source. Whereas, the addition of aucubin reduced the rate of GP at dose level, potentially due to its bactericide activity. Therefore, acteoside would have a greater positive effect than aucubin on ruminant animals. © 2016 Elsevier B.V. All rights reserved.

Abbreviations: A, potential gas production; A:P, acetate propionate ratio; ac, acteoside; ADF, acid detergent fibre; au, aucubin; BCVFA, branched chain volatile fatty acids; CH, Chicory; C+au, Chicory plus 10 mg aucubin/g DM; C+2au, chicory plus 20 mg aucubin/g DM; C+ac, chicory plus 40 mg acteoside/g DM; CO2 , carbon dioxide; CP, crude protein; DM, dry matter; GP, gas production; HPLC, high performance liquid chromatography; HWSC, hot water soluble carbohydrates; ME, metabolisable energy; MeOH, methanol; N, nitrogen; N2 O, nitrous oxide; NDF, neutral detergent fibre; NH3 , ammonia; OM, organic matter; OMD, organic matter digestibility; PL, plantain; P+au, plantain plus 10 mg aucubin/g DM; P+ac, plantain plus 36 mg acteoside/g DM; R1/2A , fermentation rate at T1/2A ; RFC, readily fermentable carbohydrates; SC, structural carbohydrate; T1/2A , half time when the potential GP was reached; V24 h, volume of gas produced after 24 h incubation; VFA, volatile fatty acids. ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Navarrete). http://dx.doi.org/10.1016/j.anifeedsci.2016.10.008 0377-8401/© 2016 Elsevier B.V. All rights reserved.

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1. Introduction Plantain (Plantago lanceolata L.) is a herb containing secondary, bioactive, compounds that may influence N cycling in pastoral livestock systems (Pacheco and Waghorn, 2008). The most well-known bioactive compounds in plantain are the iridoid glycosides: aucubin and catalpol; and the phenylpropanoid glycoside, acteoside (Syn. verbascoside) (Stewart, 1996; Tamura and Nishibe, 2002). These compounds have been reported to have antimicrobial and antifungal effects (Andary et al., 1982; Davini et al., 1986; Kim et al., 2000), and it has been suggested that these compounds may influence the rumen micro flora of grazing ruminants and ultimately their nutrient utilisation (Burke et al., 2000; Swainson and Hoskin, 2006); however, their impact on rumen fermentation is unclear. The presence of bioactive compounds in plantain with the potential to affect rumen fermentation is likely to have important implications for rumen N efficiency (Stewart, 1996). A reduction in the NH3 concentration in the rumen is desirable because it could decrease N losses to the environment, including via urine (Attwood et al., 1998). Dairy cows grazing, at the same N intake, either diverse pasture containing plantain or perennial ryegrass Lolium perenne L. had a substantially lower N concentration in their urine (Totty et al., 2013). Totty et al. (2013) did not determine if this lower N concentration in the urine was by improvement of N efficiency or N dilution in the urine. However, greater urine flows have been recorded in animals grazing plantain (Wilman and Derrick, 1994), consistent with the diuretic effect of iridoid glycosides (Tamura and Nishibe, 2002). Urine N is immediately available for leaching down the soil profile and volatilisation resulting in significant nitrous oxide (N2 O) emissions. The antimicrobial effect of aucubin, but not for acteoside (Andary et al., 1982), has been well documented (Davini et al., 1986; Bartholomaeus and Ahokas, 1995; Kim et al., 2000), and the effect of these bioactive compounds on rumen fermentation appears to not have been assessed. Our hypothesis is that the bioactive compounds in plantain will decrease NH3 production and affect volatile fatty acids (VFA) production in the rumen. This study examined the seasonal concentration of these bioactive compounds in plantain cv. ‘Ceres Tonic’ and evaluated the in vitro fermentation of plantain and compared it to chicory (Cichorium intybus L.), a perennial herb known not to contain these bioactive compounds, by the exogenous addition of aucubin and acteoside. 2. Materials and methods 2.1. Experimental site of plant material The plantain evaluated in this study was obtained from a grazing trial at Dairy 1, Massey University, Palmerston North, New Zealand (40◦ 22 S, 175◦ 36 E) from October, 2011 until May, 2013. The grazing trial evaluated plantain (cv. ‘Ceres Tonic’) and chicory (cv. ‘Grassland Choice’) pastures grazed every two and four weeks throughout two growing seasons (December, 2011–May, 2012 and August, 2012–May, 2013). The pastures of plantain and chicory were established in October, 2011 in plots (300 m2 ) arranged in a randomised block factorial design with five replicates. Plantain and chicory were grazed exactly every two and every four weeks with dairy cows immediately after the morning milking (0600 h) and until the swards achieved a residual height of 70–100 mm (approximately 5 h). 2.2. Plant material sampling The herbage from the plantain and chicory pastures was collected by taking a hand plucked sample from multiple sites in both the plantain and chicory plots at different dates through both growing seasons. During the first growing season (2011–2012), the herbage from all plots was collected at two dates: (i) summer (December, 2011) and (ii) autumn (May, 2012). During the second growing season herbage samples were taken on three dates: (i) spring (October, 2012), (ii) summer (January, 2013), and (iii) autumn (May, 2013). Samples were stored at −20 ◦ C until later analysis. 2.3. Laboratory analysis of bioactive compounds Catalpol, aucubin and acteoside in plantain and chicory were determined by high-performance liquid chromatography (HPLC). Plantain and chicory samples were stored at −20 ◦ C, then freeze-dried and ground to pass through a 1 mm diameter sieve. A 100 mg aliquot from each of the ground samples was taken for extraction of aucubin, catalpol, and acteoside, with 10 mL of methanol (MeOH) in 15 mL tubes and shaken for 2 h at room temperature. The solid plant material was filtered out using grade 41 quantitative filter papers (Whatman Co., Ltd., England). Then, 2 mL of the filtrate was diluted in 8 mL of ultra-pure water, and further filtered using 0.2 ␮m syringe filters (Whatman Co., Ltd., England) and a 20 ␮L aliquot used for HPLC analysis for the simultaneous determination of catalpol and aucubin. Whereas for acteoside, 2 mL of the filtrate undiluted was filtered using 0.2 ␮m syringe filter (Whatman Co., Ltd., England) and a 20 ␮L of aliquot used for HPLC analysis. Commercially available catalpol, aucubin, and acteoside (99% pure; Extrasynthese S.A, France) were used as standards. The standard solution contained 2 mg each of catalpol and aucubin in 50 mL of 20% MeOH and 1 mg of acteoside in 5 mL of pure MeOH. High-performance liquid chromatographywas performed at 40 ◦ C using a 100 mm × 6.0 YMC pack ODS-A column protected by a YMC guard pack (YMC America, Inc). The mobile phase was 1% acetronitrile in water for catalpol and aucubin and 29% MeOH in water (containing 5% acetic acid) for acteoside. The flow rate was 1 mL/min. For catalpol and aucubin, wavelength detection was performed at 240 nm and for acteoside at 330 nm. The HPLC system consisted of a Dionex

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Table 1 Summary of the aucubin (au) and acteoside (ac) concentration (mg/g DM) tested in chicory (CH) and plantain (PL) treatments. Aucubin and acteoside concentration (mg/g DM) Substrate

Treatments

natural level

level added

Total

CH

CH

CH

C+au

CH

C+2au

CH

C+ac

PL

PL

PL

P+au

PL

P+ac

0 aucubin 0 acteoside 0 aucubin 0 acteoside 0 aucubin 0 acteoside 0 aucubin 0 acteoside 7 aucubin 36 acteoside 7 aucubin 36 acteoside 7 aucubin 36acteoside

0 aucubin 0 acteoside 10 aucubin 0 acteoside 20 aucubin 0 acteoside 0 aucubin 40 acteoside 0 aucubin 0 acteoside 10 aucubin 0 acteoside 0 aucubin 36 acteoside

0 aucubin 0 acteoside 10 aucubin 0 acteoside 20 aucubin 0 acteoside 0 aucubin 40 acteoside 7 aucubin 36 acteoside 17 aucubin 36 acteoside 7 aucubin 72 acteoside

UltiMate 3000 HPLC system equipped with an UltiMate 3000 Pump, an UltiMate 3000 Autosampler Column Compartment, an UltiMate 3000 variable wavelength detector and Chromeleon software (version 6.8) for data processing. 2.4. In vitro experimental design The in vitro fermentation assay was undertaken over 24 h using an incubator with the capacity for 32 bottles (Contherm Scientific Limited) (Muetzel et al., 2014). Commercially available aucubin and acteoside ( > 90% and >99% pure, respectively; Extrasynthese S.A, France) were added to chicory, which does not contain detectable levels of these compounds as a negative control, and to plantain, which naturally contained both compounds, as a positive control. The higher concentration found in plantain samples established the concentrations to evaluate both compounds. To test for a dose response to aucubin in chicory two concentrations (10 and 20 mg/g DM) were used. The capacity of the incubator was insufficient for a dose response to aucubin in plantain and for acteoside in chicory and plantain. The treatments evaluated were: (i) chicory negative control, 0 mg aucubin or acteoside/g DM (CH); (ii) chicory + 10 mg aucubin/g DM (C+au); (iii) chicory + 20 mg aucubin/g DM (C+2au); (iv) chicory + 40 mg acteoside/g DM (C+ac); (v) plantain with existing natural levels of 7 mg aucubin/g DM and 36 mg acteoside/g DM as positive control (PL); (vi) plantain plus an additional 10 mg aucubin/g DM (P+au); and (vii) plantain plus an additional 36 mg acteoside/g DM (P+ac). The treatments are further summarised in Table 1. All treatments were incubated in two sets of duplicate bottles, where one set of bottles was used to record GP and the other set used to measure end products of fermentation, NH3 and VFA produced over time. Three incubation runs were carried out with each evaluating one replicate of herbage sample from separate plots in the field (n = 3). 2.4.1. In vitro incubations Plantain and chicory herbage used as substrate in this in vitro study were from the plots grazed every four weeks taken in autumn, 2013. Approximately 300 mg DM of substrate (1 mm ground of plantain or chicory) was weighed into a 125 mL serum bottle. Aucubin or acteoside were then added to the chicory and plantain bottles at the dose outlined in Table 1. Three fistulated cows were used as rumen fluid donors with rumen fluid obtained in the morning from multiple sites within the rumen and transferred to the laboratory in pre-warmed thermos flasks. Once in the laboratory, the rumen fluid was strained through a double layer of cheesecloth, pooled in equal proportions, and mixed with McDougal’s buffer (McDougall, 1948) having Na2 S (100 mg/L) as reducing agent, at a 1:4 ratio, under continuous flushing with carbon dioxide (CO2 ) in a water bath at 39 ◦ C to obtain rumen fluid to be used as the source of inoculum. Then, 30 mL of this buffered rumen fluid was injected into each bottle containing substrate with or without addition of bioactive compounds and sealed with rubber stoppers, manually agitated and place inside the incubator at 39 ◦ C for 24 h. 2.4.2. Determination of gas production and end fermentation products The headspace gas pressure inside each bottle was measured automatically using a pressure sensor connected to a needle in the cap of the incubation bottle (40PC015G1A, Honeywell, International Inc., Morris town, NJ, USA). Pressure was recorded every minute and when pressures exceeded 10 kPa the accumulated gas was released. The set of bottles for products of fermentation were repeat sampled at 1.5, 3, 5, 8, 12 and 24 h, taking 1.8 mL of medium from each bottle at each sampling time. The samples were transferred to micro tubes and centrifuged at 21,000 x g for 10 min at 4 ◦ C. Then, duplicate 0.9 mL aliquots of the supernatant were transferred into micro-tubes with 0.1 mL of an internal standard (19.87 mM ethyl butyric acid, 20% v/v ortho-phosphoric acid) and stored at −20 ◦ C until later analysis of NH3 and VFA. The pH in the medium was measure in all bottles after 24 h of incubation.

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2.4.3. Laboratory analysis for in vitro incubations Plantain and chicory substrates were analysed for total DM by drying the samples at 105 ◦ C in a conventional oven (AOAC, 2000; method 930.15, 925.10) and for organic matter (OM) by ashing the samples for 16 h at 550 ◦ C in a furnace (AOAC, 2000; method 942.05). Total N was determined by combustion using a Leco analyser (AOAC, 2000; method 968.06), and crude protein (CP) was estimated by multiplying the N content by 6.25. Neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin were analysed in a Tecator Fibretec System (Foss Fibretec, Höganäs, Sweden) by the detergent procedures of Robertson and Van Soest (1981). Cellulose was calculated as ADF less lignin, and hemicellulose as NDF less ADF. Hot water soluble carbohydrates (HWSC) were measured by the reducing sugar method (Nelson, 1944). Organic matter digestibility (OMD) and metabolisable energy (ME) were measured by following the Roughan and Holland (1977) method. The samples for VFA and NH3 analysis were thawed and centrifuged at 21,000 x g for 10 min at 4 ◦ C, and 0.8 mL of the supernatant was transferred into a crimp cap vial to determine individual VFA (acetate, propionate, butyrate, isobutyrate, and isovalerate) by gas chromatography (HP 6890, Santa Clara, CA, USA). Part of the remaining supernatant (approximately 0.1 mL) was used to determine NH3 concentration by the colorimetric method described by Weatherburn (1967). 2.5. Calculations and statistical analysis The GP profiles of each bottle were fitted to the model described by Wang et al. (2011), using the following formula: V = (A(1-exp(-kt))/(1 + exp(ln(1/d)-kt))) Where: V: total GP at time t (mL/g DM) A: is the potential GP (mL/g DM) k = is the fractional rate of GP per hour (/h) d = is a shape parameter indicating a sigmoidal or no sigmoidal shape of the gas curve. The half time of when the potential GP was reached (T1/2A ) and the fermentation rate (R1/2A ) at T1/2A were calculated as follow: T1/2A = (ln(2 + 1/d))/k R 1/2A = (k(d + 0.5))/(1 + d) Data were analysed using the PROC MIXED procedure of SAS (version 9.3, SAS Institute Inc., Cary, NC) . The concentrations of catalpol, aucubin, and acteoside in plantain, were analysed as a factorial randomised complete block (n = 5) design with date and grazing frequency (2 or 4 week intervals) as fixed effects and block treated as a random effect. Data from the in vitro experiment were analysed as a complete randomised block design with the average of the duplicate bottles as analytical replicates. The model parameters (A, T1/2A , R1/2A and V24 h) after 24 h of incubation of all in vitro treatments were analysed as a complete randomised design with incubation day (n = 3) treated as a random effect. Repeated measures analysis was performed for net NH3 production and individual VFA production over time of acetate, propionate, butyrate, branched chain VFA (BCVFA: isobutyrate and isovalerate) and total VFA in a design with in vitro treatments and incubation time (1.5, 3, 5, 8, 12 and 24 h) as fixed effects. The inclusion of incubation day as a random effect provided true replication. Chicory and plantain in vitro treatments were analysed separately. Means were compared using the least squares means test and significance declared at P < 0.05. 3. Results 3.1. Bioactive compounds in plantain The concentration of bioactive compounds found naturally occurring in the plantain samples harvested from the two growing seasons and two grazing frequencies are presented in Table 2. Catalpol was detected at very low concentrations, ranging from 0.01 to 0.06 mg/g DM, with no differences observed across the growing season, harvest dates or due to grazing frequency on both growing seasons (Table 2). Aucubin and acteoside concentrations increased over the growing season in both years (Table 2). These bioactive compounds were not detected in chicory. There was an interaction (P < 0.0001) between harvest date and grazing frequency observed for aucubin concentration in the first growing season only. In December, 2011 the aucubin concentrations were similar when grazing every two (1.8 ± 0.35) or every four weeks (1.8 ± 0.10), but they were lower when grazing every two weeks (2.2 ± 0.21) rather than every four weeks (5.4 ± 0.26) towards the end of the growing season (May, 2012). No effect of grazing frequency (P = 0.21) was observed in relation to acteoside concentration, and nor was any interaction (P = 0.85) observed during the second growing season. Acteoside concentration was not affected by grazing frequency and was nor interaction in both growing seasons.

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Table 2 Concentration (mg/g DM) of catalpol, aucubin and acteoside in plantain (Plantago lanceolata L.) cv. ‘Tonic’. Catalpol

Aucubin

Acteoside

First growing season (2011–2012) Date Dec-2011 May-2012 SEM

0.03 0.02 0.01

1.8 3.8 0.52

23.6 35.4 3.50

Grazing frequency 2 weeks 4 weeks SEM

0.03 0.02 0.01

2.0 3.6 0.59

25.7 33.3 4.12

P value Date Grazing frequency Interaction

0.447 0.378 0.211

<0.0001 < 0.0001 <0.0001

0.005 0.051 0.663

Second growing season (2012–2013) Date Oct-2012 Jan-2013 May-2013 SEM

0.01 0.09 0.04 0.02

0.4c 2.2b 6.9a 0.35

0.6c 20.0b 41.1a 3.21

Grazing frequency 2 weeks 4 weeks SEM

0.03 0.06 0.03

2.7 3.4 1.29

20.2 19.5 8.47

P value Date Grazing frequency Interaction

0.128 0.264 0.499

<0.0001 0.219 0.858

<0.0001 0.421 0.269

a,b,c Superscripts indicate values within columns that are significantly different (P < 0.05). SEM, standard error of the mean.

Table 3 Nutritive analysis of plantain and chicory from the four weeks grazing frequency plots, harvested in May, 2013 and used as substrate. Nutritive value traitsa

Plantain

Chicory

SEMb

P value

OM (g/kg DM) Ash (g/kg DM) CP (g/kg DM) ME (MJ/kg DM) NDF (g/kg DM) ADF (g/kg DM) Cellulose (g/kg DM) Hemicellulose (g/kg DM) Lignin (g/kg DM) HWSC (g/kg DM) OMD Ratio (HWSC:SC)

856.6 143.4 298.9 12.15 188.7 148.3 74.5 40.3 73.8 85.5 0.74 0.75

850.9 149.1 322.5 12.34 168.1 136.5 67.5 31.5 69.0 83.4 0.75 0.91

4.97 4.97 9.13 0.26 5.39 4.98 9.83 1.12 6.94 4.61 1.61 0.14

0.48 0.48 0.16 0.01 0.05 0.16 0.70 0.007 0.70 0.82 0.01 0.003

a DM, dry matter; OM, organic matter; CP, crude protein; ME, metabolizable energy; NDF, neutral fibre detergent; ADF, acid fibre detergent; HWSC, hot water soluble carbohydrates; OMD, organic matter digestibility; HWSC:SC, the ratio of HWSC to structural carbohydrates. b SEM, standard error of the mean.

3.2. In vitro incubations The nutritive analyses of plantain and chicory used as substrates are presented in Table 3. Chicory and plantain from plots harvested in May, 2013 had similar CP and HWSC concentrations. Plantain had higher NDF and hemicellulose concentrations and a lower ME, OMD and the ratio of HWSC to structural carbohydrate (SC) compared to chicory. 3.2.1. Gas production parameters after 24 h of incubation The in vitro GP parameters after 24 h of incubation of aucubin and acteoside addition in CH or PL substrate are reported in Table 4. Addition of acteoside to CH and PL (C+ac and P+ac) increased potential GP and V24 h in comparison with the respective control, whereas aucubin addition to either CH or PL did not affect potential GP or V24 h of CH and PL. Acteoside in P+ac increased R1/2A compared to PL, but not in C+ac when compared to CH. The T1/2A was not modified by acteoside

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Table 4 in vitro gas production parameters of chicory (CH) and plantain (PL) with addition of aucubin (au) or acteoside (ac) after 24 h of incubation. Gas production parameters1 Substrate

2

Treatments

A (mL/g DM) b

T1/2A (h) b

R1/2A (mL/h)

V24 h (mL/g DM)

CH

CH C+au C+2au C+ac SEM P value

221.41 219.12b 215.13b 237.50a 4.11 0.01

4.39 4.57b 5.01a 4.47b 0.14 0.01

21.08a 20.16b 18.28c 21.46a 0.84 0.002

220.34b 217.92b 213.30b 235.43a 3.95 0.01

PL

PL P+au P+ac SEM P value

210.95b 214.24b 233.43a 5.93 0.03

4.71a 4.43a 4.51a 0.25 0.18

17.04b 18.08ab 18.81a 0.91 0.03

207.53b 211.13b 228.24a 3.05 0.01

a,b,c

Superscripts indicate values within columns that significantly differ (P < 0.05). SEM, standard error of the mean. Model parameters: A, potential gas production (mL/g DM); T1/2A , time (h) at which half of A was reached; R1/2A , rate (mL/h) at which T1/2A was reached; V24 h, total gas produced at 24 h (mL/g DM). 2 In vitro treatments: CH, chicory; C+au, chicory + 10 mg aucubin/g DM; C+2au, chicory + 20 mg aucubin/g DM; C+ac, chicory + 40 mg acteoside/g DM; PL, plantain (containing endogenous levels of 7 mg aucubin/g DM and 36 mg acteoside/g DM); P+au, plantain+ extra 10 mg aucubin/g DM; P+ac, plantain + extra 36 mg acteoside/g DM. 1

addition in CH or PL compared to the controls. However, increasing aucubin concentration (C+au vs C+2 au) in CH decreased the R1/2A and increased the T1/2A compared to CH, but aucubin in P+au maintained similar R1/2A and T1/2A to that of PL. 3.2.2. Volatile fatty acid and ammonia production over time The VFA production resulting from the addition of aucubin and acteoside to CH or PL treatments over 24 h of in vitro fermentation are reported in Table 5. Volatile fatty acid production (mmol/g DM) of acetate, propionate, butyrate, BCVFA and total VFA increased (P < 0.0001) over time in all in vitro treatments (Table 5). Over 24 h of incubation, addition of acteoside (C+ac) or aucubin (C+au, C+2 au) to CH substrate did not modify acetate, butyrate, BCFA, nor total VFA production compared to CH, and there were no interactions between CH treatments and incubation time. However, addition of acteoside (C+ac), but not of aucubin (C+ac and C+2au), increased propionate production compared to CH. There was an interaction for propionate production between CH treatments and incubation time. After 1.5 h of incubation time propionate production was higher in C+ac than CH at every incubation time, whereas addition of the two aucubin concentrations (10 and 20 mg/g DM) maintained a similar propionate production as CH throughout all incubation times. There was also a trend (P = 0.06) for acetate and total VFA production between CH treatments and incubation time, which showed that addition of the higher concentration of aucubin (C+2au) reduced acetate and total VFA production at 5 and 8 h of incubation time compared to CH. Addition of either acteoside (C+act) or the lower aucubin concentration (C+au) did not change either acetate and total VFA production compared to CH. In PL, addition of acteoside (P+ac) or aucubin (P+au) did not modify acetate, propionate, butyrate, BCFA, nor total VFA production compared to PL. There was no interaction between PL treatment and incubation time. Both aucubin (C+au; C+2au) and acteoside (C+ac) addition into CH had the same impact, decreasing the net NH3 production over time when compared to CH (Table 6). However, the net NH3 production by the two aucubin concentration treatments (10 vs 20 mg/g DM) only tended (P = 0.07) to be lower in the C+2au than in the C+au treatment. In plantain, addition of aucubin (P+au) maintained a similar net NH3 production over time to that of PL, whereas acteoside addition (P+ac) decreased the net NH3 production in comparison to PL. The net NH3 production (mmol NH3 /g DM) increased over time in both chicory and plantain in vitro treatments (Table 6). Acteoside (C+ac and P+ac) and the higher aucubin dose (C+2au) in CH treatments had a similar net NH3 production until 5 h of incubation time, and NH3 production started to accumulated after 8 h onwards of incubation time (Table 6). Whereas, the net NH3 production from the C+au and P+au treatments was similar during the first 3 h of incubation time, after which the NH3 production started to increase at each incubation time. There was no interaction between in vitro treatments and incubation time either in CH and PL treatments for the net NH3 production (Table 6). 4. Discussion 4.1. Bioactive compounds in plantain Acteoside, aucubin, and very low levels of catalpol were detected in plantain cv. ‘Ceres Tonic’ (Table 2). The concentration of these bioactive compounds in plantain is influenced by genetic and environmental factors (Fajer et al., 1992; Tamura and Nishibe, 2002). Of these compounds, catapol was almost absent in plantain cv. ‘Ceres Tonic’ (Al-Mamun et al., 2008). Catalpol is an iridoid glycoside biologically synthesised from its precursor aucubin (Damtoft, 1994). During the selection of the cultivar ‘Ceres Tonic’, the pathway from aucubin to catalpol appears to have been reduced to a very low basal rate of synthesis (Tamura and Nibishe, 2002). Catalpol is found in all natural ecotypes of plantain and in the cultivar ‘Grassland Lancelot’ (Tamura and Nibishe, 2002).

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Table 5 Individuals and total volatile fatty acid (VFA) production (mmol/g DM) of chicory (CH) and plantain (PL) with addition of aucubin (au) or acteoside (ac) in vitro treatments over time. incubation time (h) Substrate

1

Treatments

Acetate (mmol/g DM) CH CH C+au C+2au C+ac PL PL P+au P+ac Propionate (mmol/g DM) CH CH C+au C+2au C+ac PL PL P+ au P+ ac Butyrate (mmol/g DM) CH CH C+au C+2au C+ac PL PL P+au P+ac BCVFA (mmol/g DM) CH CH C+au C+2au C+ac PL PL P+au P+ac Total VFA (mmol/g DM) CH CH C+au C+2au C+ac PL PL P+au P+ac

P value 2

1.5

3

5

8

12

24

SEM

Treatment

Time

Interaction

1.77 1.74 1.72 1.94 1.62 1.78 1.74

2.83 2.92 2.76 3.12 2.45 2.77 2.51

4.48 4.40 4.12 4.71 3.77 3.86 3.82

6.09 5.90 5.59 6.31 5.09 5.20 5.20

6.80 7.01 6.87 7.16 6.12 6.47 6.56

8.27 7.87 7.81 8.17 7.43 7.55 7.35

0.102 0.080 0.140 0.097 0.055 0.176 0.122

0.16

<0.0001

0.06

0.50

<0.0001

0.56

0.48 0.49 0.46 0.58 0.48 0.56 0.53

0.84 b 0.87 b 0.80b 1.03a 0.83 0.94 0.94

1.27 b 1.26 b 1.16 b 1.42a 1.23 1.31 1.38

1.54 b 1.53 b 1.47 b 1.75a 1.53 1.58 1.66

1.72 b 1.75 b 1.71 b 1.92a 1.73 1.83 1.93

1.97 b 1.95 b 1.93 b 2.13a 1.98 2.04 2.09

0.024 0.013 0.048 0.038 0.026 0.059 0.053

0.02

<0.0001

0.04

0.19

<0.0001

0.74

0.11 0.11 0.11 0.12 0.18 0.21 0.19

0.19 0.21 0.21 0.23 0.29 0.33 0.30

0.34 0.35 0.33 0.37 0.42 0.46 0.44

0.54 0.55 0.53 0.59 0.56 0.60 0.58

0.70 0.73 0.72 0.74 0.70 0.75 0.74

0.92 0.92 0.93 0.94 0.90 0.95 0.90

0.036 0.035 0.045 0.037 0.017 0.032 0.021

0.92

<0.0001

0.97

0.54

<0.0001

0.75

0.12 0.12 0.12 0.13 0.11 0.13 0.12

0.21 0.22 0.20 0.23 0.18 0.21 0.19

0.36 0.36 0.32 0.35 0.25 0.26 0.25

0.47 0.48 0.45 0.49 0.31 0.33 0.30

0.69 0.72 0.65 0.73 0.42 0.46 0.42

1.45 1.44 1.33 1.47 0.98 1.04 0.98

0.005 0.004 0.012 0.009 0.006 0.012 0.008

0.67

<0.0001

0.22

0.76

<0.0001

0.99

2.41 2.39 2.33 2.70 2.32 2.61 2.51

3.95 4.08 3.84 4.47 3.65 4.13 3.84

6.24 6.16 5.76 6.66 5.54 5.77 5.76

8.40 8.21 7.79 8.89 7.35 7.56 7.61

9.59 9.86 9.63 10.23 8.81 9.30 9.47

11.77 11.36 11.25 11.92 10.78 11.02 10.85

0.123 0.075 0.235 0.167 0.084 0.270 0.196

0.13

<0.0001

0.06

0.37

<0.0001

0.62

a,b

Superscripts letters indicate values within columns that are significantly different (P < 0.05). In vitro treatments: CH, chicory; C+au, chicory + 10 mg aucubin/g DM; C+2au, chicory + 20 mg aucubin/g DM; C+ac, chicory + 40 mg acteoside/g DM; PL, plantain (containing endogenous levels of 7 mg aucubin/g DM and 36 mg acteoside/g DM); P+au, plantain+ extra 10 mg aucubin/g DM; P+ac, plantain + extra 36 mg acteoside/g DM. 2 SEM, standard error of the mean. 1

Table 6 Net ammonia production (mmol NH3 /g DM) of chicory (CH) and plantain (PL) with addition of aucubin (au) or acteoside (ac) in vitro treatments over time. Incubation time (h)

P value

Substrate

Treatments1

1.5

3

5

8

12

24

mean

SEM2

Treatment

Time

Interaction

CH

CH C+au C+2au C+ac

−3.57a −6.46a −6.78a −8.93a

3.75ab −0.06ab −0.25a −2.20a

9.45b 7.00b −0.59a −0.55a

23.72c 20.42c 13.63b 14.92b

39.80d 28.67c 24.45c 31.18c

61.97e 56.75d 49.40d 58.62d

22.52x 17.72y 13.31y 15.51y

3.363 3.671 3.931 4.279

0.02

<0.0001

0.92

PL

PL P+au P+ac

−1.55a −3.01a −5.48a

3.28ab 2.06a 0.47a

6.20bc 0.27a −2.68a

10.19c 8.19b 7.46bc

18.65d 14.59b 10.78c

37.44e 31.34c 33.04d

12.37x 8.91xy 7.27y

1.230 3.359 2.749

0.03

<0.0001

0.92

a,b,c,d,e Superscripts letters indicate values within rows that are significantly different (P < 0.05) and x,y superscripts letters indicate values within column that are significantly different (P < 0.05). 1 In vitro treatments: CH, chicory; C+au, chicory + 10 mg aucubin/g DM; C+2 au, chicory + 20 mg aucubin/g DM; C+ac, chicory + 40 mg acteoside/g DM; PL, plantain (containing endogenous levels of 7 mg aucubin/g DM and 36 mg acteoside/g DM); P+au, plantain+ extra 10 mg aucubin/g DM; P+ac, plantain + extra 36 mg acteoside/g DM. 2 SEM, standard error of the mean.

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The concentrations of aucubin found in this study were lower than those reported earlier by Tamura and Nishibe (2002), but there were differences in sampling method and environmental conditions. Tamura and Nishibe (2002) cut their plantain at 5 cm above ground whereas hand plucked sampling was used in this study. The cooler night temperatures in this study compared with those in Tamura and Nishibe (2002) will have most likely kept the aucubin concentration lower (Tamura and Nishibe, 2002). High concentrations of up to 30 mg aucubin/g DM, depending on the genotype (Stewart, 1996), and from 10 to 27 mg aucubin/g DM for the cultivar ‘Ceres Tonic’ have been reported by Tamura and Nishibe, (2002). Acteoside can be present from 60 to 90 mg/g DM in natural ecotypes (Fajer et al., 1992) and from 15 to 41 mg/g DM in the cultivar ‘Ceres Tonic’ (Tamura and Nishibe, 2002), which are similar concentrations to those found in the present study. In contrast, Al-Mamun et al. (2008) reported concentrations as low as 3.2 mg/g DM of acteoside in the cultivar ‘Ceres Tonic’. The increase in aucubin and acteoside concentrations in plantain swards throughout the growing season has been reported by others (Bowers and Stamp, 1992; Stewart, 1996; Tamura and Nishibe, 2002). The concentrations of aucubin and acteoside increased similarly throughout the growing seasons in both years and the highest concentration of both compounds was in autumn (May, 2012 and May, 2013). 4.2. In vitro fermentation Both acteoside and aucubin decreased the net NH3 production demonstrating their potential for reducing urea N concentration in the urine and minimising N excretion to the environment. Both acteoside and aucubin are glycosides with bioactive properties (Kim et al., 2000; Tamura and Nishibe, 2002). Acteoside added to chicory and plantain increased potential GP and V24 h, in in vitro fermentation, to the same extent in C+ac and P+ac, but they did not affect total VFA production relative to CH and PL alone. Acteoside is a ␤-(3 ,4 -dihydroxyphenyl)ethyl-O-␣-l-rhamnopyranosyl(1 → 3)-␤-d-(4-O-caffeoyl) glucopyranoside (Andary et al., 1982). This compound has been reported as antimicrobial (Andary et al., 1982) and antifungal (Shoyama et al., 1986), but the mechanism of acteoside action has not been clearly described. The increase in GP from acteoside fermentations suggested that the sugars, glucose and rhamnose, present in acteoside provided carbohydrate that was a readily fermentable energy source for rumen microorganisms (Rønsted et al., 2000). The greater GP in P+ac was consistent with an increase in the rate (R1/2A ) at which half of the potential GP was generated compared to PL. However, addition of acteoside increased propionate production from chicory, yet according to the stoichiometry of gas production, propionate production is associated with decreased gas production (Araujo et al., 2011). That the R1/2A for C+ac was similar to that for CH could have been counterbalanced by the higher propionate production. The lack of difference in total VFA production as a result of the addition of acteoside may also indicate that acteoside contributed more nutrients to microbial growth than VFA production. The total VFA has been reported to be inversely correlated with microbial mass by Hungate (1966), but microbial mass was not measured in our study. This study demonstrated acteoside had positive effects on rumen fermentation and antimicrobial action appeared less likely from acteoside. The decrease of net NH3 production with acteoside is likely due to the increase of fermentable carbohydrates. Ammonia utilisation by microbes in the rumen is primarily carbohydrate-limited (Russell et al., 1992). Since the majority of rumen bacteria use NH3 as their N source, the availability of NH3 is also important for microbial protein production and the microbial yield may be restricted by NH3 insufficiency (Raab et al., 1983). The NH3 concentration in all our incubations was always above the minimum requirement (5 mg/dL) for microbial growth (Satter and Slyter, 1974). Therefore, the greater fermentation from additional acteoside indicated that rumen microbes may be capable of degrading and using acteoside as an energy source due to them containing non-specific glycosidase (Getachew et al., 2002). A lower NH3 production with increased energy availability indicates more NH3 is incorporated into microbial protein (Raab et al., 1983). Acteoside fermentation delayed the time of NH3 accumulation, and during the first 5 h of incubation the negative NH3 production from acteoside indicated active microbial growth. Acteoside seemed to provide an increase in energy availability in the rumen and to improve the efficiency of microbial utilisation of NH3 . The presence of acteoside in plantain capable of affecting the rumen fermentation process is likely to have important implications for rumen N efficiency (Stewart, 1996), and suggests that selecting plantain for higher acteoside concentration would have positive animal effects by reducing N losses in the urine. In contrast, the addition of aucubin did not modify potential GP, V24 h, or total VFA production either in CH or PL. Aucubin is an iridoid glycoside with an O-linked glucose at C-1; the sugar can be hydrolysed by ˇ-glucosidase forming the aglycone of aucubin, aucubigin (Berenbaum and Rosenthal, 1992). The aucubin aglycone has been identified as affecting bacteria and fungi (Davini et al., 1986). The two concentrations of aucubin (10 vs 20 mg/g DM) added to CH reduced R1/2A as a dose response but did not affect the potential GP. This effect suggested that rumen microbes might be able to degrade aucubin and tolerate the antimicrobial effect of aucubigin (Getachew et al., 2002). The higher aucubin concentration (20 mg/g DM) required more time to produce half of potential GP (T1/2A ) compared to CH which indicated that the effect of aucubin on rumen fermentation is a dose response. Aucubin addition at two concentrations (10 vs 20 mg/g DM) reduced NH3 production. Several mechanisms have been suggested for how bioactive compounds can reduce NH3 concentration in the rumen. These include: (i) reduction in peptidolysis and deamination; (ii) direct inhibition of rumen microbial growth; and (iii) inhibition of rumen hyper-ammonia producing bacteria (Durmic and Blache, 2012). A reduction in NH3 by deamination is associated with a reduction of the BCVFA (isobutyrate and isovalerate) (Chalupa et al., 1980; Horton et al., 1980). An inhibition of amino acid deamination has practical implications because it may increase ruminal escape of dietary protein and improve the efficiency of N use as has

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been shown with thymol (Broderick and Balthrop, 1979). However, in this study, the BCVFA production was not affected by the addition of aucubin or acteoside to either substrate. The lower NH3 production with the addition of aucubin appears to be a consequence of the bactericide effect of aucubin (Tamura and Nishibe, 2002). Aucubin aglycone has been reported to bind to free amino acids making them unavailable, and contributing to its biological and toxic effect (Davini et al., 1986; Kim et al., 2000). The higher aucubin concentration (20 mg/ g DM) maintained a negative NH3 production during the first 5 h of incubation. This lower NH3 production in C+2au coincided with the T1/2A and the trend of lower acetate and total VFA production at 5 and 8 h of incubation time compared to CH suggesting that aucubin had an inhibitory effect on microbial fermentation. 5. Conclusions Both acteoside and aucubin had the potential to reduce the NH3 production on rumen fermentation. 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