www.world-food.net Journal of Food, Agriculture & Environment Vol.11 (2): 576-582. 2013
WFL Publisher
Science and Technology
Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland
e-mail: info@world-food.net
Received 28 January 2013, accepted 30 April 2013.
Effect of several saponin containing plant extracts on rumen fermentation in vitro, Tetrahymena pyriformis and sheep erythrocytes
Alexandre Budan 1, 2*, Nicolas Tessier 2, Monique Saunier 1, Louisa Gillmann 1, Jérôme Hamelin 3, Pierre Chicoteau 2, Pascal Richomme 1 and David Guilet 1
1 Université d’Angers, laboratoire SONAS, SFR Quasav, 16 bd Daviers, 49045 Angers, France. 2 Nor-Feed Sud, 3 rue Amedeo Avogadro, 49070 Beaucouzé, France. 3 INRA, UR50, Laboratoire de Biotechnologie de l’Environnement, Av des Etangs, 11100
Narbonne, France. *e-mail: alexandre.budan@gmail.com
Abstract
Among the nutritional solutions developed since in-feed antibiotics were banned by the European Union in 2006, extracts from plants with high saponin content have shown the capacity to modulate rumen fermentation. Most previous studies have focused on the effects of Yucca schidigera and Quillaja saponaria. This study was designed to evaluate i) the effects of extracts obtained from 11 saponin-containing plants and monensin on rumen fermentation patterns in vitro at 417 and 2.0 mg/L, respectively, and ii) the capacity of hemolytic test and inhibition of Tetrahymena pyriformis to model the effect of saponin-containing plant extracts on rumen protozoa. Plants belonging to 8 different families were investigated to evaluate a high diversity of saponin compounds. Total gas and methane productions were numerically lower with extract of Saponaria officinalis compared to control (-3.0 and -9.0%, respectively). The effect was more pronounced with monensin (-25.5 and -31.7%, p<0.05, respectively). Ammonia (NH3) and protozoa patterns varied among the extracts during in vitro incubations, e.g. from -22.6% (p<0.05) for Y. schidigera extract to -50.7% (p<0.05) for Primula veris extract, with respect to the protozoa number. Extracts from Primula veris, Chenopodium quinoa and Gypsophila paniculata mitigated significantly NH3 production by more than 26% (p<0.05) which, in our experimental conditions, was better than the results obtained with monensin and extracts from Y. schidigera and Q. saponaria. Statistically significant positive correlations were found between hemolytic capacity and inhibition of T. pyriformis (Pearson coefficient = 0.55, p<0.05) and between protozoa number and NH3 concentration during in vitro rumen incubation (Pearson coefficient = 0.56, p<0.05). Neither hemolytic capacity, nor inhibition of T. pyriformis, nor the content in total saponins estimated by gravimetric method was correlated to the inhibition of rumen protozoa. These parameters did not model the effect of extracts from different saponin containing plants on rumen protozoa number. However, the results suggest that by-products containing saponins from food (C.
quinoa) and horticultural (G. paniculata) industries could be investigated as feed additives to improve nitrogen utilization by ruminants.
Key words: Saponins, rumen fermentation, ciliate protozoa, methane, ammonia, hemolytic capacity, Tetrahymena pyriformis.
Introduction
Unlike monogastrics, ruminants are able to metabolize cellulose and non-protein nitrogen sources, such as urea, through complex symbiosis with microorganisms occurring mainly in the rumen.
However, this feature has some drawbacks. Methane production from feed fermentation in the rumen is responsible for 10-15%
metabolisable energy loss 1 and contributes significantly to anthropogenic greenhouse gas emissions worldwide 2. From 8 to 35% of the nitrogen ingested by producing herbivores is incorporated into animal proteins 3. The remaining nitrogen is excreted, inducing ammonia, nitrate and nitrous oxide emissions.
From 10 ppm in dairy cow housing, gaseous ammonia affects the animal welfare 4. Nitrate produced from nitrification is detrimental to water quality 5. Nitrous oxide produced e.g. from denitrification is the third most important greenhouse gas 6 and the dominant ozone-depleting substance emitted in the 21st century 7.
Saponins are a very diverse group of compounds which occur in plants as secondary metabolites. These heterosides are composed of a polar sugar moiety glycosidically linked to a non-
polar aglycone (terpenoid or steroid), as shown in Fig. 1. Saponins induce specific membrane disruption of eukaryotic cells, including erythrocytes, fungi and protozoa 8. These properties allow to modulate the fermentation in a complex microbial environment such as the rumen, decreasing ammonia (NH3) concentration and methane (CH4) production 9. Yucca schidigera and Quillaja saponaria are the major sources of saponins used in feed for ruminants and their effects on NH3 and CH4 productions are well documented 10.
Little is known about the effects of saponins from other sources even though these compounds are contained in many different plants. This study was aimed at comparing the effects of aqueous and hydro-ethanolic extracts from 11 saponin containing plants, including Y. schidigera and Quillaja saponaria but also less studied botanical species on total gas, CH4, NH3 and protozoa patterns during in vitro rumen incubation in a screening based approach. Plants belonging to 8 families were investigated so as to evaluate extracts containing different chemical structures of
saponins. A complementary objective was to compare the capacity of extracts rich in saponins from different plants to lyse erythrocytes, to inhibit the growth Tetrahymena pyriformis, a model protozoon, and to decrease the rumen protozoa number.
Materials and Methods
Plants and reagents: Saponin-containing plants were harvested between June 2009 and August 2010 in the experimental fields of the ITEIPMAI technical institute in Melay, France, to produce aqueous extracts of floral heads of Calendula officinalis L., roots of Saponaria vaccaria L. and seeds of Trigonella foenum- graecum L. Commercial powders from Nor-Feed Sud, Beaucouzé, France, were used to obtain hydro-ethanolic extracts from wood of Quillaja saponaria Molina (Q. Saponaria wood) and roots of Saponaria officinalis L., aqueous extracts from roots of Primula veris L. and hulls of Chenopodium quinoa L.. Syrup from Yucca schidigera Roezl ex Ortgies and aqueous by-products from concentrated Medicago sativa L. extracts were also provided by Nor-Feed Sud. Aqueous extracts were obtained from cakes of Argania spinosa L. and roots of Gypsophilla paniculata L. kindly provided by Prof. Max Henry from the Université de Nancy, France.
Quillaja saponaria Molina extract, standardized at 100 g kg-1 sapogenin (Q. saponaria Sigma) and monensin sodium salt 900–
950 g kg-1, were purchased from Sigma-Aldrich, Steinheim, Germany. Dimethyl sulfoxide (DMSO) and phosphate buffer saline (PBS) were purchased from Fisher Scientific UK Limited, Loughborough, UK. Distilled water was purified with a Milli-Q water purification system.
Preparation of extracts and determination of saponin content:
Five g of powdered samples were extracted under stirring in 100 mL of either Milli-Q water or 1:1 ethanol/water for 1 h at room temperature. Then the solutions were centrifuged for 10 min at 3000 rpm and the supernatants were collected. Ethanol was evaporated under vacuum with a rotary evaporator and a water bath at 40°C to obtain the liquid extracts. The liquid extracts were freeze-dried to obtain the dried extracts used for the in vitro rumen incubation. Dried extracts were dry-stored in the dark at room temperature in a vacuum-dessicator.
A gravimetric method was modified from Yao et al. 11 to estimate the total saponin content. Fifty mL of liquid extract was transferred into a 250 mL separatory funnel and supplemented with 15 mL of ethyl acetate before the mixture was shaken. The aqueous layer was recovered and partitioned again twice with ethyl acetate. The purification process was then repeated three times using the water soluble portions and 10 mL of n-butanol. The saponin content was calculated as the mass of dried butanolic extract.
In vitro rumen incubation: The experiment was conducted at the laboratory of natural origin substances and structural analogues (SONAS), Université d’Angers, France, according to European guidelines for animal welfare. The Hohenheim syringe-based in vitro gas method 12 was used for rumen fermentation. Briefly, rumen fluid was collected before the morning feeding from two rumen- fistulated dry Holstein cows, fed twice daily at maintenance on a roughage-concentrate diet (70-30 w/w) at 7 kg dry matter (DM) per day (98 g kg-1 crude protein DM). Rumen fluid was filtered through a 1 mm sieve prior to being transferred to preheated thermos bottles. All sampling was quickly performed under vacuum. Rumen fluid was then added at a 1:2 ratio to a buffer medium kept at 39°C temperature to obtain the incubation medium.
The buffer medium was composed of bicarbonate buffer, macromineral, micromineral, resazurin and reducing solutions 13. All handling was performed under continuous flushing with CO2. Dry ray grass roughage and wheat seeds were ground in a Wiley mill to pass through a 1 mm screen and blended (70-30, w/
w DM) to prepare the basal feedstuff (960 g kg-1 DM content).
Dietary concentrations of crude proteins, crude fat, acid detergent fibers, neutral detergent fibers, starch, total sugars and minerals were 89, 21, 250, 492, 172, 102 and 56 g kg 1 DM. The fermentation substrates were prepared by homogeneously blending the saponin containing plants extracts and basal feedstuff (5-95 w/w DM) six hours before the incubations. The concentrations of plant extracts and monensin were set at 417 mg L-1 and 2.0 mg L-1 (3.3 µM) of incubation medium, respectively, so as to be at suitable levels for livestock production and to reach adequate concentration in active compounds according to previous findings 14, 15. Monensin is an in-feed antibiotic used to improve growth promotion and health in livestock. As mechanisms of action of monensin 16 and saponins10 include disintegration of cell membrane of specific rumen microorganisms, such as Gram positive bacteria and protozoa, monensin was a relevant reference for in vitro fermentation in this study. A control was composed of basal feedstuff only. Of each substrate 250 mg was weighed and placed into a 100 mL glass syringe (Fortuna, Poulten & Graf GmbH, Wertheim, Germany) equipped with a luer lock valve. The syringe headspaces were flushed with CO2 before incubation. Two syringes without substrate (blanks) were also prepared.
Incubation medium (30 mL) was dispensed through the valve of preheated (39°C) syringes using a peristaltic pump. Then all gas was expelled and the syringes were placed for 24 h at 39°C in an incubator shaker (KS 4000i control, IKA Werke, Staufen, Germany) at 50 rpm using a randomized-repeated measures design (triplicates). Aliquots incubation medium were sampled and preserved with 1 ml of mercury chloride (II) at -20°C for quantification of NH3. Fermentation gases were sampled in evacuated glass vials equipped with a gas-tight septum (Exetainer®,
Triterpene saponins Steroidal saponins
Quillaic acid glycoside
Protoprimulagenin glycoside
Sarsasapogenin glycoside
Diosgenin glycoside
Figure 1. Generic structures of saponins. R= sugar moiety.
Labco Limited, Buckinghamshire, England) for quantification of CH4.
Ammonia and methane and total gas production: Aliquots were analyzed for NH3 concentration by spectrophotometry 17. The total gas volume produced in the syringes was recorded after incubation. The CH4 proportion was determined using a micro- gas chromatograph coupled with a thermal conductivity detector (CP-4900, Varian Inc., Palo Alto, California, USA). Separation was performed on a molecular sieve column, detection threshold of 10 ppm. The CH4 production was calculated as:
CH4 production (ml) = CH4 proportion (%) x Total gas production (ml)
Total gas and CH4 productions, CH4 proportion and NH3 concentration are expressed as % difference with respect to control.
In vitro rumen incubation experiments were carried out in three replicates per extract.
Enumeration of rumen protozoa: Samples from incubation media were homogenized and mixed with a methyl green-formalin solution (50-50, v/v). A generic protozoa profile was microscopically determined before and after incubation using a 10 µl Agasse Lafont counting chamber (Preciss, France). Entodinium spp., large entodiniomorphids (e.g. Diplodinium spp., Eudiplodinium spp., Epidinium spp., Polyplastron spp.), Dasytricha spp. and Isotricha spp. were identified according to Ogimoto and Imai 18. Total rumen protozoa number was summed from the generic profile and expressed as % difference with respect to control. Sampling for enumeration of rumen protozoa was done twice per replicate.
Tetrahymena pyriformis inhibition assay: Antiprotozoal activity of saponin-containing plants was evaluated on T. pyriformis, with a protocol modified from Sauvant et al. 19. T. pyriformis was cultured under axenic conditions at 28°C, in Erlenmeyer closed by a bakelite cap containing a growing medium composed of proteose peptone, yeast extracts and inorganic salts. Inoculums consisted in solutions of T. pyriformis at a cell concentration of 40.0x103 mL-1. Extracts were dissolved in a mixture of 1:1:13 DMSO/sterile water/
PBS, filtered at 0.22 µm and stored into sterile test tubes to obtain stock solutions. Working solutions at 1mg mL-1 were prepared by blending growing medium, inoculums and stock solution in a ratio of 7:2:1. After 9 hours, 10µL of a mixture of working solution/
natural mineral water (Volvic, France)/formol 4% 1:8:1 was mounted on a glass slide for cell count using a light microscope. The standard deviation associated to T. pyriformis enumeration was 0.72x103 mL-1.
Determination of hemolytic activity: Hemolytic activity of saponins was evaluated on sheep erythrocytes as described by Gauthier et al. 20 with slight modifications. The hemolytic activity was expressed as the concentrations in extract inducing 50% of erythrocytes hemolysis (HD50). Extracts were dissolved in a mixture of 2:2:1 DMSO/Water/PBS. Concentration gradient was prepared within a range of 10 to 4000 µg mL-1 in 96-well mi-croplates, and 30 µL of a 100 g kg-1 sheep erythrocytes solution (bioMérieux, Craponne, France) was added to each well. Microplates were incubated in an oven for 60 min at 37°C and then centrifuged at 3000 rpm for 5 minutes. Afterwards 100 µL from the supernatant
containing liberated hemoglobin was transferred to empty 96-well microplates. Absorbance (Abs) of the supernatant was measured at 540 nm with a microplate reader (Infinite M2000, TECAN, Zürich, Switzerland). Each experiment included a negative control (PBS, 0% hemolysis) and a positive control (quillaja bark from Sigma- Aldrich at 435 µg mL-1, 100% hemolysis). The % hemolysis was calculated as:
HD50 was then determined by a linear interpolation. Absorbance at 540 nm of each extract was checked and considered as negligible when diluted in PBS at 4000 µg mL-1. Hemolytic test was carried out twice in triplicates per extract. An aqueous extract from the basal feedstuff of in vitro incubation experiments was used as the control.
Statistical analysis: Means were compared by two-way unbalanced variance analysis (ANOVA) with subsequent post- hoc multiple comparison test of Tukey-HSD (honestly significant difference) using XLSTAT (version 2011.2.04, Addinsoft, USA).
A standard Pearson coefficient (R) was used to study correlations between variables. The differences were considered significant at p<0.05 and trends were accepted at 0.05<p<0.10.
Results
Saponin content measured by the gravimetric method ranged from 95.3 to 373.5 mg g-1 DM in the extracts (Table 1), except for M.
sativa that had a lower value (13.0 mg g-1 DM).
The biological activities of extracts from saponin containing plants on in vitro rumen fermentation are presented in Table 2.
Total gas production, CH4 production, CH4 proportion, NH3 concentration, total rumen protozoa number and pH were 67.9 mL, 9.5 mL, 14%, 188.2 mg L-1, 2.92x105 mL-1 and 6.8, respectively, for the control. Monensin decreased the total gas production (-25.5%, p<0.05) and the CH4 production (-31.7%, p<0.05). The CH4 production was numerically lower with Y. schidigera (-4.7%), S.
vaccaria (4.2%), C. quinoa (-6.5%) and S. officinalis (-9.0%).
Differences were observed in CH4 proportion for monensin (-8.4%), Y. schidigera (-7.5%) and S. officinalis (-4.5%) but these drops were not significant. P. veris and C. quinoa significantly decreased NH3 concentration by 30.9 and 29.8% (p<0.05). In contrast, extract from C. officinalis increased NH3 concentration (+27%, p<0.05). Total rumen protozoa number was significantly lower than the control for seven extracts: Y. schidigera, Q.
saponaria (Sigma), Q. saponaria (wood), P. veris, S. vaccaria, A.
spinosa and C. quinoa (Table 2). P. veris was the extract showed the highest anti-rumen protozoa effect (-50.7%) and monensin had a more pronounced effect ( 67.0%).
The initial T. pyriformis number was 6.1x103 mL-1. After 9 hours of incubation, 50.1x103 mL-1 protozoa were enumerated in the control (absence of saponins). At 1000 µg mL-1, all the extracts decreased significantly the number of protozoa. The ranking order of the investigated plant extracts on the basis of their inhibition on the growth of T. pyriformis was Y. schidigera > S. officinalis = G. paniculata > C. quinoa > C. officinalis > T. foenum graecum >
S. vaccaria > Q. saponaria (Sigma) > A. spinosa.
No significant difference was found between hemolytic activity of the control (without saponins) and extracts from C. officinalis,
Absextract - Absnegative control
Abspositive control - Absnegative control
% hemolysisextract =
( )
× 100T. foenum-graecum and M. sativa. The ranking order of the investigated hemolytic plant extracts was Q. saponaria (Wood) >
S. officinalis > G. paniculata > Y. schidigera > P. veris > S. vaccaria
> A. spinosa > C. quinoa.
Discussion
The objective of this work was to compare the effects on rumen fluid, on T. pyriformis and on erythrocytes of extracts from saponin containing plants to a control (substrate not supplemented in saponins) and a synthetic antibiotic (monensin). Taking all the extracts into consideration, a high diversity of saponins was investigated. The variability in chemical structures came from sapogenins, number and nature of osidic units and number of acetate groups (Fig. 1). Monensin decreased total gas production (-25.5%, p<0.05) and CH4 production (-31.7%, p<0.05), which was consistent with Araujo et al.14. S. officinalis decreased numerically CH4 production by -9.0%. The concentration of the saponins present in the extracts at 417 mg L-1 (equivalent to around 70 g of
extract/dairy cow/day) was not suitable for mitigating the rumen CH4 production significantly. Y. schidigera and monensin, with - 7.5% and -8.4%, respectively, modulated CH4 proportion corroborating previous findings 14, 35. The absence of statistically significant effect on CH4 proportion may have been due to the experimental design (triplicates) set up to screen a large number of plant extracts rather than collect ultimate data on one or two plants.
Extracts from P. veris, C. quinoa, G. paniculata, Q. saponaria and Y. schidigera had a significant effect on total rumen protozoa number and NH3 concentration. As reported by Makkar et al.23 with a similar substrate (70% hay, 30% concentrate), the defaunation was more pronounced with Q. saponaria (-50.7%) than with Y. schidigera (-22.6%). Both plants decreased NH3 concentration (from -22 to -23%), but less than extracts from P.
veris, C. quinoa and G. paniculata (from -27 to -31%), which was in line with previous findings 15, 36-39. It is likely that the effect on the NH3 concentration would have been more substantial with a
Extracts R.prot (% Diff.)
NH3
(% Diff.) CH4
(% Diff.)
Total gas (% Diff.)
CH4/TG (% Diff.)
T.pyr.
(103 ml-1) HD50
(µg mL-1) Control 100.0a 100.0b 100.0a 100.0ab 100.0a 50.1a 2815.5ab S. officinalis 97.9ab 104.1b 91.0ab 97.0ab 95.4a 3.1g 48.6f C. officinalis 94.6ab 127.0a 99.0a 98.7ab 101.6a 11.6e 2636.1b G. paniculata 88.9ab 73.5bc 97.2a 98.6ab 98.7a 3.1g 51.2f M .sativa 86.5ab 85.4bc 104.6a 97.6ab 107.3a 18.9c 3010.6a T. foenum-graecum 83.8ab 88.8bc 109.1a 100.0ab 109.2a 12.6de 2785.9b Y. schidigera 77.4b 76.5bc 95.3ab 103.1a 92.5a 1.9g 167.2ef A. spinosa 76.6b 93.0b 102.4a 99.5ab 102.9a 45.6a 846.0d S. vaccaria 75.2b 84.0bc 95.8ab 97.4ab 98.4a 14.3d 718.9d C. quinoa 58.9c 70.2c 93.5ab 94.7b 100.5a 7.4f 1205.4d Q. saponaria (Sigma) 50.7cd 76.7bc 99.0a 99.0ab 100.0a 27.3b ND Q .saponaria (wood) 49.9cd 77.8bc 103.3a 98.8ab 104.6a ND 48.3f P. veris 49.3cd 69.1c 96.6a 94.7b 101.9a ND 312.1e Monensin 36.0d 88.7bc 68.3b 74.5c 91.6a ND ND SEM 2.7 3.3 2.0 1.0 1.4 2.5 140.3
Table 2. Effect on total gas, CH4, NH3 and protozoa from in vitro rumen fermentations, T. pyriformis number and hemolytic activity of extracts from saponin containing plants.
a, b, c, d, e, f, g: Means in the same column with different superscripts differ (p < 0.05). % Diff.: difference with respective control value;
CH4: methane; TG: total gas; NH3: ammonia; R.prot: rumen protozoa; T.pyr: Tetrahymena pyriformis; HD50: half hemolytic dose; ND:
not detected.
DM: dry matter; ND: not determined. Saponin content (mg g-1 DM) as determined by gravimetric method. All extracts were aqueous except those followed by
*, which were hydro-ethanolic extracts. No supplementary extraction was done on commercial and by-products.
Saponin content (mg g-1 DM)
Family Specie Main sapogenins
Plant Extract Asteraceae Calendula officinalis Oleanolic acid 21, 22 28.0 112.0 Caryophyllaceae Saponaria officinalis Gypsogenin, quillaic acid,
gypsogenic acid 23, 24 17.3 160.7
Caryophyllaceae Gypsophila paniculata Gypsogenin, gypsogenic acid 25
33.7 95.0
Fabaceae Medicago sativa Medicagenic acid, zhanic acid,
soyasapogenol B 26 ND 13.0
Fabaceae Trigonella foenum-graecum Diosgenin, yamogenin, (neo) ti-
gogenin, (neo) gitogenin 27, 28 34.0 208.9 Asparagaceae Yucca schidigera Schidigeragenins, sarsasapogenin,
smilagenin 29 ND 340.0
Sapotaceae Argania spinosa Protohydroxybassic acid, phyto-
laccagenic acid 30 50.6 116.5
Caryophyllaceae Saponaria vaccaria Quillaic acid, gypsogenic acid 31 23.0 373.5 Chenopodiaceae Chenopodium quinoa Hederagenin, oleanolic acid, phy-
tolaccagenic acid 32 95.3 351.6
Quillajaceae Quillaja saponaria Quillaic acid, gypsogenic acid 33 20.0 277.8 Primulaceae Primula veris Protoprimulagenin A,
priverogenin B 34 36.2 168.9
Table 1. Saponin content in plants and extracts.
Variables T.pyr. R.prot. NH3 CH4 Sap.c.
HD50 0.55* 0.36 0.48+ 0.29 -0.27 T.pyr. 0.10 0.19 0.16 -0.08 R.prot. 0.57* 0.45+ -0.34
NH3 0.01 -0.31
CH4 -0.14
Table 3. Pearson correlation coefficients between CH4, NH3 and protozoa from in vitro rumen fermentations, T. pyriformis number and hemolytic activity of extracts from saponin containing plants.
Pearson correlation coefficients followed with a symbol differed from 0: *p ≤ 0.05; + 0.05 < p ≤ 0.10. CH4: methane; NH3: ammonia; R.prot.: rumen protozoa;
T.pyr.: Tetrahymena pyriformis; HD50: half hemolytic dose; Sap.c.: Saponin content as determined by gravimetric method.
higher level of crude protein in the basal feedstuff.
The total rumen protozoa number in negative control increased from 1.43x105 mL-1 to 2.92x105 mL-1 during the 24 hours of incubation. This is probably due to favourable conditions to the growth of rumen protozoa as relatively high pH, high levels of cellulose fibers and starch grains in substrate.
Total rumen protozoa number and NH3 concentration from in vitro fermentation showed a significant positive linear correlation (R = 0.56, p < 0.05) between variables (Table 3). Protozoa play a key role in the metabolism of NH3 in the rumen, which probably explains this correlation. They indeed engulf bacteria, and consequently inhibit the utilization of NH3 for microbial protein synthesis 10. By lowering the predation activity of protozoa, saponins from Y. schidigera, A. spinosa, S. vaccaria, C. quinoa, Q. saponaria and P. veris may promote indirectly bacterial growth.
Hence, NH3 utilization for microbial protein synthesis increases while proteolysis of protozoan origin decreases 24, 40. To the contrary compounds from aerial parts of C. officinalis increased NH3 concentration. Analogous observations have been reported with another saponin containing plant, Enterolobium cyclocarpum 41. As no effect was observed on rumen protozoa with C. officinalis, it was hypothesised that this extract exercised either a stimulating effect upon proteolysis and/or an inhibitory effect upon proteosynthesis from bacteria and/or fungi.
Q. saponaria (wood) had a HD50 in the same order of magnitude than dialyzed Q. saponaria extract (9 µg mL-1), as reported by Chwalek et al. 42. No significant statistically difference was found between HD50 of the control (aqueous extract of the basic feedstuff without saponins) and extracts of C. officinalis, T. foenum-graecum and M. sativa. The basic feedstuff did not contain saponins, consequently its HD50 was certainly due to osmotic hemolysis, rather than a hemolytic activity of saponins. A similar phenomenon probably occurred with extracts of C. officinalis, T. foenum- graecum and M. sativa. Extracts from Q. saponaria, G. paniculata, Y. schidigera, P. veris, C. quinoa, A. spinosa and S. vaccaria were considered as hemolytic. The hemolytic activity and the inhibition of T. pyriformis in presence of extracts of saponin-containing plants were linearly and significantly correlated (R = 0.55, p <
0.05), as shown in Table 3. However, no correlation was found between HD50 or T. pyriformis inhibition and total protozoa from rumen fermentation. Molecular mechanisms of saponins towards membranes of erythrocytes have been reviewed by Augustin et al. 43. Three modes of action are described, all of them implicating interactions between saponins and sterols from the membrane.
Sterol composition of membranes are reported to be 8% cholesterol for sheep erythrocytes 44, 0.5% tetrahymanol for T. pyriformis 45
and 1-3% stigmastanol, campestanol and/or cholestanol for rumen protozoa 46. Therefore either composition in sterols of the membranes, or composition of the incubation medium (erythrocytes in PBS and DMSO, T. pyriformis with proteose peptone, yeast extracts and inorganic salts and protozoa in rumen fluid) may influence the activity of saponins. No correlation was found between saponin content in extracts and inhibition of rumen protozoa, corroborating the importance of the chemical structure in the activity of saponins. Further research is required to study the mechanism of action of saponins towards rumen protozoa.
Conclusions
Chemical and biological analyses were consistently performed in a similar way on several plant extracts. This global approach allowed to evaluate the activity of saponin-containing plant extracts under standard conditions. Neither HD50, nor inhibition of T. pyriformis, nor the content in total saponins modeled the effect of extracts from different species of saponin containing plants on rumen protozoa. Despite a lower activity than monensin, extracts of S. officinalis, C. quinoa and Y. schidigera were the most interesting for CH4 mitigation at 417 mg L-1. Potential of monensin, extract of Yucca schidigera and Quillaja saponaria to decrease NH3 concentration, and so probably to improve nitrogen efficiency in the rumen, was confirmed, but extracts from P. veris, C. quinoa and G. paniculata were found to be more efficient in vitro. If these results are confirmed on long-term in vivo trials, new uses for by-products containing saponins from food (C.
quinoa) and horticultural (G. paniculata) industries could be investigated as feed additives in ruminants, especially during the turn out to grass, when ammonia concentration is the highest in rumen. Decreasing ammonia concentration in the rumen engenders lower urea in the urines 47 and potentially reduced emissions of ammonia and nitrous oxide in the atmosphere. In vivo trials are required to investigate these environmental benefits from some saponin containing plants as feed additives.
Acknowledgements
Thanks to Denis Bellenot and Matthieu Wident from ITEPMAI technical institute for providing extracts of C. officinalis, S.
vaccaria and T. foenum-graecum, and Oriane Partenay, student from the University of Angers, for help with protozoa counting.
This research was supported by funding from Région Pays de la Loire, France and ANR (National Research Agency) through the project SAPONINES vs GES approved by the cluster Végépolys (agreement 2008-00286).
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