Saponins, non-protein amino acids, polysaccharides and proteins like lectins and enzyme inhibitors act as plant protection factors(Reference Gatehouse, Minney, Dobie, Fujii, Gatehouse, Johnson, Mitchel and Yoshida1). Among these compounds, alfalfa has relatively high levels of saponins. Saponins, named after their foam-producing properties(Reference Francis, Kerem and Makkar2, Reference William, Perez-Mellado and Vitt3), are widely distributed in plants, including some foods such as beans, soyabeans, peas, spinach, tomatoes, potatoes, onions, garlic, alliums, asparagus and other plants like alfalfa (Medicago sativa L.). The kind and amount of saponins are different in each species(Reference Francis, Kerem and Makkar2, Reference Applebaum, Marco and Birk4–Reference Fenwick, Price, Tsukamoto, FelixD'Mello, Duffus and Duffus6). Plant saponins can be orally toxic towards animals when present in large amounts. Alfalfa saponins were also found to be nutritionally undesirable in poultry, rats, rabbits and swine(Reference Hostettmann and Marston7–Reference Klita, Mathison and Fenton10). These compounds have been assumed to be degradable by rumen micro-organisms and exert little biological activity in ruminants(Reference Cheeke11). The effects of saponins on snails, fungi, viruses, protozoa, rat hepatoma cells, malignant cells, fish respiratory epithelia, cell membranes, animal growth and feed intake, nutrient uptake, protein digestion, oxidation reactions, cholesterol metabolism, animal reproduction, immune system and nervous system have been investigated. The mentioned activities have been reviewed as the biological roles of saponins in birds, animals – even in single-stomached animals – and cold-blooded organisms(Reference Francis, Kerem and Makkar2). The investigation of the biological activity of saponins on insects was a good idea to be carried out although different studies have proved these actions of saponins(Reference Maxwell12–Reference Mazahery-Laghab17).
Alfalfa saponins are triterpenoids composed of a C30 aglycone linked to one or more sugar groups as shown in Fig. 1(Reference Navarro, Giner and Recio18). More than thirty-three different saponins containing one or more sugar chain units have been identified in alfalfa. Medicagenic acid, hederagenin, zanhic acid and soyasapogenols A and B are the main aglycones of alfalfa aerial parts(Reference Massiot, Lavaud and Guillaume19, Reference Peri, Mor and Heftmann20). Medicagenic acid is the first sapogenin synthesised in germinating seeds and the other sapogenins are formed from medicagenic acid(Reference Nowacki, Jurzysta and Dietrych-Szstak21). The biosynthesis of sapogenins from [2-14C]mevalonic acid was investigated in alfalfa and soyabean(Reference Peri, Mor and Heftmann20). The addition of sugar moieties to sapogenins to form saponin glycosides varies during plant growth or development and results in changes in the overall saponin composition, which can have antibiotic or probiotic effects. Hydrolysis of a toxic or non-toxic saponin may also alter the biological properties of the compound. For example, Osbourn et al. (Reference Osbourn, Bowyer and Lunness22) stated that the enzyme avenacosidase in oat activates the foliar oat saponins avenacosides A and B by the removal of C-26 glucose. This protein belongs to a family which also contains other plant enzymes involved in the activation of defensive secondary metabolites by the hydrolysis of glycosidic linkages, e.g. myrosinase (glucosinolates) and linamarinase (cyanogenic glycosides). Such compounds have been associated with a variety of biological activities, including allelopathy, poor digestibility in ruminants, enzyme activity inhibition, deterrence to foraging by insects and beneficial antifungal properties(Reference Kocacaliskan, Unver and Terzi23–Reference Agrell, Oleszek and Stochmal25). Activation resulting in alteration of metabolites is normally prevented by enzyme(s) and substrate(s) being separated in different cellular compartments, but is triggered by damage to tissues resulting from wounding or pathogen attack.
Fig. 1 Chemical skeleton structure of sapogenins.
Saponins from alfalfa are also variable due to different factors. For example, when alfalfa foliages are damaged by an insect pest, a response will be induced in the plant and, consequently, more saponins will be either qualitatively or quantitatively induced(Reference Agrell, Oleszek and Stochmal25, Reference Oleszek26). Due to the presence of medicagenic acid glycosides which are found both in roots and in shoots, saponins in alfalfa are able to inhibit or reduce damage by insects, and soyasapogenol, and hederagenin glycosides found in the roots, which are rich in medicagenic acid derivatives, were markedly toxic to the flour beetle (Tribolium castaneum)(Reference Fenwick, Price, Tsukamoto, FelixD'Mello, Duffus and Duffus6). Alfalfa saponins are also natural feeding barriers for phytophagous insects, and they were found to be toxic compounds to many insects(Reference Goławska27). Alfalfa saponins have been described as effective compounds interfering with aphid feeding behaviour(Reference Goławska, Leszczyński and Oleszek28). An antibiosis mechanism of alfalfa saponins was the factor for this resistance(Reference Agrell, Oleszek and Stochmal25, Reference Goławska, Leszczyński and Oleszek28). Golawska extracted saponins 1, 2 and 3 from alfalfa with toxicity towards the pea aphid. Their toxicity potentials can be used as alfalfa resistance factors(Reference Goławska27).
Variety, season, cultivar, environment(Reference Stochmal and Oleszek29–Reference Burgos and Talbett32), field drying(Reference Alzueta, Rebole and Barro33), plant ensilage(Reference Kalac, Price and Fenwick34), age and plant part(Reference Kapusta, Janda and Stochmal35–Reference Oleszek and Stochmal38) are the other factors responsible for allelochemical variations in alfalfa and other plants. The amounts of saponins and also other secondary metabolites in plant tissues are variable and biologically affect insect pests such as the pea aphid (Acyrthosiphon pisum Harris)(Reference Goławska, Leszczyński and Oleszek28, Reference Goławska, Lukasik and Leszczynski39, Reference Goławska and Łukasik40).
We were interested to investigate the biological role of saponins in the response of alfalfa (as a preferred host) to spotted alfalfa aphid (Therioaphis maculata Buckten) feeding, particularly in relation to changes in saponin composition during alfalfa development. This aphid, as an economically important pest of alfalfa, is also specific for different host plants such as onions, sainfoin, broad beans, clover, etc.(Reference Blackman and Eastop41). In this research, we were also interested to know whether resistance to this pest could be produced by increasing the saponin levels or altering the saponin composition in the plant.
Materials and methods
Plant and insect materials
Alfalfa seeds (cultivar Euver) were sown in pots which were filled to the height of 10 cm with washed sea sand. The seeds were then covered with a 5 mm sea sand layer. The pots were placed in a greenhouse at the average temperature of 27°C under 16/8 h light–dark and irrigated every other day. Each pot was composed of two sections of a plastic drink bottle, with pores for air conditioning and preventing insects from escaping. For the evaluation of the effects of feeding on experimental alfalfa, for each treatment, four pots were used as four replicates. Plants were also transferred into bigger pots and moved outside the greenhouse for further experiments in developed growth stages.
Spotted alfalfa aphids were first reared on a present cultivation alfalfa cv. Hamedan. For more uniformity of the aphid colony, aphids were transferred to Blackman boxes containing alfalfa stem cuttings. Boxes were placed in a plastic tray containing water to the height of about 5 mm and then placed in the greenhouse at 27°C and 16/8 light–dark illumination. Produced neonates were used for bioassays at different stages of plant growth.
Tissues were also cut off from shoots at different growth stages, frozen in liquid N2 and kept in a freezer at − 20°C for saponin extraction.
Aphid bioassay
The response of the spotted alfalfa aphid to shoot tissues at different growth stages was investigated under greenhouse and field conditions in the Agriculture Faculty of Bu-Ali Sina University in Hamedan, Iran. In order to produce a colony of aphids, three adult aphids were removed from host plant leaves and placed on cutting stems in Blackman boxes. Five nymphs (1 d of age) produced by these adults were transferred from boxes and placed onto the leaves of the experimental plants in pots using a fine silk brush. Aphid survival, accumulative nymph production and the onset of nymph production were measured by daily observation on the experimental plants over a 2-week bioassay period. Treatments were analysed in the pattern of a completely randomised design with four replicates as Yazdi-Samadi et al. (Reference Yazdi-Samadi, Rezaei and Vali-Zadeh42) suggested.
Extraction of crude saponins from alfalfa
Saponin extraction was carried out using the method described by Mazahery-Laghab & Gatehouse(Reference Mazahery-Laghab and Gatehouse16) and Mazahery-Laghab(Reference Mazahery-Laghab17). A quantity of 10 g of shoot (including leaves) and partly root tissues (in order to identify the sources of individual saponins) from alfalfa at different growth stages (seedling (Sdg) in greenhouse, after flowering (Af) and before flowering (Bf) outside the greenhouse in pots) was collected, frozen in liquid N2, weighed out and placed in a cooled mortar. The tissues were finely ground using liquid N2 to prevent any enzymatic degradation. The powdered tissue was then transferred to a conical flask and 50 ml of 80 % methanol (MeOH, 5 ml/g tissue) was added to the extracted saponins by stirring overnight at room temperature. The solution was filtered through a fine glass sinter, and the filtrate was evaporated under vacuum in a rotary evaporator at 40°C. Finally, 1·5 ml distilled water per g tissue was added to dissolve the residue. The resulting solution was stored and frozen at − 20°C as crude saponin extract until required.
Chemical purification of saponins
A volume of 15 ml of crude saponin extract was transferred to a separating funnel and was mixed with 10 ml water-saturated n-butanol (BuOH). Two distinguishable phases, an upper BuOH layer and a lower aqueous layer, were formed after mixing and settling. Sometimes, an interface layer was also present depending on the sample being partitioned. The upper BuOH layer was first taken off and stored. The inter-phase layer was transferred into a centrifuge tube and centrifuged at >3600 rpm and the upper layer was added to the BuOH fraction. Aqueous supernatant was added to the lower H2O layer and then the precipitate was removed. The combined aqueous layers were re-extracted twice with 10 ml BuOH as described above. The three BuOH layers were combined, evaporated in vacuum at 55°C, and then the residue was dissolved in MeOH. This method was scaled down for smaller amounts of material.
The saponin solution after extraction with BuOH was filtered through a glass micro-fibre filter CF/G. MeOH (5 ml) was used to wash the residue. The filtrate was mixed with five volumes of diethyl ether. The suspension, in a beaker covered by para film, was shaken and then left under a laboratory hood until a precipitate of saponins was formed, which was separated by centrifugation at >3600 rpm for 10 min. The precipitate was washed with diethyl ether until the diethyl ether wash solution was colourless after centrifugation. Residue pellets containing a pure mixture of saponins were dissolved in MeOH to use for TLC.
TLC
TLC was carried out on Alltech and Merck Silica Gel 60 F254 (Milano, Italy) 20 × 20 cm plates or as cut plates in 10 × 10 or 10 × 20 cm sizes. Pure saponin mixtures were analysed using TLC. After the centrifugation of the sample solution for 2 min, 10 μl of each sample was pipetted on TLC plate. The spotted samples were dried down with a hair dryer. TLC plates were then left inside a TLC tank lined with a filter paper and pre-equilibrated with solvent. The solvent system for saponin separation contained ethyl acetate–distilled water–acetic acid in the ratio 7:2:2 (by vol.). Plates were removed from the tank when the solvent front had reached approximately 1 cm from the top of the TLC plate. TLC plates were allowed to dry in air and then were sprayed with a reagent system, containing MeOH–acetic anhydride–H2SO4 in the ratio 10:1:1 (by vol.), freshly made. After spraying with this reagent, plates were transferred into an oven at 104°C for 15 min. Sprayed plates were observed under UV illumination (300 nm).
Identification of saponins using reference compounds
Either 1 mg of reference saponins or 10 mg of diethyl ether-precipitated purified saponin mixtures from alfalfa shoot and root tissues were dissolved in 1 ml of MeOH, micro-centrifuged, and after optimisation, 8·5 μl of solution of reference compounds (0·1 %) and 20 μl of purified saponin mixture (10 %) solutions were spotted on a glass TLC plate. As mentioned above, the plate was developed in a TLC tank containing the ethyl acetate solvent system. Different saponins in purified saponin mixture were compared with references (both under normal and UV light) after the staining of the TLC plate with saponin (H2SO4–acetic anhydride) reagent. Migration distances of band spots on TLC (R f) of major spots were measured for comparison.
For the identification of alfalfa saponins, the total four saponins presented by Professor Dr Georges Massiot (Faculté de Pharmacie, URA CNRS 492, 51 Reims-Cedex, France) and Professor Dr W. A. Oleszek (Department of Biochemistry, Institute of Soil Science and Plant Cultivation, Pulawy, Poland) were used as reference compounds(Reference Massiot, Lavaud and Guillaume19, Reference Oleszek26, Reference Massiot, Lavaud and Men-Oliver43).
Results and discussion
Identification of alfalfa saponins using reference compounds
Saponin mixtures from alfalfa shoot and root tissues were spotted on glass TLC plate. A series of standard saponins were obtained, and were also used to tentatively identify saponin spots on TLC, using similar R f and similar spot colour after spraying with the acidic reagent as criteria for identification. Results are presented in Fig. 2. Purification and identification of saponins allowed specific components to be identified in comparison with reference standards.
Fig. 2 Identification of saponin in alfalfa seedling roots (Rt) and shoots (Sht) by TLC using reference compounds. Migration distances of band spots R f on TLC are given on the Y-axis of the figure. OR, origin of sample movement; SF, solvent front; Soya1, soyasaponin1; Med I, medicoside I; Med G, medicagenic acid glycoside; Med J, medicoside J.
A monodesmoside contained soyasapogenol B as the aglycone; 3-O-α-l-rhamnopyranosyl (1 → 2)-β-d-galactopyranosyl (1 → 2)-β-d-glucuronopyranosyl soyasapogenol B (soyasaponin1)(Reference Massiot, Lavaud and Men-Oliver43) gave a red-brown spot at R f 0·33 (Fig. 2). A comparison between root and shoot saponin mixtures showed that this compound was purified in fractions eluted with 45–56 % iso-propanol and appears to be the major saponin present in both tissues, previously described as having R f 0·43(Reference Mazahery-Laghab and Gatehouse16) (result not shown here).
A bisdesmoside 3-O-β-d-glucopyranosyl-28-O-(β-d-glucopyranosyl (1 → 4)-(α-l-rhamnopyranosyl (1 → 2)-α-l-arabinopyranosyl) medicagenic acid (medicoside J)(Reference Massiot, Lavaud and Guillaume19) gave a dark olive-coloured spot at R f 0·29, but did not appear to be present in the root or shoot saponin mixtures in the present work.
Another bisdesmosidic saponin 3-O-β-d-glucopyranosyl (1 → 2)-β-d-glucopyranosyl(-28-O-(β-d-xylopyranosyl(1 → 4)-(α-l-rhamnopyranosyl(1 → 2)-α-l-arabinopyranosyl) medicagenic acid(Reference Massiot, Lavaud and Guillaume19) produced an olive green spot at R f 0·19. An additional, fainter, purple spot at R f 0·59 was also present in this sample, which could be identified as a 3-O-β-d-glucopyranosyl medicagenic acid in comparison with the other reference compounds. The additional spot had probably resulted from partial hydrolysis of the bisdesmoside. The shoot saponin mixture contained this compound as a major component; it was also present in a fraction eluted with 44 % iso-propanol(Reference Mazahery-Laghab and Gatehouse16).
The third bisdesmoside component, structurally identified as 3-O-(α-l-arabinopyranosyl (1 → 2)-β-d-glucopyranosyl (1 → 2)-α-l-arabinopyranosyl(-28-O-β-d-glucopyranosyl medicagenic acid (medicoside I)(Reference Massiot, Lavaud and Guillaume19), was detected as a blue-grey spot at R f 0·26. This compound appeared to be a minor component of the root saponin mixture, present in fractions which eluted with 52–56 % iso-propanol(Reference Mazahery-Laghab and Gatehouse16).
Although tentative identification of saponins can be made on the basis of R f values on TLC, and values given in the literature, it is not valid to identify saponins on the basis of R f values only, since mobility of components on TLC plates is subject to a high level of random variation between different TLC runs. However, the use of standard saponins of known structures, which can be run on the same TLC plate as unknowns, allows specific components to be identified. This identification is still not conclusive, since correspondence of spots on TLC does not prove identity, and single spots may be composed of more than a single saponin or aglycone, depending on the sample and the TLC system, but is sufficient as a working definition if other evidence is taken into account. In agreement with this conclusion, on the basis of TLC analysis, it was possible to identify major components of the crude saponin extracts in comparison with authentic compounds(Reference Tava, Oleszek and Jurzysta44).
Changes of alfalfa saponins extracted from shoot tissues
Crude saponins were extracted from the shoot tissue of alfalfa in three different growth stages. Subsequently, the extracts were chemically purified and analysed by TLC to show changes in the saponin content that take place as the plant develops. The results of changes in alfalfa saponins during the development are shown in Fig. 3.
Fig. 3 TLC analysis of saponins extracted from alfalfa shoots in different growth stages. Migration distances of band spots on TLC (R f) are given on the Y-axis of the figure. O, origin movement of solvent; Fm, final movement of solvent; 1, saponin extract from seedling tissues; 2, saponin extract from tissues before flowering stage; 3, saponin extract from tissues after flowering stage.
The saponin extract showed both quantitative and qualitative changes in components, as the alfalfa plant developed, with the intensity of spots increasing with plant age, suggesting that saponin content also increased. In addition, spots present in Sdg (e.g. a green-dark spot at R f approximately 0·50) were not present in older tissues, showing that the chemical nature of the saponin fraction changed with plant age. The major changes were detected in the range of R f 0·23–0·50 during the three growth stages (Fig. 3), and the colour and R f values are listed in Table 1. It has also been stated that immature plants of a species have higher saponin contents than mature plants of the same species(Reference Francis, Kerem and Makkar2). However, it seems to be dependent on the kind of species and also on the kind of saponin(s). A number of factors, such as physiological age, environmental and agronomic factors, have been shown to affect the saponin content of plants(Reference Francis, Kerem and Makkar2, Reference Yoshiki, Kudou and Okubo45). Reports reviewed by Francis et al. (Reference Francis, Kerem and Makkar2) indicated that saponins increase on sprouting in some plants such as soyabean, alfalfa, mung beans and peas but decrease in others such as moth beans. Changes have also been observed in canavanine when alfalfa plants were developed. Saponin concentration rose to 8·7 % in roots and 1·8 % in shoots on the 8th day and slowly decreased to lower levels on the 24th day. No saponin in alfalfa seeds but 1 % canavanine was reported(Reference Gorski, Miersch and Ploszynski46). Quantitative and qualitative differences in alfalfa shoot saponins have been reported by Bagheri et al. (Reference Bagheri, Yazdi-Samadi and Mazahery-Laghab47). The average amount of crude extract of saponins from twenty-two cultivars was 0·81 %. Saponin concentrations in different alfalfa varieties have also been reported and ranged from 0·8 to 2 %(Reference Pedersen and Wang48, Reference Majak, Fesser and Goplen49). In the present study, the concentration of saponins was not measured; however, saponin contents varied with alfalfa development. For example, Soya1 in both root and shoot tissues of alfalfa with an R f of 0·29 (Fig. 4), also detected at R f 0·50 on another TLC (Fig. 3), showed a relative decline in the amount as the plant developed, while a component of lower mobility at R f 0·37 displayed an increase (Fig. 3). Some reports refer to the variations of secondary metabolites like saponins during plant development stages and some other reports refer to variations due to environmental factors(Reference Agrell, Oleszek and Stochmal25–Reference Burgos and Talbett32, Reference Kapusta, Janda and Stochmal35–Reference Oleszek and Stochmal38, Reference Gorski, Miersch and Ploszynski46).
Table 1 Migration distance of band spots on TLC (R f) values and colours of spots detected in crude saponin extracted from the shoots of alfalfa during development under normal light on TLC
+, Compound with very weak intensity; *, low intensity; **, medium intensity; ***, high intensity; –, unclear; F12, final accumulative hydrolysed spots.
Fig. 4 TLC of pure saponin mixture from root (R) and shoot (Sh) tissues. Migration distances of band spots on TLC (R f) are given on the Y-axis of the figure. A, B and F, presented standard saponins: soyasaponin1, medicoside J and medicoside I, respectively. O, origin movement of solvent; Fm, final movement of solvent.
As indicated in Fig. 4, an intense brown spot at R f 0·26 was detected on TLC and identified as medicoside J. This compound had also been identified by Massiot et al. (Reference Massiot, Lavaud and Guillaume19) as a bisdesmoside of medicagenic acid. Medicoside J did not appear to be present in the purified root or shoot saponin mixtures from Sdg tissues (Fig. 4) but appeared to produce a more intensive spot in mature alfalfa plants in Af (R f between 0·37 and 0·50) as shown in Fig. 3.
Another saponin in both root and shoot tissues during the Sdg stage in alfalfa as medicoside I was visualised as an intensive spot at R f 0·24 on TLC (Fig. 4) under UV light whereas this spot visualised at R f 0·37 was given in Fig. 3. The colour of this compound was brown in shoot tissues under normal light (Table 1). Considering the reference component of F, this compound could be a kind of medicagenic acid which is identified as medicoside I, a bisdesmosidic compound (Fig. 4)(Reference Massiot, Lavaud and Guillaume19). The intensity of the spot of medicoside I on TLC increased as the plant developed.
Final accumulative spots at R f 1·00 are hydrolysates of different components during extraction and purification. When the hydrolysis of components takes place, the products either disappear on silica gel or stop at higher R f on the TLC plate depending on the kind of hydrolysed saponin or produced sapogenin (saponins with no sugar moiety). Disappearing components are probably sapogenins which are the hydrolysate of saponins. The latter components are probably different kinds of saponins on which one or two sugar moieties have been separated from aglycone moiety. So, it can be concluded that there are probably hydrolysing enzyme(s) in alfalfa tissues capable of producing new saponins (as resistance or non-resistance factors towards bio-stresses, i.e. toxic or non-toxic to insects or microbes). Mazahery-Laghab & Gatehouse(Reference Mazahery-Laghab and Gatehouse16) extracted a hydrolysing enzyme from alfalfa shoots and confirmed its responsibility for alfalfa saponin hydrolysis using TLC. Not only in the present study but also in previous studies, TLC was a potent technique to analyse alfalfa saponins qualitatively(Reference Mazahery-Laghab and Gatehouse16, Reference Mazahery-Laghab17) as also Oleszek(Reference Oleszek50) reported that TLC has a good potential to provide excellent qualitative information for determining plant saponins.
Analytical technique
Although many different procedures have been used for saponin analysis, such as foam production, haemolytic activity, inhibition of fungal growth, insecticidal or piscicidal activity, gravimetry, spectrophotometry, TLC, GC and HPLC(Reference Hostettmann and Marston7, Reference Price, Johnson and Fenwick51), TLC was used as the principal analytical technique in the present study. While this technique has a number of drawbacks, in that it is semi-quantitative at best, and R f values of specific components are not routinely reproducible among different TLC plates, these drawbacks are outweighed by its advantages.
Centrifugation was used in the preparation of samples to remove the insoluble materials that would distort the spot pattern. It was found that desalted samples gave a good chromatogram of spots, in agreement with Plummer(Reference Plummer52), who stated that before chromatography, biological samples should be desalted using electrolysis or electro-dialysis. The presence of excess salts in the chromatography medium causes the spreading of spots on the plate and changes in their R f values. To obtain reproducible results, it is also necessary to ensure a constant atmosphere in the solvent container. For this reason, during the development of the chromatogram, not only any exhausting of the evaporated solvent from the tank but also any importing of air from outside to the tank should be prevented. The tank should also be lined with filter paper, dipped in the solvent; this paper will keep the container saturated with the vapour of the solvent and will aid the ascent of the solvent front(Reference Oleszek and Jurzysta53).
The stationary phase here is relatively polar, while the mobile phase is non-polar and acidic (and thus ensures that saponins containing carboxylic acid groups are uncharged). Saponins should thus separate on the basis of polarity; the less polar the saponin, the further it should migrate. For example, zanhic acid glycoside, a major triglycoside compound with a high polarity, appeared as the first spot at a low R f with a high intensity after initial movement. High polarity of this compound makes it enter the water phase layer during water-saturated BuOH extraction. Previously, in shoot extracts, the 10 and 20 % MeOH fractions using column chromatography gave two green spots at R f 0·14 and 0·20 on TLC(Reference Mazahery-Laghab and Gatehouse16). It has been suggested that these spots may be zanhic acid tridesmosides; the presence of three glycosides makes this saponin relatively polar, leading to early elution from the reverse-phase column, and a low R f value on TLC(Reference Nowacka and Oleszek54, Reference Oleszek55).
The coloured spots produced can be viewed under normal or UV light; the latter has the advantage that phenolic compounds give fluorescent spots, and thus can be distinguished from saponins. In the present study, by extraction with water-saturated BuOH, phenols were removed and disappeared on TLC.
Aphid bioassay
The survival and fecundity of the spotted alfalfa aphid that feeds on shoots of alfalfa cv. Euver at different plant developmental stages over a 2-week bioassay were measured.
After 14 d, no significant difference in the survival of aphids among alfalfa plants of different ages was detected (Table 2). However, examination of the survival curves (Fig. 5) shows that survival remains high up to 8 d, but declines subsequently for aphids feeding on mature plants Bf, whereas survival on Sdg and mature plants Af remains high over the whole 14 d bioassay. However, there was no significant difference between the survivals of aphids in alfalfa growth stages (Table 2). There was no correlation between aphid survival and saponin contents as estimated by TLC, suggesting that this aphid is able to detoxify or is insensitive to the defensive compounds produced by alfalfa as a natural host. The aphid may be able to detoxify saponins by their hydrolysis to sapogenins. For example, it has been found that a hydrolysing avenacinase coded in Gaeumanomyces graminis removes β,1-2 and β,1-4 linked to d-glucose molecules from avenacin A-1 to give products with a lower toxicity to fungal growth(Reference Osbourn, Bowyer and Lunness22, Reference Crombie, Crombie and Green56). However, sapogenins have been shown to be insecticidal in their own right(Reference Mahato, Sarkar and Poddar57). The results of the investigation of the biological activity of medicagenic acid sodium salt and medicagenic acid glycosides from alfalfa Sdg on the growth of Amaranthus, Lepidium and tomato (Lycopersicon) cell growth showed that in contrast to medicagenic acid glycosides, medicagenic acid as sapogenin had stronger inhibition of plant and cell growth(Reference Gorski, Miersch and Ploszynski46). However, it is possible for some insects to have a potential to cleave one or more sugar moieties from saponins and change their biological activities(Reference Jain and Tripathi58).
Table 2 ANOVA for feeding effects from different growth stages on the biology of Therioaphis maculata
SOV, sources of variations; MS, mean of squares.
*, ** Calculated F values were significantly different at 5 and 1 %, respectively.
Fig. 5 Spotted alfalfa aphid survivals feeding on alfalfa plants at different growth stages. –♦–, Survivals on seedling stage; –■–, survivals on before flowering stage; –▲–, survivals on after flowering stage.
Accumulative nymph production on shoot tissues of alfalfa was significantly different at the level of < 1 % probability in different growth stages (Table 2). The earliest onset of nymph production and the highest nymph production took place in mature plants at Bf stage (Fig. 6). Higher nutrient availability in phloem sap(Reference Potter and Kimmerer59), production of new saponins (Fig. 3) and other metabolites such as flavonoids(Reference Goławska, Lukasik and Leszczynski39) and also lower concentration of Soya1 in Bf are probably some of the factors which have positive effects on the fertility of aphids. However, other than saponins, when plants contain other compounds, e.g. free sugars, phenolics and polar lipids(Reference Mazahery-Laghab and Gatehouse16), the effects could not be ascribable to any one compound. These is a combination of probiotic and antibiotic effects caused by different components(Reference Mazahery-Laghab and Gatehouse16, Reference Applebaum, Gestetner and Birk60) like saponins, which may express synergistic interactions in alfalfa, other plants and also insects(Reference Goławska27). A significant reduction in aphid fecundity at the level of 5 % in Sdg and Af (Table 2 and Fig. 6) indicates that although saponin contents are comparably low in Sdg, the antibiotic effect of saponins are significantly too high, probably due to their quality and bio-activity. Therefore, an increase in the amounts of saponins in Af (compared to Bf) would result in an increase in the synergistic activity and the expression of antibiotic effects of saponins. In both the stages, the onset of nymph production was 2 d later than in Bf. Alfalfa saponins have also shown to reduce fertility of Spodoptera littoralis (Reference Adel, Sehnal and Jurzysta61).
Fig. 6 Fecundity of spotted alfalfa aphid feeding on alfalfa plants at different growth stages. –▲–, After flowering stage nymph number; –■–, before flowering stage nymph number; –♦–, seedling stage nymph number.
According to Fig. 6, although the fecundity of aphids feeding on alfalfa in Sdg and Af was similar and started on day 9, nymph number on Sdg was less than what was observed in other stage, i.e. Bf stage. This could be due to the high levels of saponins and the frequency of nutrients in the young leaves of Sdg tissues(Reference Mazahery-Laghab and Gatehouse16, Reference Potter and Kimmerer59). Young leaves of Ilex opaca Aiton with high level of nutrients were also unsuitable for the red mite Oligonychus ilicis McGregor due to the high levels of saponins(Reference Potter and Kimmerer59). However, the presence of Soya1 as the most effective compound in shoot tissues of alfalfa Sdg against the potato aphid (Aulacorthum solani), as Mazahery-Laghab & Gatehouse(Reference Mazahery-Laghab and Gatehouse16) reported in 1997, could be the main reason of the delay on onset of nymph production and a medium fecundity of spotted alfalfa aphids (Fig. 6). As shown in Fig. 5, the increased nymph production at the Bf stage corresponded to lower survival. Previous studies showed that Soya1 caused 60 % mortality of potato aphids, delayed the onset of nymph production by 5 d and decreased aphid sizes by 29 % compared with the control ones(Reference Mazahery-Laghab and Gatehouse16). The onset of nymph production was significantly different at the level of < 5 % probability in three growth stages (Table 2). The fecundity of adults on alfalfa Sdg started on day 10, approximately 2 d later than aphids feeding on the shoots at developed ages in Bf. Again, this may reflect either a lower nutritional availability in Sdg or more toxicity of saponins during the Sdg stage as stated above. Exposure of the potato aphid to Soya1 from alfalfa Sdg caused a delay in nymph production (15 d compared with the control in which it occurred at day 12)(Reference Mazahery-Laghab and Gatehouse16).
In general, although saponins were quantitatively increased during plant development, less of a biological role was performed in the plant Bf in relation to both Sdg and Af stages. So, it can be concluded that the quantity of saponins can be overshadowed by their quality, e.g. presence of Soya1 in shoot tissues of alfalfa Sdg.
The data from these bioassays show that unlike the potato aphid, the spotted alfalfa aphid is able to tolerate the presence of saponins in its ‘normal’ host and that neither aphid survival nor development is significantly affected by the increased levels of saponins in older plants.
Conclusion
Data from bioassays carried out on plants at different developmental stages with different amounts of saponins present suggest that saponins are not effective as a defence against spotted alfalfa aphid attack, with neither survival nor fecundity showing a correlation with saponin content. This result contrasts with the insecticidal effects of alfalfa saponins on the potato aphid and shows that the spotted alfalfa aphid is adapted to the secondary defensive compounds present in its host plant. No significant difference in the survival of aphids, from neonate to adult, was observed, but two differences were found in the onset of nymph production and cumulative nymph production. The results also showed that the saponin composition in alfalfa changes with plant development and this, in turn, can affect the fecundity of even specific insect pests such as the spotted alfalfa aphid, concluding a possible biological role of alfalfa saponins.
Acknowledgements
The present study was funded by the Bu-Ali Sina University, and we are grateful to all colleagues in the university and the Agriculture faculty. We thank B. Y. S. and M. B. for their contribution in saponin quality determination and A. R. B. for his contribution in insect bioassay and plant material preparation. Peter Green, a former Durham University educational psychologist, H. M.-L., a PhD student at Freiburg University, for final language editing and W. A. Oleszek, the professor working in the Department of Biochemistry, Institute of Soil Science and Plant Cultivation, Pulawy, Poland and Dr G. Massiot, the professor working in the Faculté de Pharmacie, URA-CNRS, France, for presenting standard saponins, Dr Kamal Ghadrdan, the staff of Tehran University, for helping in aphid bioassays, A. A. Azizian Sorush and Mr P. Ostad Ahmadi, the staff of the Bu-Ali Sina University, for the preparation of plants and insects are acknowledged. There are no conflicts of interest among the authors of this research work.
