Medicagenic Acid Glycosides Research Articles

Antifeedant and insecticidal effects of alfalfa saponins in the management of the Japanese beetle Popillia japonica

AbstractPopillia japonica is a quarantine pest of priority interest for the EU, given its potentially important economic, social and environmental impacts. Alternative strategies to chemical methods are essential to limit its spread in newly infested areas with favourable climatic and environmental conditions. Saponins are biologically active molecules widely distributed in plants, displaying a well‐known repellent activity combined with a mortality effect against insects. In this context, saponins were extracted from alfalfa Medicago sativa, where medicagenic and zanhic acid glycosides and Soyasaponin I were the most abundant compounds and used in the laboratory and semi‐field experiments for treating leaves of susceptible host plants for P. japonica. Under laboratory conditions, a food deterrence effect and a significant mortality rate were observed using Corylus avellana leaves treated at increasing saponin concentrations, ranging from 1% to 5% w/v. Semi‐field condition experiment supported the food deterrence effect, as a significant food preference was observed for untreated plants of Vitis vinifera compared to treated plants. The promising results obtained suggest that alfalfa saponins could represent a potential eco‐friendly approach for Japanese beetle control.

Precaecal digestibility of crude protein and amino acids from alfalfa (Medicago sativa) and red clover (Trifolium pratense) leaves and silages in broilers

The aim of the present study was to determine the precaecal (pc) digestibility of crude protein and amino acids from organically produced alfalfa and red clover leaves and whole plant silages by a linear regression approach in slow-growing male Hubbard JA-757-broilers. Dried alfalfa leaves (AL; 219 g crude protein/kg dry matter (DM)), dried red clover leaves (RCL; 262 g crude protein/kg DM), alfalfa silage (AS; 240 g crude protein/kg DM) and red clover silage (RCS; 190 g crude protein/kg DM) were included in the diets at the levels of 100, 150 and 200 g/kg respectively at the expense of maize starch. Titanium dioxide was used as an indigestible marker for digestibility estimation. On day 41/42, digesta was sampled pen-wise from the terminal two thirds of the intestine section between Meckel’s diverticulum up to 2 cm anterior to the ileocaeca-colonic junction. Feed intake did not differ significantly between feeding groups. All broilers grew during the experimental phase of digestibility estimation (day 30−41/42). The pc crude protein and amino acid digestibility of alfalfa and red clover leaves was lower than of alfalfa and red clover silages. Methionine was less digestible in AL (0.61) and RCL (0.73) than in AS (0.84) and RCS (0.99). This tendency was also observed for lysine (AL 0.49, RCL 0.61, AS 1.00, RCS 0.88) as well as for other amino acids. Anti-nutritional factors, e.g. saponins, were suspected of being responsible for the lower pc digestibility of the leaf products. The saponin analysis showed a higher content of medicagenic acid glycosides in AS than in AL, whereas a higher content of a zanhic acid glycoside was found in AL than in AS. Differences in saponin contents suggest that certain individual saponins, e.g. zanhic acid glycosides, might have a more negative effect on crude protein and amino acid digestibility than other saponins.

Compound Characterization and Metabolic Profile Elucidation after In Vitro Gastrointestinal and Hepatic Biotransformation of an Herniaria hirsuta Extract Using Unbiased Dynamic Metabolomic Data Analysis.

Herniaria hirsuta L. (Caryophyllaceae) is used for treatment of urinary stones and as a diuretic. Little is known about the active compounds and the mechanism of action. The phytochemical composition of H. hirsuta was comprehensively characterized using UHPLC-UV-HRMS (Ultrahigh-Performance Liquid Chromatography-Ultraviolet-High Resolution Mass Spectrometry) data. An in vitro gastrointestinal model was used to simulate biotransformation, which allowed the monitoring of the relative abundances of individual compounds over time. To analyze the longitudinal multiclass LC–MS data, XCMS, a platform that enables online metabolomics data processing and interpretation, and EDGE, a statistical method for time series data, were used to extract significant differential profiles from the raw data. An interactive Shiny app in R was used to rate the quality of the resulting features. These ratings were used to train a random forest model. The most abundant aglycone after gastrointestinal biotransformation was subjected to hepatic biotransformation using human S9 fractions. A diversity of compounds was detected, mainly saponins and flavonoids. Besides the known saponins, 15 new saponins were tentatively identified as glycosides of medicagenic acid, acetylated medicagenic acid and zanhic acid. It is suggested that metabolites of phytochemicals present in H. hirsuta, most likely saponins, are responsible for the pharmaceutical effects. It was observed that the relative abundance of saponin aglycones increased, indicating loss of sugar moieties during colonic biotransformation, with medicagenic acid as the most abundant aglycone. Hepatic biotransformation of this aglycone resulted in different metabolites formed by phase I and II reactions.

Hepatoprotective activity of a purified methanol extract and saponins from the roots of Chenopodium bonus-henricus L.

An ultra-high-performance liquid chromatography-high-resolution mass spectrometry based profiling of a purified MeOH extract (PME) from the roots of Chenopodium bonus-henricus L. (Amaranthaceae) tentatively identified 15 saponins of six sapogenins. The PME exerts hepatoprotective and antioxidant activities comparable to those of flavonoid complex silymarin in in vitro (1 and 10 μg/mL) and in vivo (200 mg/kg/daily for 7 days) models of hepatotoxicity, induced by CCl4. The main constituents of PME, respectively saponins bonushenricoside A (1), 3-O-β-D-glucuronopyranosyl-bayogenin-28-O-β-D-glucopyranosyl ester (2), 3-O-β-D-glucuronopyranosyl-medicagenic acid-28-O-β-D-xylopyranosyl (1→4)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl ester (3), 3-O-β-D-glucuronopyranosyl-2β-hydroxygypsogenin-28-O-β-D-glucopyranosyl ester (4), 3-O-α-L-rabinopyranosyl-bayogenin-28-O-β-D-glucopyranosyl ester (6) and bonushenricoside B (8) (3 μg/mL each), compared to silymarin (5 and 50 μg/mL), significantly reduced the cellular damage caused by CCl4 in rat hepatocytes, preserved cell viability and glutathione level, decreased lactate dehydrogenase leakage and reduced lipid damage. The experimental data suggest that the glycosides of phytolaccagenin, bayogenin, medicagenic acid, 2β-hydroxygypsogenin, 2β-hydroxyoleanoic acid and oleanoic acid are a promising and safe class of hepatoprotective agents.

Structural and quantitative changes of saponins in fresh alfalfa compared to alfalfa silage.

Alfalfa (Medicago sativa) is an important legume used in animal feed. The primary objective of the present study was to analyze and trace the individual saponins in fresh and ensiled alfalfa of ten varieties, with the aim of evaluating the structural changes that saponins undergo during ensiling. A secondary objective was to examine whether any of the changes in saponin content were associated with changes in the basic nutrient composition of the ensiled alfalfa. The total saponin concentration increased when the fresh alfalfa was processed into silage. Three of the zanhic acid glycosides were degraded substantially, whereas the other two increased in all the tested varieties. Five of the seven medicagenic acid glycosides were not changed, whereas the remaining saponins completely disappeared. Finally, two glycosides of soyasapogenol B displayed an inverse relationship to each other. The nutrient content of fresh material and silages remained relatively constant, with some variations in the crude protein content of the selected alfalfa varieties. The total concentration of saponins increased two- to three-fold after ensilation over the levels present in fresh alfalfa material. The increases in saponin concentration showed a negative linear relationship between protein on a dry matter basis and saponin content. These findings are significant because no known published data have shown this transformation in the ensiling of alfalfa. © 2018 Society of Chemical Industry.

Artefact formation during acid hydrolysis of saponins from Medicago spp.

Artefact compounds obtained during acid hydrolysis of saponins from Medicago spp. (Fabaceae), have been monitored and evaluated by GC-FID. Their identification has been performed by GC-MS and 1H and 13C NMR. Saponins with different substituents on the triterpenic pentacyclic aglycones were considered, and their hydrolysis products were detected and quantified during 10 h of time course reaction. From soyasapogenol B glycoside the well known soyasapogenols B, C, D and F were obtained together with a previously undescribed sapogenol artefact identified as 3β,22β,24-trihydroxyolean-18(19)-en and named soyasapogenol H. From a zanhic acid saponin two major artefact compounds identified as 2β,3β,16α-trihydroxyolean-13(18)-en-23,28-dioic acid and 2β,3β,16α-trihydroxyolean-28,13β-olide-23-oic acid were obtained, together with some zanhic acid. Other compounds, detected in very small amount in the reaction mixture, were also tentatively identified based on their GC-MS and UV spectra. The other most characteristic saponins in Medicago spp., hederagenin, bayogenin and medicagenic acid glycosides, under acidic condition of hydrolysis, released instead the correspondent aglycones and generated a negligible amount of artefacts. Nature of artefacts and mechanism of their formation, involving a stable tertiary carbocation, is here proposed and discussed for the first time.

Isolation and chemical characterization of saponins from lucerne seeds (Medicago media Pers.)

Saponins of lucerne seeds have been prepared by two different methods. It was found by thin-layer chromatography that these glycosides consist of at least four components with one dominant. Soyasapogenols B, C and E were identified in acid hydrolysate of these saponins and in carbohydrate moiety glucose, galactose, rhamnose, small quantity of arabinose and xylose and glucuronic acid. It was established that saponins of lucerne seeds do not haemolyse red blood cells, contrary to top, root and blossom saponins. That may be due to the lack of medicagenic acid glycosides.

Effect of saponins and apigenin mixtures on feeding behavior of the pea aphid, Acyrthosiphon pisum Harris

Electrical penetration graphs (DC EPG) were used to monitor the feeding behavior of the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) exposed to mixtures of saponins: 3GlcA, 28AraRhaXyl medicagenic acid glycoside (MAG) and zanhic acid tridesmoside (ZAD) with flavonoid apigenin, on agarose-sucrose gels. In general, mixtures of saponins with apigenin incorporated into agarose-sucrose gels resulted in a reduction in both the number of aphid probes and delayed their duration. Aphid feeding on gels impregnated with mixtures of the tested chemicals also showed a reduction in salivation into the gels and elongation of passive ingestion from the gels (EPG patterns g-E1 and g-E2, respectively). There were significant differences among the feeding behaviors of the pea aphids on gels containing mixtures of either MAG or ZAD with apigenin. These differences were also depended on the concentration and proportions of these compounds.

Triterpene saponins of Trigonella monspeliaca L.

The triterpene saponin fraction was isolated from <em>Trigonella monspeliaca</em> tops. It consisted of ten saponosides, three of which were determined to be medicagenic acid glycosides, the other seven - soyasapogenol glucosides. Acid hydrolysis of the saponin fraction yielded medicagenic acid, soyasapogenol B and its artifacts soyasapogenol C, D and F. These data consitute the first report on the occurence of medicagenic acid and soyasapogenol glycosides in the genus <em>Trigonella</em>.

Isolation, chemical characterization and biological activity of alfalfa (Medicago media Pers.) root saponins

Saponins from alfalfa (<em>Medicago media</em> Pers.) roots were isolated and their acid hydrolysis revealed several aglycones that were obtained in crystalline form and characterized. Medicagenic acid, hederagenin and soyasapogenols A. B, C. D, E and F were found. Crude saponins were separated into cholesterol-precipitable and nonprecipitable fractions. The precipitable fraction consisted of medicagenic acid glycosides: glucose, arabinose, xylose and rhamnose were found as their sugar chain components. The nonprecipitable fraction was a mixture of hederagenine and soyasapogenol glycosides, and glucose arthinose. xylose. galactose and glucuronic acid were found in the sugar component,. 7 he medicagenic acid glycosides made up 6% of root dry matter and showed high biological activity. They cuased red blood cells lysis (haemolytic index 3000), completely inhibited <em>Trichoderma viride</em> growth at the concentration of 2.5 Mg. 100 cm<sup>-3</sup> of growth medium and retarded wheat seedling growth at concentrations as low as 100 ppm. The cholesterol-nonprecipitable fraction caused no blood cell lysis and fungus growth inhibition, although it did inhibit seedling growth, but to a much lesser extent than medicagenic acrd glycosides. The detrimental effect of alfalfa root saponins on winter wheat crop after alfalfa is discussed.

Studies on Medicago lupulina saponins. 2. Isolation, chemical characterization and biological activity of saponins from M. lupulina tops

Two pure saponin fractions from the tops of <em>M. lupulina</em> were isolated and characterized. The fractions named Ma and Ss varied in composition and biological activity. It was shown by TLC that fraction Ma contained 5 components. In the acid hydrolysates of that fraction only one aglycone (medicagenic acid) and 4 sugars: rhamnose, xylose, arabinose, glucuronic acid were found. The Ss fraction contained 11 components. In its acid hydrolysates, soyasapogenols B, C, D, E, F and two new aglycones N and An were found. The same sugars as above and additionally glucose and galactose were identified. It was found that the Ma fraction of saponins (medicagenic acid glycosides) was highly fungistatic, haemolytic and toxic to fishes. The Ss fraction of saponins (soyasapogenol glycosides) showed no such activity.

Studies on Medicago lupulina saponins. 6. Some chemical characteristics and biological activity of root saponins

The saponin fraction of black medic trefoil (<em>Medicago lupulina</em>) roots was isolated and purified. Its hydrolysis afforded several aglycones that have been identified by spectral and chemical properties as medicagenic acid, hederagenine and soyasapogenols: B, C, D, E and F. They made up, respectively. 46.9 and 45% of total sapogenins isolated. Two-dimensional TLC of <em>M. lupulina</em> root saponins revealed fourteen compounds, two of which were medicagenic acid glycosides. The haemolytic, antifungal and allelopathic activities of <em>M. lupulina</em> and <em>M. media</em> roots are compared and discussed.

Studies on Medicago lupulina saponins. 3. Effect of M. Iupulina saponins on the growth and feed utilization by mice

The effect of <em>M. lupulina</em> saponins on the growth of mice and feed utilization was investigated. The nutritive value of diets containing different amounts of <em>M. lupulina</em> meal or saponins was evaluated using a diet containing clover meal as the control. Feeding expermients revealed that <em>M. lupulina</em> meal in concentrations higher than 5% in the diet caused significant growth depression in mice. The diet containing 30% <em>M. lupulina</em> meal was toxic to mice. It was shown that the fraction of saponins which contains medicagenic acid glycosides was responsible for the antinutritional properties of <em>M. lupulina</em> meal. The diet containing more than 0.1% of toxic <em>M. lupulina</em> saponins caused significant growth depression in experimental mice. A high dose of those saponins in the diet (0.5%, 'had a large inhibitory effect on mause growth and feed utilization.

Studies on Medicago lupulina saponins. 5. Isolation and chemical characterization of blossom saponins

The separation of saponins derived from <em>Medicago lupulina</em> L. flowers yielded two saponin fractions. The first one, made up of crystalline saponins, readily precipitable from water solution, was a mixture of three glycosides of soyasapogenol B. Acid hydrolysis of these saponins yielded soyasapogenol B and its three artifacts: soyasapogenols C. D and F. Xylose, rhamnose, galactose, glucose and glucuronic acid were found as sugar constituents. The second fraction obtained by cholesterol precipitation consisted of seven haemolytically active medicagenic acid glycosides. Their hydrolysis furnished medicagenic acid and glucose, xylose, rhamnose and traces of glucuronic acid.

Antifungal (Gaeumannomyces graminis var. tritici) activity of various glycosides of medicagenic acid

Different concentrations of medicagenic acid and five glycosides of this acid isolated from alfalfa (<i>Medicago sativa</i>) were added to agar medium (corn meal agar, CMA) inoculated with cultures of <i>Gaeumannomyces graminis</i> var. <i>tritici</i> (Ggt). After 7 days of incubation at 25<sup>o</sup>C colony radius was measured and % of inhibition calculated in relation to the control medium (CMA enriched with the solvent of the tested compounds). Within the tested concentrations, only 3-O-<i>β</i> -D -glucopiranoside medicagenate (monoglucoside) significantly reduced the growth of Ggt on CMA medium. This compound at 0.05 mM concentration completely inhibited the development of the fungus and the effect was shown to be fungi-toxic.

Relationship Between Saponin Content in Alfalfa and Aphid Development

We studied the effect of qualitative and quantitative variation of saponin content in foliar tissues of four European alfalfa (Medicago sativa L.) cultivars (Radius, Sapko, Sitel, Radius line 1) on pea aphid (Acyrthosiphon pisum Harris) development, and the effect of aphid infestation on alfalfa saponin content. Aphids (adult apterae, larvae, and adult alatae) were counted on 3-, 6- and 9-month-old plants (before the first, second and third cutting). Thin-layer chromatography was used to detect and estimate the quantity of the following saponins: 3GlcA, 28AraRhaXyl medicagenic acid; 3Glc, 23Ara, 28AraRhaXylApi zanhic acid (zanhic acid tridesmoside); and 3RhaGalGlcA soyasapogenol B (soyasaponin I). Radius, Sapko, and Sitel contained all three saponins but Radius line 1 did not contain zanhic acid tridesmoside or medicagenic acid glycoside. Saponin content was highest in Radius and lowest in Radius line 1. Regardless of the cultivar, saponin content was higher in aphid-infested than uninfested plants. For all sampling dates, aphid numbers were highest on Radius line 1 and lowest on Radius; that is, aphid numbers were inversely related to saponin content. Alfalfa has a herbivoreinduced defense. Saponin levels increase in the foliage of infested alfalfa. Attempts of plant breeders to reduce saponin content in order to increase alfalfa digestibility for livestock might make the plants more susceptible to aphids and other pests.

Control of plant parasitic nematodes with active saponins and biomass from Medicago sativa

Medicago sativa L., alfalfa, is the most known plant species within the Medicago genus. The plant has been extensively studied for its content of saponins, mainly consisting of triterpene glycosides of medicagenic acid, possessing several biological properties including a biocidal activity on different soil microorganisms. Phytoparasitic nematodes are responsible for heavy economic damages to numerous agricultural crops and, due to their large distribution, they are among the most difficult crop pests to control. Attention on environmental safety and human and animal health has led to the progressive dismission of many synthetic formulations for the control of those pests and to the search of alternative strategies, including the use of natural metabolites from plants. Saponins from M. sativa may be good candidates for natural nematicide formulations, as in our in vitro studies the saponin mixtures from M. sativa tissues have been found effective in vitro against the virus-vector nematode Xiphinema index, the root-knot nematode Meloidogyne incognita and the potato cyst parasite, Globodera rostochiensis. A structure–activity relationship among saponins and related prosapogenins and sapogenin, respectively, has also been analyzed. The nematicidal efficacy differed among the three assayed nematode species, G. rostochiensis being the most susceptible to the active compounds from alfalfa. The in vitro results were also confirmed by experiments in potting mixes infested by M. incognita or G. rostochiensis and amended with dry top and root material from M. sativa, and in field trials on M. incognita and carrot cyst nematode Heterodera carotae with M. sativa pelleted meal. All amendments reduced root and soil population densities of target nematode species compared to non-treated and chemical controls, with a general improvement of plant growth and yield performances.

Effect of alfalfa saponins and flavonoids on pea aphid

AbstractWe studied the development and feeding behaviour of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), on the Radius and Sapko alfalfa (Medicago sativa L.) (Fabaceae) cultivars. Three saponins and flavones were identified in the alfalfa cultivars after thin layer chromatography separation. Cultivar Radius differed from Sapko in that it had a higher level of saponins, including zanhic acid tridesmoside and 3‐GlcA,28‐AraRhaXyl medicagenic acid glycoside. The flavones identified, including 7‐O‐β‐D‐glucuronopyranosyl‐4′‐O‐[2′‐O‐E‐feruloyl‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside] apigenin, 7‐O‐{2‐O‐E‐feruloyl‐[β‐D‐glucuronopyranosyl(1→3)]‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside} apigenin, and 4′‐O‐[2′‐O‐E‐feruloyl‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside] apigenin, occurred in tissues of both alfalfa cultivars. However, cv. Radius had very low mean flavonoid concentrations in comparison to cv. Sapko. Pea aphids that fed on cv. Radius plants showed a reduction in reproduction and survival. The aphid pre‐reproductive period on cv. Radius was prolonged and the reproductive and post‐reproductive periods on cv. Radius were reduced, compared to those on cv. Sapko. Cultivar Radius also negatively influenced aphid probing behaviour. This was especially the case during the initial period of the pathway phase. The results suggested that alfalfa cv. Radius, which had a higher level of saponins and a lower level of flavonoids, was less accepted by the pea aphid.

Deterrence and Toxicity of Plant Saponins for the Pea Aphid Acyrthosiphon Pisum Harris

Plant saponins are widely distributed among plants and have a wide range of biological properties. Three alfalfa saponins - zanhic acid tridesmoside, 3GlcA, 28AraRhaXyl medicagenic acid glycoside, and 3GlcA, 28AraRha medicagenic acid glycoside - were tested for their settling inhibition effects on feeding behavior of the aphid Acyrthosiphon pisum using the electrical penetration graph method. Application of saponins to artificial diets affected the insects' probing behavior. In general, saponins incorporated into sucrose-agarose gels significantly reduced the number of aphid probes and extended their duration. Lower saponin concentrations (50 ppm) extended aphid activity and corresponded to phloem sap ingestion. In contrast, higher concentrations (100 ppm) strongly reduced aphid ability to ingest phloem and xylem sap.

Effect of low and high-saponin lines of alfalfa on pea aphid

Pea aphid fed on a high-saponin line of alfalfa showed reduction of reproduction and survival, and disturbance in development of its population. This line negatively influenced aphid probing behaviour, particularly prolonging the non-probing period and probing of the peripheral tissues (epidermis and mesophyll) and shortening the period of phloem sap ingestion. The high-saponin line of alfalfa differed from the low-saponin one by the presence of zanhic acid tridesmoside and a higher level of 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The saponins incorporated into sucrose–agarose gels significantly reduced number of the aphid probes into the gels and extended their duration in comparison to the control gels (without tested compounds). Role of zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside as potential factors for partial resistance of alfalfa towards the pea aphid is discussed.