Mariusz Kowalczyk

Qualitative and quantitative analysis of steroidal saponins in crude extract and bark powder of Yucca schidigera Roezl.

Steroidal saponins in commercial stem syrup and in extract of a bark of Yucca schidigera were identified with high-performance liquid chromatography ion trap mass spectrometry and quantitated using ultraperformance liquid chromatography with quadrupole mass spectrometric detection. Fragmentation patterns of yucca saponins were generated using collision-induced dissociation and compared with fragmentation of authentic standards as well as with published spectrometric information. In addition to detection of twelve saponins known to occur in Y. schidigera, collected fragmentation data led to tentative identifications of seven new saponins. A quantitation method for all 19 detected compounds was developed and validated. Samples derived from the syrup and the bark of yucca were quantitatively measured and compared. Obtained results indicate that yucca bark accumulates polar, bidesmosidic saponins, while in the stem steroidal glycosides with middle- and short-length saccharide chains are predominant. The newly developed method provides an opportunity to evaluate the composition of yucca products available on the market.

New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans.

The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa (aerial parts and roots) and Saponaria officinalis (used as a well-known source of plant saponins) against Candida albicans reference and clinical strains, their yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated. Direct fungicidal/fungistatic properties of the tested phytochemicals used alone, as well as their synergy with azoles (probably resulting from yeast cell wall instability) were demonstrated. Here, to the best of our knowledge, we report for the first time the ability of saponin-rich extracts of M. sativa and S. officinalis to inhibit C. albicans germ tube formation, limit hyphal growth, reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the range of concentrations which were active modulators of Candida virulence factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These properties seem to be very promising in the context of using plant-derived SFs as potential novel antifungal therapeutics supporting classic drugs or as ingredients of disinfectants.

Saponin Inventory from Argania spinosa Kernel Cakes by Liquid Chromatography and Mass Spectrometry

INTRODUCTION Argania spinosa kernel cakes, obtained from argan oil extraction process, are known to contain large amounts of saponins. Only a few have been characterised previously, due to the use of pure ethanol as extracting solvent. The use of aqueous 50% ethanol improved the extraction of more polar saponins. OBJECTIVE Identification of polar saponins in kernel cakes of Argania spinosa by liquid chromatography-mass spectrometry and NMR techniques. METHODS Defatted kernel cakes were first extracted with ethanol and then twice with 50% aqueous ethanol. Individual crude extracts were analysed with an ion-trap mass spectrometer in negative mode electrospray MS and MS/MS modes. NMR experiments were run under standard conditions at 300 K on a Bruker DRX-600 spectrometer. RESULTS The LC-MS base peak chromatogram of saponins from pure ethanol extract was dominated by 11 large and several small peaks but the UV chromatogram showed only two peaks, corresponding to the main neutral saponins found previously in Argania: arganine A and B. In 50% aqueous ethanol extracts, numerous other saponins were detected. Many of them were glucuronide oleanane-type triterpene carboxylic acid 3,28-O-bidesmosides (GOTCAB saponins). The assignments of (1) H- and (13) C-NMR spectra of the four most abundant GOTCAB saponins confirmed the MS results. CONCLUSION Four GOTCAB saponins were structurally identified by NMR analysis in the 50% aqueous ethanol extract. Furthermore, LC-MS analyses showed the presence of at least 19 additional polar saponins in these kernel cakes.

Yunnaneic Acid B, a Component of Pulmonaria officinalis Extract, Prevents Peroxynitrite-Induced Oxidative Stress in Vitro

Our work reveals that the aerial parts of Pulmonaria officinalis L. are a new source of yunnaneic acid B. We studied antioxidant activity and cytotoxicity of this compound (1-50 μg/mL) and its contents in various plant extracts. This is the first study confirming the presence of yunnaneic acid B in P. officinalis L. and Pulmonaria obscura Dumort and hence in the Boraginaceae family. Determination of 1,1-diphenyl-2-picrylhydrazyl radical reduction and peroxynitrite-scavenging efficacy in inorganic experimental systems provided EC50 values of 7.14 and 50.45 μg/mL, respectively. Then we examined the antioxidant action of yunnaneic acid B in blood plasma under peroxynitrite-induced oxidative stress in vitro. Yunnaneic acid B effectively diminished oxidative damage to blood plasma proteins and lipids. Furthermore, it was able to prevent the peroxynitrite-induced decrease in nonenzymatic antioxidant capacity of blood plasma. Additionally, cytotoxicity of yunnaneic acid B (at concentrations ≤50 μg/mL) toward peripheral blood mononuclear cells was excluded.

LC–ESI-MS/MS profile of phenolic and glucosinolate compounds in samh flour (Mesembryanthemum forsskalei Hochst. ex Boiss) and the inhibition of oxidative stress by these compounds in human plasma

Samh flour (Mesembryanthemum forsskalei) is a foodstuff with high protein content, which can be used as a replacement for wheat flour. It is often consumed by Bedouin tribes of northern Saudi Arabia. Very little is known about bioactive molecules present in samh flour, therefore we analyzed its extracts to evaluate the contents of secondary metabolites. A total of 43 secondary metabolites present in 60% MeOH extract of samh flour were tentatively identified using LC-ESI-MS/MS. These compounds represented five major categories: glucosinolates, sinapic acid and sinapoylglycosides, acylated flavonoids, flavonoids, and amide derivatives. Their effect on oxidative damage of proteins and lipids was determined in vitro by assessing levels of protein thiol groups and concentrations of thiobarbituric acid reactive species (TBARS) in human plasma. Obtained results indicated that samh flour is a rich source of compounds with antioxidant activity.

Triterpene Saponins from the Aerial Parts of Trifolium medium L. var. sarosiense

Seven previously unreported triterpene glycosides (1-7) were isolated from methanol extract of the aerial parts of Trifolium medium var. sarosiense (zigzag clover). Their structures were established by the extensive use of 1D and 2D NMR experiments along with ESI-MS and HRMS analyses. Compounds 1-7 are oleanane derivatives characterized by the presence of a keto group at C-22 of an aglycone and a primary alcoholic function at C-24 and differing functions at C-30. Among these, compounds 1-3 and 6 showed a secondary alcoholic function at C-11, which is methoxylated in compounds 4 and 7. Compound 5 was shown to possess a known aglycone, wistariasapogenol A; however, it is described here for the first time as a saponin constituent of the Trifolium genus. Some aspects of taxonomic classification of zigzag clover are also discussed.

Effect of Saponaria officinalis L. or Panax ginseng C.A Meyer triterpenoid saponins on ruminal fermentation in vitro.

Abstract This study examined the potential effects of Saponaria officinalis (SO) or Panax ginseng (PG) saponins supplemented to diets differing in the proportion of forage to concentrate on rumen microbial fermentation and methane production in vitro. Two experiments were carried out using the rumen simulation technique (RUSITEC). In the first experiment the substrate was comprised of a mixture of meadow hay and corn meal in a ratio of 60:40 dry matter (DM; high forage diet; HF). In the second experiment low forage diet (LF) consisting of meadow hay and corn meal in the ratio of 40:60 DM was used. Diets were supplemented with dried roots of SO or PG to provide 1% of triterpenoid saponins in dietary dry matter. All triterpenoid sources significantly (P≤0.05) decreased number of protozoa, by 50% and 72% respectively when HF diet was analysed (first experiment). There were no changes in methane production. In the second experiment (LF), the potential to mitigate methane production was reported for both SO and PG addition, however only in PG a decrease in the protozoal population was detected. Supplementation of plants rich in triterpenoid saponins has been identified as a diet dependent potential factor which has an important role in modulation of rumen fermentation processes. However, further studies are needed to evaluate their effect in animal production. STRESZCZENIE Celem przeprowadzonych badań było określenie wpływu ekstraktu saponin triterpenowych, Saponaria officinalis (SO) lub Panax ginseng (PG), w dawkach różniących się poziomem i rodzajem węglowodanów, na proces fermentacji w żwaczu i produkcję metanu w warunkach in vitro. Przeprowadzono dwa doświadczenia, wykorzystując dynamiczny system symulujący pracę żwacza (RUSITEC). W pierwszym doświadczeniu zastosowana dawka podstawowa składała się z siana łąkowego i śruty kukurydzianej w stosunku 60:40 suchej masy (dawka z wysokim udziałem pasz objętościowych - HF). W drugim eksperymencie przeanalizowano dawkę pokarmową z niskim udziałem pasz objętościowych (LF; siano łąkowe i śruta kukurydziana w stosunku 40:60 suchej masy). Do dawek podstawowych dodano saponiny triterpenowe, które stanowiły 1% suchej masy dawki. W pierwszym doświadczeniu (HF), zarówno SO, jak i PG znacząco (P≤0,05) ograniczyły liczebność pierwotniaków, odpowiednio o 50% i 72%. Nie odnotowano natomiast zmian w poziomie koncentracji metanu. W drugim doświadczeniu (LF) potencjał do ograniczania produkcji metanu wykazał zarówno dodatek SO, jak i PG, jednak tylko w przypadku PG zaobserwowano zmniejszenie liczby pierwotniaków. Na podstawie przeprowadzonych badań stwierdzono, iż rośliny bogate w saponiny triterpenowe odgrywają istotną rolę w regulacji procesów fermentacji w żwaczu. Jednak aby ocenić ich wpływ na produkcję zwierzęcą, konieczne jest przeprowadzenie dalszych badań.

ScBx gene based association analysis of hydroxamate content in rye (Secale cereale L.)

Hydroxamates (HX) are major secondary metabolites synthesized by rye and are responsible for some of the unique properties of this cereal, including good tolerance of biotic and abiotic stresses and allelopathy. Recently, five genes encoding enzymes taking part in HX biosynthesis have been sequenced and characterized, which was the starting point to undertake the present study. Association analysis of the content of six HX–HBOA (2-hydroxy-1,4-benzoxazin-3-one), GDIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one glucoside), DIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one), GDIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one glucoside), DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) in the above-ground parts of plants and roots was performed on a population consisting of 102 and 121 diverse inbred lines, in 2013 and 2014, respectively. Altogether, 48 single nucleotide polymorphisms (SNPs) were found to be associated with the content of at least one HX: 20 SNPs were associated with HX synthesized in the above-ground parts of rye plants (AG-SNP), and 28 were associated with HX synthesized in the roots (R-SNP). The highest number of SNPs was present in genes ScBx1 (9) and ScBx5 (11). The majority of SNPs were affected by environmental factors, except for two: ScBx4_1702 associated with GDIBOA and MBOA contents, and ScBx5_1105 associated with HBOA content in roots.

Three new triterpene saponins from roots of Eryngium planum

Saponin composition of the roots of Eryngium planum L. was investigated. Triterpene saponins found in E. planum and also present in Eryngium maritimum were different from those described previously in Eryngium campestre L. Three primary saponins were isolated and their tentative identifications, based on the electrospray MS/MS fragmentation patterns, were subsequently confirmed by 1D and 2D NMR analyses. Their structures were established as 3-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl-21-O-acetyl-22-O-angeloyl-R1-barrigenol (1) and 3-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl-22-O-angeloyl-A1-barrigenol (2) and 3-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl-22-O-angeloyl-R1-barrigenol (3). Concentrations of the newly identified compounds in aerial parts and roots of both species were estimated using the liquid chromatography–mass spectrometry method.

New Bufadienolides Isolated from the Roots of Kalanchoe daigremontiana (Crassulaceae)

An aqueous extract from the roots of Kalanchoe daigremontiana turned out to be a rich source of bufadienolides. The existing literature data relate mainly to the aerial parts of Kalanchoe but there is no information about the metabolic profile of the roots, which are also used in traditional medicine. Our investigation concerning the roots of K. daigremontiana led to the isolation and characterization of eight new bufadienolides, namely 1β,3β,5β,14β,19-pentahydroxybufa-20,22-dienolide (1), 19-(acetyloxy)-1β,3β,5β,14β-tetrahydroxybufa-20,22-dienolide (2), 3β-O-α-l-rhamno-pyranosyl-5β,11α,14β,19-tetrahydroxybufa-20,22-dienolide (3), 19-(acetyloxy)-3β,5β,11α,14β-tetrahydroxybufa-20,22-dienolide (4), 3β,5β,11α,14β,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (5), 19-(acetyloxy)-3β,5β,11α,14β-tetrahydroxy-12-oxo-bufa-20,22-dienolide (6), 19-(acetyloxy)-1β,3β,5β,11α,14β-pentahydroxy-12-oxo-bufa-20,22-dienolide (7) and 1β-(acetyloxy)-3β,5β,11α,14β,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (8), together with seven known compounds: 11α,19-dihydroxytelocinobufagin (9), bersaldegenin-1-acetate (10), daigredorigenin-3-acetate (11), bersaldegenin-1,3,5-orthoacetate (12), bryotoxin B (13), bryophyllin B (14) and bersaldegenin (15). The structures were established applying extensive 1D- and 2D-NMR and MS spectroscopic analyses.