Enhancement of saporin cytotoxicity by Gypsophila saponins—More than stimulation of endocytosis

Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that saporin exerts no cytotoxicity on seven human cell lines. However, the combination of saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein.In this study we investigated whether the enhancement of the saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged saporin (^hissaporin) in ECV-304 cells. For this purpose ^hissaporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of ^hissaporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic ^hissaporin was significantly higher in saponin-treated cells than in cells, which were only incubated with ^hissaporin. This indicates a saponin mediated endosomal escape of saporin.     

In-depth characterization of the GOTCAB saponins in seven cultivated Gypsophila L. species (Caryophyllaceae) by liquid chromatography coupled with quadrupole-Orbitrap mass spectrometer

Roots of Gypsophila L. (Caryophyllaceae) have been shown to accumulate bidesmosides of triterpenoid carboxylic acids, also called GOTCAB saponins (Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3, 28-Bidesmosides). The study aimed at in-depth characterization of GOTCABs from root extracts of cultivated Gypsophila scorzonerifolia Ser., G. acutifolia Stev. ex Spreng., G. altissima L., G. pacifica Kom., G. paniculata L., G. oldhamiana Miq. and G. zhegualensis Krasnova using ultra high-performance liquid chromatography coupled with hybrid quadrupol-Orbitrap high resolution mass spectrometry (UHPLC-HRMS). Based on the accurate mass measurements, elemental composition, isotopic peak profiles, fragmentation pattern in tandem mass spectrometry (MS/MS) and literature data, a total of 53 GOTCABs were tentatively identified. In addition, 29 core structures, forming between 2 and 12 isobaric isomers were described. They possess gypsogenin, quillaic and gypsogenic acid as sapogenin, substituted at C-3 with O-β-d-galactopyranosyl-(1 → 2)-[pentosyl-(1 → 3)]-β-d-glucuronopyranoside (β-chain). According to the C-28 ester-bonded oligosaccharide (α-chain) saponins were classified into four groups: GOTCABs with C-28 tetra- and pentasaccharide (type I), GOTCABs with C-28 oligosaccharide substituted with methoxycinnamoyl group (type II), GOTCABs with mono- and diacetylated C-28 oligosaccharide (type III) and GOTCABs with C-28 oligosaccharide substituted with both acetyl and methoxycinamoyl groups (type IV). The possible fragmentation pathways of saponins were proposed. Eleven core structures forming between 2 and 7 isobars are undescribed in the literature. To examine the differences between the assayed Gypsophila species at the same environmental conditions, the variation of saponins was estimated by hierarchical clustering on isobaric fingerprints of GOTCABs. The clustering of the studied species revealed three well-defined clusters. The first cluster comprises G. scorzonerifolia (G1) and G. altissima (G3), characterized by GOTCABs from type III. G. acutifolia (G2) and G. pacifica (G4) formed the second cluster accumulating saponins from types II and III. The third cluster grouped G. paniculata (G5), G. oldhamiana (G6) and G. zhegualensis (G7) sharing GOTCABs from types IV in addition to II and III. This is the first report on the saponins from G. scorzonerifolia and G. zhegualensis. An in-depth depiction of the GOTCAB saponin composition of seven cultivated Gypsophila species was achieved. Therefore, saponins are worth investigating for better understanding of the potential use of Gypsophila roots for pharmaceutical purposes.     

Application of preparative high-speed counter-current chromatography/preparative high-performance liquid chromatography mode in rapid separation of saponins

Combined with preparative high-performance liquid chromatography, high-speed counter-current chromatography was employed for isolation and purification of saponins from Gypsophila paniculata L. n-Hexane-n-butanol-methanol-0.02% TFA (1:9:1:9, v/v) was employed as solvent system and 210 nm was chosen as the wavelength of ultraviolet detection for the first time. The research tried to compare HSCCC with prep-HPLC, and further integrated their advantages to improve separation efficiency. Five known triterpene saponins were identified by 13C NMR and ESI-MS and their purities were all above 96%. The results demonstrated that adopted method was a feasible, economical and efficient technique for rapid preparative isolation of saponins.     

Mycoplasma-Like organisms associated with stunting ofGypsophila paniculata L.

A new undescribed yellows-type disease ofGypsophila paniculata L. was found. Since a stunted growth is the predominant symptom the disease has been termed stunting. Electron micrographs of ultrathin sections revealed numerous mycoplasma-like organisms in diseased phloem tissues. Attempts to maintain the diseasedGypsophila plants for further investigation of this disease failed for the present in spite of all applied modifications in nutrient media.     

Nebulosides A–B,novel triterpene saponins from under-ground parts of Gypsophila arrostii Guss. var. nebulosa

A bioassay-guided phytochemical analysis of the triterpene saponins from under ground parts of Gypsophila arrostii var. nebulosa allowed the isolation of two triterpene saponins; nebuloside A, B based on gypsogenin and quillaic acid aglycone. Two new oleanane type triterpenoid saponins (nebuloside A, B) and three known saponins (1–3) were isolated from the root bark of Gypsophila arrostii var. nebulosa. The structures of the two new compounds were elucidated as 3-O-β-d-galactopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl quillaic acid 28-O-β-d-glucopyranosyl-(1→3)-[β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)]-α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranosyl ester (nebuloside A) and 3-O-β-d-xylopyranosyl-(1→3)-[β-d-galactopyranosyl(1→3)-β-d-galactopyranosyl-(1→2)]-β-d-glucuronopyranosyl gypsogenin 28-O-β-d-glucopyranosyl-(1→3)-[β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)]-α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranosyl ester (nebuloside B), on the basis of extensive spectral analysis and chemical evidence. Nebuloside A and B showed toxicity enhancing properties on saporin a type-I RIP without causing toxicity by themselves at 15 μg/mL.     

The saponin-mediated enhanced uptake of targeted saporin-based drugs is strongly dependent on the saponin structure

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.     

Aboveground and Belowground Impacts Following Removal of the Invasive Species Baby's Breath (Gypsophila paniculata) on Lake Michigan Sand Dunes

The removal of invasive species is often one of the first steps in restoring degraded habitats. However, studies evaluating effectiveness of invasive species removal are often limited in spatial and temporal scale, and lack evaluation of both aboveground and belowground effects on diversity and key processes. In this study, we present results of a large 3‐year removal effort of the invasive species, Gypsophila paniculata, on sand dunes in northwest Michigan (USA). We measured G. paniculata abundance, plant species richness, plant community diversity, non‐native plant cover, abundance of Cirsium pitcheri (a federally threatened species endemic to this habitat), sand movement, arbuscular mycorrhizal spore abundance, and soil nutrients in fifteen 1000 m² plots yearly from 2007 to 2010 in order to evaluate the effectiveness of manual removal of this species on dune restoration. Gypsophila paniculata cover was greatly reduced by management, but was not entirely eliminated from the area. Removal of G. paniculata shifted plant community composition to more closely resemble target reference plant communities but had no effect on total plant diversity, C. pitcheri abundance, or other non‐native plant cover. Soil properties were generally unaffected by G. paniculata invasion or removal. The outlook is good for this restoration, as other non‐native species do not appear to be staging a “secondary” invasion of this habitat. However, the successional nature of sand dunes means that they are already highly invasible, stressing the need for regular monitoring to ensure that restoration progresses.     

New triterpenoid saponins from the roots of Gypsophila pacifica Kom.

Six new triterpenoid saponins (1-6) have been isolated from the roots of Gypsophila pacifica Kom. Their structures were established on the basis of extensive NMR (¹H, ¹³C, TOCSY, HSQC, and HMBC) and ESIMS studies.     

Characterization of quillaja bark extracts and evaluation of their purity using liquid chromatography–high resolution mass spectrometry

In the course of development of semi-preparative liquid chromatographic methods for the isolation of individual quillaja saponins from Quillaja saponaria (L.), some commercially available quillaja bark extracts revealed a distinctive and characteristic pattern of additional peaks in the chromatogram that could not be attributed to saponins commonly present in quillaja. To identify these peaks, analytical procedures based on HPLC coupled with high resolution MS detection were optimized which allowed the identification of the additional saponins Mi saponin A, Mi saponin B, Mi saponin C, madhucoside A and madhucoside B. These compounds are known to be the main saponins of the Indian plant Madhuca longifolia (L.). Tandem MS experiments were performed for the unambiguous assignment of the sapogenin. Madhuca saponins yielded a characteristic fragment of protobassic acid, whereas quillaja saponins showed a fragment of quillaic acid as expected. In addition, samples from madhuca seed kernels were analysed to verify the origin of the characteristic chromatographic peak pattern observed frequently in commercially available quillaja bark extracts.     

The co-occurrence of ecdysones with bufadienolides and steroidal saponins in the genus Helleborus

A method is described for the detection of ecdysones in small quantities of plant material. When applied to 14 taxa of Helleborus L., ecdysterone and 5 β-hydroxyecdysterone were detected and quantified in 11; the other 3, which are morphologically distinct, gave a negative result. The co-occurrence of ecdysones with bufadienolides and steroidal saponins is discussed. The isolation of the ecdysones from the aerial parts of H. orientalis hybrids is described.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

A new method to isolate lichen algae by using Percoll®gradient centrifugation

Abstract:A rapid method to isolate intact functional algae from the lichens Evernia prunastri and Ramalina farinacea has been developed. This method is based on the use of Percoll^®gradients after mechanical disruption of lichen thalli. Results obtained show that the algal preparations were virtually free of contamination by fungal hyphae. The purified algal cells were photosynthetically active and without symptoms of photoinhibition, which indicates their functional integrity. This method may be used for the isolation of intact algae from a broad range of lichen species.     

Uptake,accumulation and phytoextraction efficiency of cesium in Gypsophila paniculata

Abstract

To evaluate the phytoextraction efficiency of Gypsophila paniculata from Cs-contaminated soils and analyze the mechanism of Cs accumulation in G. paniculata, we analyzed the characteristics of Cs bioaccumulation and subcellular distribution, in addition to its chemical forms in the plant under hydroponic conditions. The results showed that total Cs content in the aboveground parts and the entire plant were as high as 6137.32?mg·kg^?1?dry weight and 7338.49?mg·kg^?1?dry weight, respectively, after 17?days in the 50?mg·L^?1 Cs treatment. The BCF was between 2.35 and 3.38. The TF was between 1.00 and 2.46 in G. paniculata. Subcellular distribution of Cs in the plant was as follows: soluble fraction?>?cell wall?>?organelles. Inorganic Cs (F-ethanol) and water-soluble Cs (F-dH2O) were the main types of Cs in G. paniculata. Further studies show that the phytoextraction efficiency can reach 10.30–11.91% planting a season of G. paniculata under potted conditions. The results suggested that G. paniculata, a perennial, drought-tolerant herb, was a high-accumulator of Cs, which may have potential uses in phytoremediation of Cs-contaminated soil.     

The Interactive Effects of Atmospheric Carbon Dioxide and Light on Stem Elongation in Seedlings of Four Species

Four species,Sinapis albaL.,Medicago sativaL.,Gypsophila paniculataL.andPicea abies(L.) Karsten, were grown in three light regimes:darkness, low light (25 µmol m^-2s^-1for 10 min d^-1) andhigh light (120 µmol m^-2s^-1for 12 h  d^-1) and fourlevels of carbon dioxide: 0, 350, 700 and 1400±50 µll^-1. Germination was not affected by any of the treatments.The effects of carbon dioxide on stem elongation were identicalin low and high light: stem length increased at a decreasingrate with level of carbon dioxide in all species. Level of carbondioxide also affected stem elongation in complete darkness,but the pattern was more complex and varied among species. Totalweight did not vary with level of carbon dioxide to any significantextent in either darkness or low light, but increased with levelof carbon dioxide at high light in all four species. Due tothe absence of any effect of carbon dioxide on growth in darknessand low light, we suggest the effects of carbon dioxide on stemelongation are independent of effects on growth and may be dueto a direct interaction with developmental processes. In contrast,level of carbon dioxide had little effect on allocation patternsin the dark and low light experiments, but had marked effectsin high light. Therefore, the effect of carbon dioxide on allocationwas probably due to the effects of carbon dioxide on growthrather than to any direct interaction between carbon dioxideand development. An understanding of the mechanisms by whichcarbon dioxide affects development may help us understand theoften variable effects of carbon dioxide upon plants.Copyright1998 Annals of Botany Company Sinapis albaL.;Medicago sativaL.;Gypsophila paniculataL. andPicea abies(L.) Karsten; elevated carbon dioxide; stem elongation; germination; allocation; phytochrome.     

Medicago sativa as a source of secondary metabolites for agriculture and pharmaceutical industry

Medicago sativa L. (lucerne or alfalfa) is a species which is generally regarded as a cheap source of valuable protein not only for animal fodder but also for nourishment provided in the poorest and developing countries. Moreover, this plant has a long tradition of use in folk medicine. Numerous studies indicate that besides protein, M. sativa synthesizes a variety of secondary metabolites. Among secondary metabolite classes produced by alfalfa, the saponins and flavonoids are of most interest and well characterized. In the current review, we have summarized their chemical structure and method of identification. The potential of alfalfa extracts in medicine and pharmacology has been highlighted. Furthermore, new ways of utilizing flavonoids as well as saponins in agriculture and horticulture are discussed. Collected data can be the starting point and inspiration for scientists of various specialties for future research on this plant. This review draws attention to the necessity of developing modern analytical tools for identification and quantification of individual M. sativa phytochemicals.     

Steroidal glycosides from Ruscus ponticus

A comparative metabolite profiling of the underground parts and leaves of Ruscus ponticus was obtained by an HPLC-ESIMSⁿ method, based on high-performance liquid chromatography coupled to electrospray positive ionization multistage ion trap mass spectrometry. The careful study of HPLC-ESIMSⁿ fragmentation pattern of each chromatographic peak, in particular the identification of diagnostic product ions, allowed us to get a rapid screening of saponins belonging to different classes, such as dehydrated/or not furostanol, spirostanol and pregnane glycosides, and to promptly highlight similarities and differences between the two plant parts. This approach, followed by isolation and structure elucidation by 1D- and 2D-NMR experiments, led to the identification of eleven saponins from the underground parts, of which two dehydrated furostanol glycosides and one new vespertilin derivative, and nine saponins from R. ponticus leaves, never reported previously. The achieved results highlighted a clean prevalence of furostanol glycoside derivatives in R. ponticus leaves rather in the underground parts of the plant, which showed a wider structure variety. In particular, the occurrence of dehydrated furostanol derivatives, for the first time isolated from a Ruscus species, is an unusual finding which makes unique the saponins profile of R. ponticus.     

Mass isolated ameba nuclei. I. Isolation procedure and determination of macromolecular composition and RNA polymerase activity

A rapid method for mass isolation of intact nuclei from Amoeba proteus has been developed. By this procedure, which includes a novel way of recovering nuclei during the filtration step, about 40% of the nuclei in the starting suspension of 4 to 5 × 10⁶ cells can be recovered within 70 min. A typical suspension of purified nuclei consists of 93% intact nuclei, 5.6% food-waste pellets, 0.4% membranes, and 1.0% extracellular debris.     

Phytoecdysteroids from Lamium spp: identification and distribution within plants

Bioassay/radioimmunoassay (RIA) analysis of the seeds of four Lamium species, L. album, L. galeobdolon, L. maculatum and L. pupureum revealed the presence of phytoecdysteroids in all of them. Bioassay/RIA-guided and photo-diode array-monitored HPLC analysis of the aerial parts of L. album and L. purpureum led to the isolation of four known ecdysteroids (abutasterone, inokosterone, polypodine B and pterosterone) from the former, and 20-hydroxyecdysone from the latter. Distribution and identities of ecdysteroids in different parts of these two species and also in the seed extract of L. maculatum have been analysed by RIA and bioassay.     

3α-Angeloyloxy-2α-hydroxycativic acid,a new diterpene from Brickellia paniculata

The investigation of Brickellia paniculata resulted in the isolation of a new diterpene of the labdane type. It was identified as 3α-angeloyloxy-