Broadening the Scope of Sapofection: Cationic Peptide-Saponin Conjugates Improve Gene Delivery In Vitro and In Vivo.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Saponaria officinalis saponins as a factor increasing permeability of Candida yeasts' biomembrane.

Saponins are a large group of compounds, produced mostly by plants as a side product of their metabolic activity. These compounds have attracted much attention over the years mostly because of their surface activity and antibacterial, anti-inflammatory and antifungal properties. On the other hand, most of the hitherto research has concerned the action of saponins against microbial cells as a whole. Therefore, knowing the possible interaction of saponins with biomembrane, we decided to check in-vitro the influence of saponin-rich extract of Saponaria officinalis on spheroplasts of two Candida sp. The obtained results show that 10 mg L- 1 of extract increased the permeability of spheroplasts up to 21.76% relative to that of the control sample. Moreover, the evaluation of surface potential has revealed a decrease by almost 10 mV relative to that of the untreated samples. Such results suggest its direct correlation to integration of saponins into the biomembrane structure. The obtained results have proved the antifungal potential of saponins and their ability of permeabilization of cells. This proves the high potential of saponins use as additives to antifungal pharmaceutics, which is expected to lead to improvement of their action or reduction of required dosage.

Development and stability of intimate soap formulations using Sapindus saponaria L. extract as a natural surfactant.

The use of synthetic surfactants reflects the high demand in the hygiene and cleaning sector for products with low-cost and good-effectiveness. These ingredients are the main components of intimate soap formulations. Sapindus saponaria L. is a plant rich in saponins, with the potential to be used as a natural surfactant due to its amphiphilic character and its foam-forming properties. Therefore, this study aimed to develop intimate soap formulations using S. saponaria extract as a natural surfactant and analyze its stability and surfactant characteristics. Preliminary and accelerated stability parameters, rheological characteristics, surface tension, foaming power, foam stability and emulsification potential were evaluated. The formulations were stable at a pH suitable for the intimate region (4.0 to 4.5), the presence of S. saponaria extract provided greater reduction of surface tension, better foaming and foam stability and greater emulsification power, desirable characteristics for an intimate liquid soap. These results demonstrate that the incorporation of S. saponaria extract into liquid soap formulations is an excellent option as a natural surfactant to reduce the use of synthetic anionic surfactants such as SLES.

Antimicrobial Activities of Saponaria cypria Boiss. Root Extracts, and the Identification of Nine Saponins and Six Phenolic Compounds.

The purpose of this study was to identify the chemical components in root extracts of Saponaria cypria, an endemic species of Cyprus. Subsequently, the synergistic bioactivity of its root extracts through different extraction procedures was also investigated for the first time. A total of nine saponins, along with six phenolic compounds, were identified and quantified using the UHPLC/Q-TOF-MS method. Additionally, S. cypria root extracts demonstrated antibacterial potential against Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Salmonella enteritidis. S. aureus presented the highest susceptibility among all bacteria tested. These findings provide the first phytochemical data regarding the saponin, phenolic content and antimicrobial activity of S. cypria extracts, indicating that the Cyprus saponaria species is a rich natural source for bioactive compounds with a potentially wider bioactivity spectrum.

Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Novel Oleanane-Type Triterpene Glycosides from the Saponaria officinalis L. Seeds and Apoptosis-Inducing Activity via Mitochondria.

Saponaria officinalis L., commonly known as "Soapwort", is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1-17), including seven new glycosides (1-7). The structures of 1-7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells.

Separation and Quantification of Selected Sapogenins Extracted from Nettle, White Dead-Nettle, Common Soapwort and Washnut.

Saponins are an important group of secondary metabolites naturally occurring in plants with important properties like: antibacterial, antiviral and antifungal. Moreover, they are widely used in the cosmetic industry and household chemistry. The sapogenins are saponin hydrolyses products, frequently used to facilitate saponin detection. In the present study, an improved methodology for isolation and separation of five sapogenins extracted from nettle (Urtica dioica L.), white dead-nettle (Lamium album L.), common soapwort (Saponaria officinalis L.) and washnut (Sapindus mukorossi Gaertn.) was developed using ultra-high-performance liquid chromatography with an evaporative light-scattering detector (UHPLC-ELSD). Based on quantitative analysis, the highest content of hederagenin (999.1 ± 6.3 µg/g) and oleanolic acid (386.5 ± 27.7 µg/g) was found in washnut extracts. Good recoveries (71% ± 6 up to 99% ± 8) were achieved for four investigated targets, while just 22.2% ± 0.5 was obtained for the fifth one. Moreover, hederagenin and oleanolic acid of whose highest amount was detected in washnut (999.1 ± 6.3 µg/g and 386.5 ± 27.7 µg/g, respectively) were subject to another approach. Consequently, liquid chromatography coupled mass spectrometry (LC/MS) with multiple reaction monitoring mode (MRM) was used as an additional technique for fast and simultaneous identification of the mentioned targets.

Soapwort (Saponaria officinalis L.) Extract vs. Synthetic Surfactants-Effect on Skin-Mimetic Models.

Our skin is continuously exposed to different amphiphilic substances capable of interaction with its lipids and proteins. We describe the effect of a saponin-rich soapwort extract and of four commonly employed synthetic surfactants: sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), ammonium lauryl sulfate (ALS), cocamidopropyl betaine (CAPB) on different human skin models. Two human skin cell lines were employed: normal keratinocytes (HaCaT) and human melanoma cells (A375). The liposomes consisting of a dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3, mimicking the cell membrane of keratinocytes and melanoma cells were employed as the second model. Using dynamic light scattering (DLS), the particle size distribution of liposomes was analyzed before and after contact with the tested (bio)surfactants. The results, supplemented by the protein solubilization tests (albumin denaturation test, zein test) and oil emulsification capacity (using olive oil and engine oil), showed that the soapwort extract affects the skin models to a clearly different extent than any of the tested synthetic surfactants. Its protein and lipid solubilizing potential are much smaller than for the three anionic surfactants (SLS, ALS, SLES). In terms of protein solubilization potential, the soapwort extract is comparable to CAPB, which, however, is much harsher to lipids.

Fatty acid composition, enzyme inhibitory effect, antioxidant and anticancer activity of extract from Saponaria prostrata WILLD. subsp. anatolica HEDGE.

This study attempts to evaluate the antioxidant, enzyme inhibitory, and anticancer properties as well as fatty acid compositions of endemic Saponaria prostrata WILLD. subsp. anatolica HEDGE. The gas chromatography-mass spectrometry (GC-MS) was used to determine the fatty acid content of methanol: dichloromethane extract from S. prostrata subsp. anatolica (SPA). Enzymatic activity was measured against acetylcholinesterase, butyrylcholinesterase and α-glucosidase. DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and Ferric reducing antioxidant power assay (FRAP) were conducted to antioxidant properties. The anticancer effect of SPA on human MCF-7 breast cancer and human HCT116 colorectal cancer cell line was evaluated by WST-1 cell viability assay, colony formation assay and wound healing assay. In addition, human VEGF Elisa method was used to determine the anti-angiogenic effect, and the quantitative real-time PCR (qRT-PCR) method on p53, Bax and Bcl-2 mRNA levels were used to evaluate apoptosis. While high amounts of palmitic acid (40.8%), linoleic acid (17.75%) and α-linolenic acid (16.84%) were detected in the SPA, the total amount of unsaturated fatty acid (51.34%) was higher than the total amount of saturated fatty acid (48.66%). SPA displayed the most promising acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and α-glycosidase (AG) inhibitory activities (AChE: IC50: 18.03 µg/mL, BuChE: IC50: 44.24 µg/mL and AG: IC50: 210.85 µg/mL). The half maximum inhibitory concentration (IC50) of SPA in MCF-7 and HCT116 cells was determined as 259.79 µg/mL and 97.24 µg/mL, respectively. In addition, it was determined that SPA suppresses colony formation and wound closure, and suppresses angiogenesis as well as triggering apoptosis at a significant level. It is true that endemic S. prostrata subsp. anatolica is a potential source of functional food ingredients, but more analytical and in vivo experiments are needed to explore further secondary metabolite diversity and pharmacological properties.

Antibacterial and Photocatalytic Properties of ZnO Nanoparticles Obtained from Chemical versus Saponaria officinalis Extract-Mediated Synthesis.

In the present work, the properties of ZnO nanoparticles obtained using an eco-friendly synthesis (biomediated methods in microwave irradiation) were studied. Saponaria officinalis extracts were used as both reducing and capping agents in the green nanochemistry synthesis of ZnO. Inorganic zinc oxide nanopowders were successfully prepared by a modified hydrothermal method and plant extract-mediated method. The influence of microwave irradiation was studied in both cases. The size, composition, crystallinity and morphology of inorganic nanoparticles (NPs) were investigated using dynamic light scattering (DLS), powder X-ray diffraction (XRD), SEM-EDX microscopy. Tunings of the nanochemistry reaction conditions (Zn precursor, structuring agent), ZnO NPs with various shapes were obtained, from quasi-spherical to flower-like. The optical properties and photocatalytic activity (degradation of methylene blue as model compound) were also investigated. ZnO nanopowders' antibacterial activity was tested against Gram-positive and Gram-negative bacterial strains to evidence the influence of the vegetal extract-mediated synthesis on the biological activity.

Human Nails Permeation of an Antifungal Candidate Hydroalcoholic Extract from the Plant Sapindus saponaria L. Rich in Saponins.

We evaluated a hydroalcoholic extract of Sapindus saponaria L. pericarps (ETHOSS), as a candidate to a topical antifungal medicine for onychomycosis. ETHOSS was produced by extracting the crushed fruits in ethanol. The saponin contents were identified and characterized by electrospray ionization mass spectrometry. We measured the in vitro antifungal activity against three dermatophyte fungi, isolated from onychomycosis: Trichophyton rubrum, T. mentagrophytes, and T. interdigitale, using broth microdilution tests. The minimum fungicide concentration of ETHOSS ranged from 195.31 to 781.25 µg/mL. The cytotoxicity of the crude extract was tested on the HeLa cell line, and its ability to permeate into healthy human nails by photoacoustic spectroscopy and Fourier transformation infrared spectrometer (FTIR) spectroscopy by attenuated total reflection. Besides its strong antifungal activity, ETHOSS showed low cytotoxicity in human cells. It was able to permeate and reach the full thickness of the nail in one hour, without the aid of facilitating vehicles, and remained there for at least 24 h. These results suggest that ETHOSS has great potential for treating onychomycosis.

Usability of soapwort and horse chestnut saponin extracts as foaming agents in foam mat drying of pomegranate juice.

BACKGROUND: This study evaluated the usability of saponin-rich extracts (soapwort and horse chestnut) as a foaming agent for foam mat drying of pomegranate juice. RESULTS: According to the foaming and stabilization studies, the optimum conditions were determined as 0.4% of soapwort extract, 0.03% of carboxymethyl cellulose as a stabilizer, and 3 min of whipping time. The foams produced using these conditions were dried at different spreading thicknesses and drying temperatures. The results showed that the thicker spreading thicknesses provided a higher antioxidant activity. On the other hand, drying temperature had a significant effect on all measured parameters except moisture content and water activity. The higher drying temperature caused a greater colour change and a lower content of total phenolics, total monomeric anthocyanins, cyanidin-3-glucoside, and delphinidin-3-glucoside. On the other hand, a higher content of ascorbic acid and better antioxidant activity was determined in the samples dried at 70 °C. CONCLUSION: According to the results obtained, spreading thickness of 2 mm and drying temperature of 70 °C were suggested for pomegranate juice powder production by foam mat drying. Overall, it was demonstrated that saponin-rich extracts are a good foaming agent alternative that provides foaming at very low concentrations and a product with high quality. © 2020 Society of Chemical Industry.

Effect of the nanocapsulated adjuvant Sapomax on the expression of some immune response genes.

The use of the nanocapsulated adjuvant Sapomax increased the expression of innate immunity genes (H2Q10, Ddx58, Tyk2, Tlr3, Tlr7, and TNF) responsible for the primary recognition of influenza virus, i.e., those belonging to the RLR and TLR families; genes involved in stimulating the production of type I and III IFN and pro-inflammatory cytokines; and Th1 and Th2 cellular immunity genes (Ccr4, Ccr5, IFNγ, IL-2, IL-4, and IL-10) responsible for triggering regulatory immune mechanisms in the cell. The high immunological activity of the plant-derived nanocapsulated adjuvant Sapomax may be used to enhance the efficacy of vaccines.

Effect of synthetic surfactants and soapwort (Saponaria officinalis L.) extract on skin-mimetic model lipid monolayers.

The effect of a saponin-rich extract from rhizomes of Soapwort (Saponaria officinalis L) and four synthetic surfactants: sodium lauryl sulphate (SLS), sodium laureth sulphate (SLES), ammonium lauryl sulphate (ALS) and cocamidopropyl betaine (CAPB) on two model lipid monolayers is analyzed using surface pressure, surface dilatational rheology and fluorescence microscopy. The following monolayers were employed: dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3 (DPPC/CHOL) and Ceramide [AP]/stearic acid/cholesterol in a molar ratio of 14:14:10 (CER/SA/CHOL). They mimicked a general bilayer structure and an intercellular lipid mixture, respectively. Both lipid mixtures on Milli-Q water were first compressed to the initial surface pressure, Π0 = 30 mN/m and then the subphase was exchanged with the respective (bio)surfactant solution at 1% (w/w). All four synthetic surfactants behaved in a similar way: they increased surface pressure to about 40 mN/m and reduced the storage modulus of surface dilational surface rheology, E', to the values close to zero. The corresponding fluorescence microscopy pictures confirmed that the lipids mimicking the stratum corneum components were almost completely removed by the synthetic surfactants under the present experimental conditions. The components of the Soapwort extract (SAP) increased surface pressure to significantly higher values than the synthetic surfactants, but even more spectacular increase was observed for the storage modulus of the SAP-penetrated lipid monolayers (up to E'= 715 mN/m).

Biodegradation of clotrimazole and modification of cell properties after metabolic stress and upon addition of saponins.

Azole fungicides constitute an extensive group of potential emerging pollutants which can be found in natural environment. This study focuses on the biodegradation of clotrimazole and the characterization of cell surface properties of microorganisms capable of degradation of this compound. The influence of long-term contact of bacteria with clotrimazole and the impact of the addition of Saponaria officinalis extract on cell surface modification was also checked. The biodegradation of clotrimazole did not exceed 70%. The presence of plant extract increased biodegradation of fungicide. The cells metabolic activity after one-month exposure to clotrimazole was the highest for each tested strain. Moreover, metabolic stress led to a strong modification of cell surface properties. The results are promising for determining the impact of clotrimazole on environmental microorganisms.

Phytochemical analysis of Saponaria officinalis L. shoots and flowers essential oils.

Phytochemical analysis by GC and GC/MS of the essential oil samples obtained from fresh shoots and flowers of Saponaria officinalis L. allowed the identification of 96 components in total, comprising 94.7% and 86.0% of the total oils compositions, respectively. Regarding the shoots essential oil, the major of 87 identified volatile compounds were phytol (14.1%), tricosane-6,8-dione (13.4%), patchouli alcohol (7.9%) and tricosane (7.2%), whereas patchouli alcohol (20.0%), heneicosane (11.5%) and tricosane (8.4%) were dominant among the 66 volatiles in the flower oil. Nonterpenoid compounds had the highest contribution in S. officinalis shoots essential oil (53.7%), while in the flower oil, constituents were almost evenly distributed between the oxygenated sesquiterpenoid (41.2%) and nonterpenoid compounds (39.5%).

Saponaria officinalis L. extract: Surface active properties and impact on environmental bacterial strains.

Plant-derived surfactants are characterised by low toxicity, high biodegradability and environmental compatibility. They therefore have many applications; for instance, they can be used in bioremediation to accelerate biodegradation processes, especially of hydrophobic pollutants. This paper analyses the properties of an extract from Saponaria officinalis L. containing saponins and its impact on bacterial strains isolated from soil, as well as its potential for application in hydrocarbon bioremediation. The tested extract from Saponaria officinalis L. contains gypsogenin, hederagenin, hydroxyhederagenin and quillaic acid aglycone structures and demonstrates good emulsification properties. Contact with the extract led to modification of bacterial cell surface properties. A decrease in cell surface hydrophobicity and an increase in membrane permeability were recorded in the experiments. An increase of up to 63% in diesel oil biodegradation was also recorded for Pseudomonas putida DA1 on addition of 1gL-1 of saponins from Saponaria officinalis L. Saponaria extract showed no toxic impact on the tested environmental bacterial strains at the concentration used in the biodegradation process.

Highly potent anti-leishmanial derivatives of hederagenin, a triperpenoid from Sapindus saponaria L.

Leishmaniasis is a neglected tropical disease (NTDs), endemic in 88 countries that affect more than 12 million people. Current drugs are limited due to their toxicity, development of biological resistance, length of treatment and high cost. Thus, the search for new effective and less toxic treatments is an urgent need. In this study, we report the synthesis of 3 new amide derivatives of hederagenin (22-24) with yields between 70% and 90%, along with 57 other derivatives of hederagenin (1-21, 25-60) carrying different groups at C-28 previously reported by our group, and the results of their in vitro ability to inhibit the growth of Leishmania infantum. Some derivatives (3, 4, 44, 49 and 52), showed activity at micromolar level and low toxicity against BGM and HepG2 cells. Moreover, the ability of hederagenin derivatives 3 (IC50 = 9.7 µM), 4 (12 µM), 44 (11 µM) and 49 (2 µM), to prevent proliferation of intracellular amastigote forms of L. infantum and their higher selectivity index and low toxicity compared to commercial positive drug control of choice (potassium antimonyl tartrate trihydrate) (IC50 = 80 µM, SI = 0.1), make these compounds promising candidates for the treatment of leishmaniasis.

Saponins from Saponaria officinalis L. Augment the Efficacy of a Rituximab-Immunotoxin.

Triterpenoidal saponins are synthesized in the roots of Saponaria officinalis L. The same plant is also a source for the toxin Saporin, which is a ribosome-inactivating protein. Triterpenoidal saponins are known to increase the cytotoxicity of Saporin by modulating its intracellular trafficking. Here, we investigated if the combinatorial effects elicited by purified saponins and Saporin can be applied to increase the therapeutic efficacy of the immunotoxin Saporin-Rituximab. First, saponins were purified by high-performance liquid chromatography. Thereafter, their intrinsic cytotoxicity was evaluated on Ramos cells with no observed effect up to 5 µg/mL, however, saponins increased the cytotoxicity of Saporin, while no influence was observed on its N-glycosidase activity. Saporin-Rituximab bound to CD20 in Ramos cells and, in the absence of saponins, had a GI50 (concentration inhibiting cell growth to 50 %) of 7 nM. However, in the presence of a nontoxic concentration of saponins, the GI50 of Saporin-Rituximab was 0.01 nM, a nearly 700-fold increase in efficacy. Moreover, two further immunotoxins, namely Saporin-anti-CD22 and Saporin-anti-CD25, were tested in combination with saponins yielding enhancement factors of 170-fold and 25-fold, respectively. All three receptors are present in Ramos cells and the differences in cytotoxicity enhancement may be explained by the differing expression levels of the cellular receptors. The application of purified saponins from S. officinalis L. is therefore a new strategy to potentially improve the cytotoxicity and therapeutic efficacy of Rituximab-immunotoxins for the treatment of B-cell lymphoma.

Transition State Structure of RNA Depurination by Saporin L3.

Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem-loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem-loop RNA (5'-GGGAGGGCCC-3') by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.