Plant Saponaria Officinalis Research Articles

Plant molluscocide for prevention of animal helminthosis

Soft-bodied are intermediate hosts of helminthiases, in the body of which a number of development stages of larval forms of helminths occur. There is the highest population density of mollusks in the areas of ruminant grazing, which leads to mass infection of animals with trematodes. To destroy the intermediate host of helminths in agricultural production, molluscicidal remedies of synthetic and plant origin are used. The aim of the work was to determine the molluscicidal effectiveness of a plant remedy based on Saponaria officinalis in conditions of natural pastures. The material for work was the green mass of the plant Saponaria officinalis obtained in the warm season from roots, leaves, stems, flowers and seeds. By grinding this plant, a powder with a particle size of 1-3 mm was obtained. Then, the powder was extracted with ethyl alcohol. The obtained product (concentrate) was an amorphous gel-like mass of dark green color with a specific smell and well soluble in water. Field work in natural pastures was carried out on 6 biotopes with an area of 5-23 m2. Three species of gastropods were recorded from freshwater mollusks in the biotopes: Physella acuta, Lymnaea truncatula and Lymnaea palustris. The results of experiments conducted in the conditions of pastures indicate a high molluscocidal activity of the studied plant agent on pond fish, intermediate hosts of trematodosis pathogens. The effectiveness of the developed molluscocide in relation to gastropods when treated with a working solution (10.0 g/l) is from 92.3 to 100%.

Saponaroxins A–C, a new 19-oxa-tricyclohenicosatetraenone and, a new dioxacyclopropacycloundecene-10-carboaldehyde and its 6,7-dihydro derivative, produced by Alternaria saponariae, a pathogen of a medicinal plant Saponaria officinalis

A new 19-oxa-tricyclohenicosatetraenone and a new dioxacyclopropacycloundecene-10-carboaldehyde and its 6,7-dihydro derivative, named saponaroxins A–C, were isolated from the culture filtrate of Alternaria saponariae a fungal pathogen of the well-known medicinal plant Saponaria officinalis. Saponaroxins A–C were characterized by spectroscopic methods. When assayed on punctured leaf segments of two weeds, Sonchus arvensis and Elytrigia repens saponaroxins B–C were found to be phytotoxic. They demonstrated moderate antimicrobial activity and low toxicity against Paramecium caudatum. Saponaroxin A was acutely toxic to P. caudatum while showed no phytotoxic nor antimicrobial activity.

Mechanism of Anti-HIV Activity of Ribosome Inactivating Protein, Saporin.

Ribosome inactivating proteins (RIPs) are a family of proteins produced by plants, bacteria and fungi. RIPs have specific N-glycosidase activity, and they cleave a specific glycosidic bond in a universally conserved stem and loop structure within the large ribosomal RNA of all organisms. Saporin, a cytotoxic RIP from the plant Saponaria officinalis has been earlier shown to manifest its cytotoxicity by a combination of its N-glycosidase and apoptosis inducing activities. Saporin, along with many other RIPs also has strong inhibitory activity towards HIV integrase. In the current study, using two in vitro model systems, it is established that saporin inhibits propagation of HIV-1 in host cells. Saporin also showed a potent anti-HIV-1 integrase activity in vitro. Using three active site mutants of saporin, which respectively lack N-glycosidase, apoptosis inducing or both activities, it is shown that saporin's in vitro anti-HIV-1 integrase activity is independent of its N-glycosidase activity. However, for the anti-HIV activity of saporin, the apoptosis inducing activity is important.

Saponins modulate the intracellular trafficking of protein toxins

Type I ribosome inactivating proteins such as saporin from the plant Saponaria officinalis L. are widely used as toxin moieties of targeted anti-tumor toxins. For exerting cytotoxicity the toxin moieties have to be released into the cytosol of tumor cells. However the cytosolic transfer of toxin molecules into the cytosol is mostly an inefficient process.In this report we demonstrate that certain saponins, which are also biosynthesized by Saponaria officinalis L., specifically mediate the release of saporin out of the intracellular compartments into the cytosol without affecting the integrity of the plasma membrane. The relevant cellular compartments were identified as late endosomes and lysosomes. Further studies revealed that endosomal acidification is a prerequisite for the saponin-mediated release of saporin. Binding analysis demonstrated an association of the saponins with saporin in a pH-dependent manner. The applicability of the saponin-mediated effect was demonstrated in vivo in a syngeneic tumor model using a saporin-based targeted anti-tumor toxin in combination with characterized saponins.

Mechanism of Saporin Entry into the Target Cell Cytosol

Saporin is a type I ribosomal inactivating protein (RIP) derived from the plant Saponaria officinalis. It kills target cells through its RNA N-glycosidase activity, but has low in vivo toxicity because of the absence of a cell-binding subunit. Saporin enters the cell by endocytosis and transverses the endosomal membrane to reach its cytosolic target by a mechanism that remains elusive. We hypothesized that saporin may behave like a metamorphic protein that undergoes conformational switching between membrane-rupturing and enzymatic states, which may be mediated by pH and endosomal membranes. To test this hypothesis, we examined saporin interactions with phospholipid membranes that mimic the charge and fluidity of the endosomes, its membrane rupturing capabilities, and pH-dependent conformational changes in saporin. Our data indicate that a) saporin does bind to membranes composed of 80% DOPC, 5% POPC, 15% POPG, as detected by resonance energy transfer experiments b) binding is stronger at pH 5.5 than 7.0 (KD = 0.4 μM and 1.1 μM, respectively), c) saporin causes calcein leakage from phospholipid vesicles in a dose-dependent manner, and d) saporin undergoes significant conformational changes upon acidification in the presence of membranes. Circular dichroism data show a moderate change in saporin secondary structure upon a pH shift from 7.0 to 5.5 in the absence of membranes, which can be interpreted as an alpha-helix to beta-sheet transition. In contrast, in the presence of membranes the protein undergoes a prominent conformational change by gaining a larger alpha-helical content upon a similar pH shift. Together, these results provide a foundation for understanding the molecular mechanisms of membrane translocation of saporin, and probably other type I RIP proteins, which may acquire distinct membrane rupturing and catalytic activities by means of conformational switching, regulated by environmental factors such as pH and membranes.

Conjugation of an anti–transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells

We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition, anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling, leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6), a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells.

The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N.

The effect of AZT and chloroquine on the activities of ricin and a saporin–transferrin chimeric toxin

This study deals with the combination of chloroquine (CQ, an anti-malaric drug) and 3′-azido-3′-deoxythymidine (AZT, anti-human immuno-deficiency virus (HIV) drug) with a chimeric toxin (TS) obtained by chemical linking of saporin (a ribosome inactivating protein from the plant Saponaria officinalis) and human transferrin, in the intoxication of the human chronic myeloid leukaemia cells (K562). Our data demonstrate that AZT, at concentrations comparable to those reached in the blood of HIV-infected patients under pharmacological treatment with this drug, can increase the toxicity of TS in cooperation with CQ inducing an increased effect on protein synthesis in K562 cells (∼50% inhibition of protein synthesis for TS alone, and TS with AZT and ∼70% with both AZT and CQ). Furthermore, pre-treatment of cells with AZT alone can induce an increase of apoptosis in K562 cells intoxicated with TS. By comparing data obtained with the model toxin ricin, we get indications that the two toxins partially differ in their intracellular routes, also suggesting that chimeric constructs containing ricin-like toxins (i.e. immunotoxins) could be coupled with the use of common and cheap drugs for the treatment of cancer in HIV-infected patients.

Cytotoxicity of ribosome-inactivating protein saporin is not mediated through α 2-macroglobulin receptor

Saporin is a single chain ribosome-inactivating protein produced by the plant Saponaria officinalis. Several isoforms of saporin have been isolated from various parts of the plant. In the present study recombinant saporin isoforms 5 and 6 were produced in Escherichia coli. Saporin-6 was found to be more active than saporin-5 in its N-glycosidase, cytotoxic, and genomic DNA fragmentation activities. Earlier, saporin has been shown to bind low-density lipoprotein receptor-related protein (LRP), however, in this study the sensitivities of LRP-negative and LRP-positive cell lines were found to be similar towards saporin-6 toxicity suggesting the internalization of saporin not to be solely dependent on the expression of LRP on eukaryotic cells.

Anti-B16-F10 melanoma activity of a basic fibroblast growth factor-saporin mitotoxin

The authors attached basic fibroblast growth factor (FGF-2), a growth factor for numerous tumors and normal cell types, to saporin (SAP), a ribosome-inactivating protein isolated from the plant Saponaria officinalis. The conjugate (FGF-SAP) then was tested for antitumor activity using B16-F10 melanoma cells. This rapidly growing murine melanoma cell line has been used classically as a model to screen antitumor agents. B16-F10 cells in culture were used for in vitro experiments or introduced into C57BL/6 mice to demonstrate the in vivo antitumor activities of FGF-SAP. FGF-SAP was found to be an extremely effective cytocidal agent in vitro with an ED50 of 30-60 pM. The effects were specific for FGF-2 receptors, as shown by the ability of FGF-2 to block FGF-SAP action. In the in vivo models, FGF-SAP was found to increase survival time, inhibit tumor growth, and decrease metastases. The authors conclude that this mitotoxin has potent in vitro and in vivo effects on B16-F10 cells, supporting the hypothesis that ligand-mediated cytotoxicity can control tumor growth.

The expression of saporin, a ribosome-inactivating protein from the plant Saponaria officinalis, in Escherichia coli.

We have isolated and sequenced genomic clones from the DNA of Saponaria officinalis using a cDNA probe that encodes proteins with high homology to saporin-6, one of the most potent of the ribosome-inactivating proteins that is currently used for the construction of immunotoxins and mitotoxins. Sequence differences in the clones suggest a multigene family of proteins. These data agree with observations of several different proteins with ribosome-inactivating protein activity and similar structure. Two of the genomic clones encode proteins that have identical sequences to two of the four isoforms of saporin-6. We have inserted the DNA from one genomic clone into an Escherichia coli expression system that encodes a signal sequence for export to the bacterial periplasmic space. Exportation is observed and the isolated gene product has ribosome-inactivation activity similar to the native protein. Sequence analysis shows differential processing of the remaining plant signal sequence. The majority of the expressed protein remains intracellular and this material also shows high specific activity and differential processing. Saporin as an immunotoxin in clinical trial and as a mitotoxin in experimental models has been extremely efficacious. These data indicate the ability to produce fusion proteins with saporin and cell-binding ligands for production of new reagents for further clinical and experimental use.

In vivo effects in mice of an anti-T cell immunotoxin.

We have evaluated both the proliferative response as well as the Thy-1 Ag expression of lymphocytes from mice treated in vivo with an anti-Thy-1 immunotoxin (IT). The IT was a rat IgG2c mAb recognizing the Thy-1 Ag, disulfide-linked to a ribosome-inactivating protein isolated from the seeds of the plant Saponaria officinalis (soapwort). Toxicity studies showed that a single i.v. injection of doses up to 20 micrograms IT/mouse was well tolerated and allowed indefinite survival. The Con A-induced proliferative response of spleen cells from mice killed 1 day after treatment with sublethal doses of IT was inhibited in a dose-dependent manner, with complete inhibition observed at doses of greater than or equal to 5 micrograms IT/mouse. Control experiments showed that the inhibition was due to the IT and not to its single components. Moreover, the IT effect was abolished by a large (100-fold) excess of anti-Thy-1 mAb alone given concurrently, but not by an unrelated, isotype-matched rat mAb. At all IT doses, the proliferative response to a B cell mitogen (LPS) was normal. Kinetic studies showed a time- and dose-dependent reconstitution of Con A responsiveness. In limiting dilution cultures of spleen cells from mice treated with 5 micrograms IT 1 or 4 days before death, a 97% depletion of T lymphocytes capable of proliferation was observed. Limiting dilution cultures showed that also the thymus of IT-treated mice was depleted by more than 90% of growth-competent T lymphocytes. Cytofluorographic studies of Thy-1+ cells from the spleens of IT-treated mice gave results which did not correlate with those obtained in functional assays. Thus, a dose-dependent reduction, followed by a time-dependent reconstitution of Thy-1+ cells was observed in this case too, but the depletion occurred at later time points and was less complete than that observed in functional assays. Moreover, the mean fluorescence intensity of the residual Thy-1+ cells decreased below normal levels.

B-cell restricted saporin immunotoxins: activity against B-cell lines and chronic lymphocytic leukemia cells

B cell-restricted immunotoxins were constructed by conjugating anti-B monoclonal antibodies to saporin, the major ribosome inactivating protein from the seeds of the plant Saponaria officinalis. HD37-SAP is directed against CD19, the broadest B cell-specific determinant. HD39- SAP and HD6-SAP recognize two different epitopes on the CD22 molecule, an antigen present on the cell surface of B cells at late stages of differentiation. All three immunotoxins inhibited DNA synthesis and protein synthesis in target B lymphoma cells with a dose-related effect, in short incubation times and in the absence of potentiators. A clonogenic assay demonstrated that all immunotoxins could eliminate more than two logs of clonogenic malignant B cells with a two-hour incubation at concentrations not toxic to cells not bearing target antigens. The immunotoxin activity was evaluated by DNA synthesis inhibition in fresh B-chronic lymphocytic leukemia cells (B-CLL) stimulated to proliferate by incubation with an antibody specific for the receptor of C3b complement component (CR1) plus B cell growth factor. B-CLL cell DNA synthesis was actively inhibited by treatment at low immunotoxin concentration without need of potentiators. Immunotoxins exerted their effect also in whole blood of CLL patients under conditions achievable in vivo. We conclude that B cell-restricted immunotoxins HD37-SAP, HD39-SAP, and HD6-SAP are good candidates for in vivo therapy of B-cell malignancies.

B-Cell Restricted Saporin Immunotoxins: Activity Against B-Cell Lines and Chronic Lymphocytic Leukemia Cells

B cell-restricted immunotoxins were constructed by conjugating anti-B monoclonal antibodies to saporin, the major ribosome inactivating protein from the seeds of the plant Saponaria officinalis. HD37-SAP is directed against CD19, the broadest B cell-specific determinant. HD39-SAP and HD6-SAP recognize two different epitopes on the CD22 molecule, an antigen present on the cell surface of B cells at late stages of differentiation. All three immunotoxins inhibited DNA synthesis and protein synthesis in target B lymphoma cells with a dose-related effect, in short incubation times and in the absence of potentiators. A clonogenic assay demonstrated that all immunotoxins could eliminate more than two logs of clonogenic malignant B cells with a two-hour incubation at concentrations not toxic to cells not bearing target antigens. The immunotoxin activity was evaluated by DNA synthesis inhibition in fresh B-chronic lymphocytic leukemia cells (B-CLL) stimulated to proliferate by incubation with an antibody specific for the receptor of C3b complement component (CR1) plus B cell growth factor. B-CLL cell DNA synthesis was actively inhibited by treatment at low immunotoxin concentration without need of potentiators. Immunotoxins exerted their effect also in whole blood of CLL patients under conditions achievable in vivo. We conclude that B cell-restricted immunotoxins HD37-SAP, HD39-SAP, and HD6-SAP are good candidates for in vivo therapy of B-cell malignancies.

Synthesis and Characterization of an Antihuman T-Lymphocyte Saporin Immunotoxin (OKT1-SAP) With in vivo Stability Into Nonhuman Primates

The authors conjugated, by a disulphide bond, the antihuman T-lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen-induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double-antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.

Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates

The authors conjugated, by a disulphide bond, the antihuman T- lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen- induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double- antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.

Activity of a monoclonal antibody-saporin-6 conjugate against B-lymphoma cells.

A monoclonal antibody reactive with the immunoglobulin heavy chain (TEC IgM) has been conjugated to saporin-6 (SAP), which is the major ribosome-inactivating protein from the seeds of the plant Saponaria officinalis. Studies with Burkitt's lymphoma cell line Bjab 113 demonstrate that this immunotoxin is capable of killing 3 logs (99.9%) of clonogenic lymphoma cells after a 2-hour incubation. The presence of human bone marrow inhibits the activity of the conjugate. However, full potency of TEC IgM-SAP immunotoxin is restored by adding 1 mM amantadine to the incubation medium. The reaction is highly specific and is inhibited by the presence of excess anti-mu-antibody or human serum. Clonal growth of other Burkitt's lymphoma cell lines is inhibited to a lesser extent by the immunotoxin. The presence of surface IgM on the different cell lines is directly correlated to target cell killing by TEC IgM-SAP. Isolation of Bjab 113 clones surviving treatment demonstrates that only a minority are truly resistant and that the others randomly escape the treatment. The highly potent and specific activity of this conjugate in the presence of bone marrow buffy coat and its exceptionally rapid onset of action make this conjugate a good candidate for the ex vivo elimination of neoplastic cells from the bone marrow of non-Hodgkin's lymphoma patients.