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Soybean saponins suppress the release of proinflammatory mediators by LPS-stimulated peritoneal macrophages.

Proinflammatory mediators such as prostaglandins (PGs), nitric oxide (NO), certain inflammatory cytokines and chemokines play a pivotal role in tumor development. The anti-inflammatory properties of naturally occurring phytochemicals are associated with significant reduction in the risk of certain cancers in vitro and in vivo. In the present study, we investigated the effects of soybean saponins on the production of proinflammatory mediators in LPS-stimulated peritoneal macrophages. Soybean saponins significantly inhibited the release of PGE(2), NO, TNFalpha and monocyte chemotactic protein-1 (MCP-1) in a dose-dependent manner. Soybean saponins also down-regulated the expression of COX-2 and iNOS at mRNA/protein levels. Moreover, soybean saponins suppressed NF-kappaB activation by blocking IkappaB-alpha degradation. Our data indicate that soybean saponins exhibit anti-inflammatory properties by suppressing the transcription of inflammatory cytokine genes through the NF-kappaB signaling pathway. The anti-inflammatory properties of soybean saponins may be useful for ameliorating inflammatory diseases as well as suppressing tumor progression.

Animals↗

Combined application of saponin and chimeric toxins drastically enhances the targeted cytotoxicity on tumor cells.

Immunotoxins have to be administered in high doses due to low cytosolic uptake with the consequence of severe side effects. Recently we found that the cytotoxic activity from Agrostemma githago seeds can be attributed to a synergistic toxicity of a triterpenoid saponin and a ribosome-inactivating protein. Here we investigated whether saponins are able to enhance the efficacy of a receptor-specific chimeric toxin consisting of saporin-3, epidermal growth factor and a molecular adapter previously shown to reduce side effects on non-target cells. Pre-applied saponin enhances the target cell-specific cytotoxic effect, dependent on the cell line, between 3560- and 385,000-fold with an IC50 up to 0.67 pM. Non-target cells are not affected at the same concentration. At the optimal concentrations of the chimeric toxin and saponin application of either one of the components shows no cytotoxicity at all proving a synergistic effect. In the presence of saponin ligand-free saporin-3 does not exhibit any cytotoxic effect up to 0.1 nM providing further evidence for an increased specificity. This synergistic effect is in the same order of magnitude as in a mouse model. Our investigations clearly demonstrate that a combined administration of saponin and chimeric toxins opens up a promising perspective for tumor therapy.

Animals↗

Triterpenoid saponins from the stem bark of Caryocar villosum.

Five triterpenoid saponins, caryocarosides II-22 (3), III-22 (4), II-23 (5), III-23 (6), and II-24 (7), have been isolated from the methanol extract of the stem bark of Caryocar villosum, along with two known saponins (1-2). The seven saponins are glucuronides of hederagenin (II) or bayogenin (III). Caryocaroside II-24 (7) is an unusual galloyl ester saponin acylated on the sugar chain attached to C-28, the 3-O-alpha-L-rhamnopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-beta-D-glucuronopyranosyl hederagenin-28-O-[2-O-galloyl-beta-D-glucopyranosyl] ester. The structures of the saponins were established on the basis of extensive NMR ((13)C, (1)H, COSY, TOCSY, HSQC, HMBC and ROESY) and ESI-MS studies. The cytotoxic activity of saponins 2 and 3 was evaluated in vitro against human keratinocytes. The DOPA-oxidase inhibition and the lipolytic activities were evaluated ex vivo using an explant of human adipose tissue.

Adipose Tissue↗

Effect of spermicides on Lactobacillus acidophilus in vitro-nonoxynol-9 vs. Sapindus saponins.

Saponins extracted from the fruit pericarp of Sapindus mukorosii were tested for their bactericidal/bacteriostatic property against Lactobacillus acidophilus. Nonoxynol-9 was used as a reference compound for the comparison of activity. Lactobacillus colonies were grown on specific medium (Rogosa SL agar) containing different concentrations of saponins and nonoxynol-9 in an atmosphere of 5% CO2/95% air at 37 degrees C for 72 h. The number and size of colonies were recorded at the end of the experiment and compared with controls. Results indicated that nearly 90% of Lactobacillus colonies with minor reduction in size thrived at 0.05% concentration of saponins whereas only 18% of colonies with approximately 75% reduction in size grew in dishes containing 0.05% nonoxynol-9. At higher concentrations of saponins, there was a gradual, dose-dependent reduction in the number and size of colonies and at 2.5% concentration there was an approximately 55% reduction in the number and 60% reduction in the size of surviving colonies. No lactobacillus colonies, however, grew in dishes containing 0.1% and higher concentrations of nonoxynol-9. The studies indicate that Sapindus saponins as compared to nonoxynol-9 are far less toxic to lactobacillus species and therefore saponins containing spermicidal preparations are likely to be more vaginal-friendly than equivalent nonoxynol-9 preparations.

In Vitro Techniques↗

Five saponins from the root bark of Aralia elata.

Five saponins, 3-O-[beta-D-glucopyranosyl (1-->2)-[beta-D-glucopyranosyl (1-->3)]-beta-D-glucopyranosyl]-oleanolic acid 28-O-beta-D-glucopyranosyl ester (aralia-saponin V), 3-O-[beta-D-glucopyranosyl (1-->2)-[beta-D-glucopyranosyl (1-->3)]-beta-D-glucopyranosyl]-echinocystic acid 28-O-beta-D-glucopyranosyl ester (aralia-saponin VI), 3-O-beta-D-glucopyranosyl (1-->2)-[beta-D-glucopyranosyl (1-->3)]-beta-D-glucopyranosyl]-hederagenin 28-O-beta-D-glucopyranosyl ester (aralia-saponin VII), 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl]-caulophyllogenin 28-O-beta-D-glucopyranosyl ester (aralia-saponin VIII), 3-O-[beta-D-glucopyranosyl (1-->2)-[beta-D-glucopyranosyl(1-->3)]-alpha-L-arabinopyranosyl]-hederagenin 28-O-beta-D-glucopyranosyl ester (aralia-saponin IX), were isolated from the root bark of Aralia elata (Miq.) Seem., together with four known compounds. Their structures were determined on the basis of chemical and spectroscopic methods.

Magnoliopsida↗

Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer.

Triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots were separated, profiled and identified using an optimized, reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). ESI source polarity and solvent conditions were compared. The effects of these parameters on mass spectral attributes were determined. Ion structures were confirmed using tandem mass spectrometry (MS/MS). Fifteen saponins were identified in alfalfa (cultivars Apollo, Radius, and Kleszczewska) based upon negative-ion HPLC/PDA/ESI/MS, HPLC/PDA/ESI/MS/MS and literature data. In addition, the identification of two new malonated saponins in alfalfa are proposed. Negative-ion HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS spectra were utilized along with HPLC retention times to profile and identify 27 saponins in M. truncatula (cultivar Jemalong, A17). M. truncatula yielded a much more complex mixture of saponins than observed for alfalfa. The authors are not aware of any previous reports identifying saponin glycosides in M. truncatula.

Chromatography, High Pressure Liquid↗

Genetic diversity and variation of saponin contents in Panax notoginseng roots from a single farm.

Radix notoginseng, the root of Panax notoginseng (Burk.) F. H. Chen, has been widely used in traditional Chinese medicine. Its main components, saponins, have been reported to have many pharmacological activities. To test the general assumption that herbs of a single species planted and harvested from a single location are uniform in chemical and genetic makeup, chemical analysis and DNA fingerprinting were carried out. High-performance TLC together with HPLC analysis were used to analyze 17 randomly sampled 3-year-old roots from a single farm for the presence of six saponins. Five roots showed distinct chemical profiles with changed ratios of ginsenosides Rd/Rg1, Re/Rg1, or Rb1/Rg1. The same samples, together with some 1- and 2-year-old samples, were also subjected to fluorescent amplified fragment length polymorphism (AFLP) analysis, and their internal transcribed spacer 2 (ITS 2) regions were sequenced. Fluorescent AFLP analysis was found to be much more polymorphic than the ITS 2 sequence and showed clear evidence of genetic diversity within the tested population. In conclusion, genetic diversity and variation of saponin contents between individual P. notoginseng roots have been detected. We suggest that genetic diversity affects the contents of the six saponins. The saponin contents variation and genetic diversity were also found among P. notoginseng root samples collected from China and Singapore markets. Since variable saponin contents may affect therapeutic efficacy, combining the use of genetic profiling with chemical profiling will help ensure greater uniformity in the quality of P. notoginseng roots. The genetic and chemical diversity within a population also provides the opportunity for breeding new cultivars with more desirable chemical constituents.

Base Sequence↗

Phyto-saponins as a natural adjuvant for delivery of agromaterials through plant cuticle membranes.

With growing use of synthetic adjuvants in modern agriculture, their impacts on the environment are being questioned. In a search for an environmentally safe phyto-adjuvant, we have investigated natural glycosidic saponin for delivery of agromaterials through plant cuticle membranes. Four saponin preparations from Quillaja saponaria bark (QE), obtained from Sigma-Aldrich, and Balanites aegyptiaca fruit mesocarp (ME), kernel (KE), and root (RE), isolated and characterized in our laboratory, were used for testing the delivery of [14C]-2,4-dichlorophenoxyacetic acid (2,4-D) across isolated intact astomatous adaxial Citrus grandis leaf cuticle membranes (CMs). The results showed that both Q. saponaria and B. aegyptiaca saponin preparations enhanced delivery of 2,4-D through CMs. Among the saponin preparations, ME exhibited a significantly higher level of delivery of 2,4-D with a concentration effect (2% being the highest). Transmission electron microscope (TEM) and dynamic light scattering (DLS) characterization of these saponin preparations in aqueous solution clearly demonstrated the formation of nanoscale vesicles. Various possibilities for a natural amphiphatic phyto-saponin as a delivery adjuvant through CMs are discussed.

2,4-Dichlorophenoxyacetic Acid↗

Triterpene saponins from the roots of Clematis chinensis.

A saponin-enriched fraction prepared from the MeOH extract of the roots of Clematis chinensis showed cytotoxic activity against HL-60 promyelocytic leukemia cells, from which five new triterpene saponins based on oleanolic acid, along with three known saponins, were isolated. The structures of the new saponins were determined on the basis of spectroscopic analysis, including extensive 1D and 2D NMR data and hydrolysis followed by chromatographic and spectroscopic analysis. Among the isolated saponins, monodesmosidic saponins exhibited cytotoxic activities against cultured tumor cells.

Antineoplastic Agents↗

Saponin adjuvant primes for a dominant interleukin-10 production to ovalbumin and to Trypanosoma cruzi antigen.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.

Adjuvants, Immunologic↗

Saponin, an inhibitory agent of carbon dioxide production by white cells: its use in the microbiologic examination of blood components in an automated bacterial culture system.

BACKGROUND: Blood components with a white cell count > 100 x 10(9) per L may cause false-positive results when the BacT/Alert system is used for the microbiologic examination. The effects of different concentrations of saponin on bacterial growth and on carbon dioxide production by blood fractions with a high white cell count, in particular peripheral blood progenitor cells and buffy coats, were investigated. STUDY DESIGN AND METHODS: The effect of saponin on carbon dioxide production was studied by adding different fractions of white cell-rich material (buffy coat or leukapheresis material) to BacT/Alert culture bottles with or without saponin and incubating these bottles. Five bacterial strains were used to inoculate the culture bottles at four levels ranging from about 1 colony-forming unit per mL to about 10(3) colony-forming units per mL. Aerobic and anaerobic bottles with and without saponin were used. RESULTS: It was demonstrated that the addition of 0.5 percent saponin to BacT/Alert culture bottles effectively inhibited carbon dioxide production, without affecting bacterial growth. CONCLUSION: Saponin at a concentration of 0.5 percent is a valuable additive to BacT/Alert culture media because it prevents false-positive results in the examination of white cell-rich blood components.

Bacteriological Techniques↗

Comparative study on triterpene saponins of Ginseng drugs.

A comparative study on the triterpene saponins of 47 samples of Ginseng drugs derived from 12 Panax taxa was conducted using a reverse-phase high-performance liquid chromatography (HPLC)method. Eleven ginsenosides, which represent 4 types of typical sapogenins, were chosen as standards for quantitative determination in order to characterize the chemical constituent pattern of each Ginseng drug and investigate the relationship between genetic varieties and chemical constituent pattern. The results showed that the ginsenoside compositions in Ginseng drugs of different origins were of considerable variability. Total saponin contents varied by 10-fold from the highest drug to the lowest one. Chikusetsu-ninjin derived from P. japonicus (Japan) was found to have the highest content (192.80 - 296.18 mg/g) and Ginseng from P. ginseng to be the lowest (5.78 - 15.63 mg/g). Two main groups (I and II) suggested by phytochemical data were clearly observed; group I mainly containing dammarane saponins consisted of P. ginseng, P. quinquefolius, P. notoginseng, P. vietnamensis and P. vietnamensis var. fuscidiscus; and group II containing a large amount of oleanolic acid saponins was com-posed of P.japonicus (apan), P. zingiberensis, P.japonicus (China),P. japonicus var. angustifolius, P. japonicus var. major, P. japonicus var. bipinnatifidus and P. stipuleanatus. The ratios of the subtotal of dammarane saponins to that of oleanolic acid saponins (D/0) were found to be > 1.9 and < 0.25 for groups I and II, respectively. The drug samples derived from the same botanical origin revealed similar constituent patterns, in other words, each Panaxtaxon showed its own characteristic chromatographic profile,which appeared in the specific shape of an 11-direction radar graph constructed on the basis of the result of quantitative analysis. Similarities of chemical constitution were seen among the closely phylogenetically-related taxa, including P. ginseng and P.quinquefolius, P. vietnamensis and P. vietnamensis var.fuscidiscus,P. japonicus (China) and its varieties were demonstrated, except P. japonicus (Japan) and P. zingiberensis.

Chromatography, High Pressure Liquid↗

Protection of rat liver microsomes against carbon tetrachloride-induced lipid peroxidation by red ginseng saponin through cytochrome P450 inhibition.

A possible role of cytochrome P450 (P450) inhibition by red ginseng saponins in carbon tetrachloride (CCl4)-induced lipid peroxidation was investigated in liver microsomes prepared from male Sprague Dawley rats. The total saponin of red ginseng standardized on ginsenosides-Rb1, -Rb2, -Rc, -Rd, -Re, and -Rg1 whose composition was studied in our previous report was used in the present study. The standardized saponin of red ginseng showed inhibitory effects on P450-associated monooxygenase activities in a dose-dependent manner, particularly p-nitrophenol hydroxylase activity which has been known to represent CCl4-activating P450 2E1 enzyme. Meanwhile, silymarin enhanced the activity of P450 2E1 enzyme in liver microsomes. When the lipid peroxidation was induced by incubating rat liver microsomes with CCl4 in the presence of NADPH, the standardized saponin significantly blocked the formation of thiobarbituric acid-reactive substances at the same concentrations showing P450 inhibition in liver microsomes. Silymarin revealed more potent protection against CCl4-induced lipid peroxidation. When the lipid peroxidation was induced by FeCl3, in which metabolic activation may not be required, only silymarin could protect the lipid peroxidation in liver microsomes. Taken together, our present results indicated that the inhibitory effects of red ginseng saponin on P450 enzymes may have a critical role in CCl4-induced lipid peroxidation in rat liver microsomes and that the mechanism of hepatoprotection by red ginseng saponin may be distinct from that of silymarin.

Animals↗

Alfalfa saponins affect site and extent of nutrient digestion in ruminants.

Alfalfa saponins isolated by ethanol extraction and partial acid hydrolysis were intraruminally administered to sheep to investigate the biological activities and nutritional implications in ruminants. Mature wethers fitted with ruminal, duodenal and ileal cannulas were fed a concentrate or roughage diet formulated to be isonitrogenous and isoenergetic. Microbial fermentation and nutrient degradation in the rumen were reduced by saponins. Total protozoal count in the rumen was reduced 34 and 66% by saponins at levels of 2 and 4% dietary dry matter, respectively. Bacterial nitrogen flow to the duodenum was reduced 20 and 30% in the same sequence. Apparent digestion coefficients of organic matter, hemicellulose and cellulose in the total digestive tract were increased by saponins in sheep fed concentrate diets. Fractional digestion coefficients of organic matter, hemicellulose, cellulose and nitrogen were reduced in the stomach while they were increased in the small intestine by saponins in both diets. Saponins inhibited microbial fermentation and synthesis in the rumen and altered the sites of nutrient digestion in sheep.

Animal Feed↗

Positive inotropic action of saponins on isolated atrial and papillary muscles from the guinea-pig.

The effects of several saponins of animal and plant origin on the contractile activity of atrial and papillary muscles of the guinea-pig were tested. In a concentration of 1 X 10(-5)M, holothurin-A (HL-A), holothurin-B, echinoside-A, echinoside-B and sakuraso-saponin (Saku) exhibited positive inotropic and chronotropic actions whereas desacyl-jego-saponin and ginsenoside-Rd did not. Saponins having a positive inotropic action caused haemolysis of rabbit erythrocytes whereas those without inotropic action did not cause haemolysis. The positive inotropic action of saponins was not affected by practolol, chlorpheniramine, cimetidine and indomethacin. Verapamil (10(-6)M) inhibited the inotropic actions due to HL-A and isoprenaline (10(-8)M) to the same extent but had a small effect on those due to ouabain (10(-7)M). In high K+ (30 mM K+) medium where the action potential and the contraction were depressed, HL-A, Saku and isoprenaline restored the action potential and the contraction of the 'slow response' type whereas ouabain failed to do so. In normal medium HL-A and Saku reduced the resting membrane potential by 15-20 mV. These results suggest that modification of the Ca channel is involved in the positive inotropic action of saponins.

Animals↗

The effects of saponin on the binding and functional properties of the human adenosine A1 receptor.

1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H-antagonist binding produced by GTP which has been observed in essentially all studies of A1 receptors and has been ascribed previously to precoupling of A1 receptors to G-proteins in the absence of agonists.

Adenosine↗

Immune responses of mice to inactivated rabies vaccine administered orally: potentiation by Quillaja saponin.

Administered orally, Quillaja saponin markedly potentiated the humoral immune responses of mice fed inactivated rabies vaccine, and significantly increased their resistance to subsequent intracerebral challenge with live rabies virus. Although mean serum neutralizing antibody titres were generally 8- to 16-fold higher when vaccine was given intraperitoneally, orally administered vaccine, with saponin, stimulated production of high protective antibody levels that were maintained for at least 6 months. The potentiating effect of saponin appeared to be mediated through several mechanisms, one of which was by increasing the permeability of the intestinal mucosa, allowing increased uptake of viral antigen. The potentiating effect was enhanced when saponin was administered in advance (up to at least 16 h) of the oral vaccine. Mice tolerated the effective saponin doses without any visible signs of distress or injury. In view of the many favourable physiological activities and low toxicity of orally delivered saponins, it is suggested that they may find more general applications in the immunopotentiation of oral vaccines.

Adjuvants, Immunologic↗

Studies on the cellular site of action of the adjuvant activity of saponin for sheep erythrocytes.

Saponin, which complexes with cholesterol in cell membranes, is a powerful adjuvant for antigens such as whole cell protozoal vaccines and SRBC. Saponin attached to SRBC ghosts potentiated the humoral response; but injection of saponin 1 or 2 days before or after SRBC at the same subcutaneous site was also effective. After the subcutaneous injection of saponin and SRBC the cellularity of the draining lymph node increases sixfold, and indirect PFC appear in it and the spleen. Splenectomy increases immunopotentiation by saponin, but T cell depletion abolishes it. The data show that attachment of the saponin to the antigen is not essential, and suggest that the adjuvant effect, which is T cell dependent, is exerted on host cells in the draining lymph node.

Adjuvants, Immunologic↗