Study of interactions between phenolic compounds and H2O2 or Cu(II) ions in B14 Chinese hamster cells

The antioxidant and prooxidant effects of tannic, ellagic and gallic acids (1-60 microM) in the presence of hydrogen peroxide (40 and 100 microM) or copper ions (50 microM) on B14 Chinese hamster cells were examined. The fluorescence probe DCFH-DA (dichlorofluorescein-diacetate) was used to analyse the levels of reactive oxygen species. This method showed the reduction in oxidation of DCF (dichlorofluorescein). It indicates that antioxidant capacity of tested polyhenols is decreased in the presence of hydrogen peroxide or copper ions. Spectrophotometric assay with Ellman's reagent was used to determine SH-groups. The experimental results revealed the oxidative modification of proteins after exposure to polyphenols at concentrations above 15 microM. Additional incubation with H2O2 or Cu(2+) ions showed the prooxidant activity of tested complexes also for polyphenols used at a concentration of 1 microM. Fluorescence method with Hoechst 33258/propidium iodide dyes was used to study apoptotic and necrotic cell death. The obtained data demonstrated that polyphenols alone, as well as in the presence of hydrogen peroxide or copper ions, can induce DNA fragmentation.     

THE KINETICS OF PENETRATION : XIV. THE PENETRATION OF IODIDE INTO VALONIA

When 0.1 M NaI is added to the sea water surrounding Valonia iodide appears in the sap, presumably entering as NaI, KI, and HI. As the rate of entrance is not affected by changes in the external pH we conclude that the rate of entrance of HI is negligible in comparison with that of NaI, whose concentration is about 10⁷ times that of HI (the entrance of KI may be neglected for reasons stated). This is in marked contrast with the behavior of sulfide which enters chiefly as H₂S. It would seem that permeability to H₂S is enormously greater than to Na₂S. Similar considerations apply to CO₂. In this respect the situation differs greatly from that found with iodide. NaI enters because its activity is greater outside than inside so that no energy need be supplied by the cell. The rate of entrance (i.e. the amount of iodide entering the sap in a given time) is proportional to the external concentration of iodide, or to the external product [N⁺]_o [I^-l_o, after a certain external concentration of iodide has been reached. At lower concentrations the rate is relatively rapid. The reasons for this are discussed. The rate of passage of NaI through protoplasm is about a million times slower than through water. As the protoplasm is mostly water we may suppose that the delay is due chiefly to the non-aqueous protoplasmic surface layers. It would seem that these must be more than one molecule thick to bring this about. There is no great difference between the rate of entrance in the dark and in the light.     

A novel 19F-NMR method for the investigation of the antioxidant capacity of biomolecules and biofluids.

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.     

Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal caco-2 cells

Oxidative stress has been linked to the development of various chronic diseases. Vegetables and fruits, which contain polyphenols, were shown to have protective effects. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol abundant in tea, has been shown to have antioxidant activities in cell-free conditions and this study focused on the effect of cellular EGCG. Using an intestinal cell model to examine the oxidative stress induced by hydroxyl radicals, we report here that physiological concentrations (0.1-1 microM) of EGCG have dose- and incubation duration-dependent cell-associated lipid antioxidant activity (measuring malondialdehyde production). Vitamin E and vitamin C at 10-40 microM also showed cell-associated lipid antioxidant activities under shorter incubation durations. When EGCG was included in the incubation with vitamin E or C, more antioxidant activities were consistently observed than when vitamins were added alone. Catechin (widely present in fruits and vegetables) at 1 microM also significantly increased the antioxidant activity of vitamins E and C. Previous studies examining cell-associated activity of EGCG mainly focused on the 10-100 microM concentration range. Our results suggest that although the physiological level (0.1-1 microM) of dietary catechins is much lower than that of vitamins, they further contribute to the total antioxidant capacity even in the presence of vitamins.     

Oxidatively modified proteins and DNA in digestive gland cells of the fresh-water mussel Unio tumidus in the presence of tannic acid and its derivatives

The oxidative effect of tannic acid and its two derivatives (ellagic and gallic acid), naturally occurring plant polyphenols, has been studied on digestive gland cells of the fresh-water mussel Unio tumidus. A spectrophotometric method was used to determine the protein thiol groups after incubation of the cells with the polyphenols at concentrations of 1, 15 and 60 microM. The results showed that the oxidative modification of proteins increased in a concentration-dependent manner but no changes were observed at the concentration of 1 microM. The comet assay (single-cell gel electrophoresis assay) with the formamido-pyrimidine glycosylase (FPG) protein was used to assess oxidative DNA base damage. The cells were treated with polyphenols at the concentrations of 30 and 60 microM and post-incubated with FPG. FPG strongly enhanced DNA damage induced by the polyphenols, indicating that N-7 guanine oxidation is responsible for the observed effect. Using the comet assay in combination with proteinase K we were able to demonstrate the presence of DNA-protein cross-links as the probable cause of the decrease in DNA migration. After treatment of the cells with tannic acid and its metabolites at concentrations of 120, 180 and 240 microM, they were post-incubated with proteinase K. After this treatment an increased DNA migration was observed, indicating the presence of DNA-protein cross-links. We have also used a fluorescence method with Hoechst 33258/propidium iodide DNA-binding dyes to study the extent of DNA fragmentation after exposure of the cells to polyphenols at concentrations of 1, 5 and 60 microM. The results demonstrate that the polyphenols can induce apoptosis and necrosis at higher concentrations (5 and 60 microM). All experimental data suggest that tannic, ellagic and gallic acids at concentrations above 1 microM are able to interact with proteins and DNA, which leads to their degradation or changes in their function.     

The increase in human plasma antioxidant capacity after apple consumption is due to the metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids

Regular fruit consumption lowers the risk of cardiovascular diseases and certain cancers, which has been attributed in part to fruit-derived antioxidant flavonoids. However, flavonoids are poorly absorbed by humans, and the increase in plasma antioxidant capacity observed after consumption of flavonoid-rich foods often greatly exceeds the increase in plasma flavonoids. In the present study, six healthy subjects consumed five Red Delicious apples (1037 +/- 38 g), plain bagels (263.1 +/- 0.9 g) and water matching the carbohydrate content and mass of the apples, and fructose (63.9 +/- 2.9 g) in water matching the fructose content and mass of the apples. The antioxidant capacity of plasma was measured before and up to 6 h after food consumption as ferric reducing antioxidant potential (FRAP), without or with ascorbate oxidase treatment (FRAPAO) to estimate the contribution of ascorbate. Baseline plasma FRAP and FRAPAO were 445 +/- 35 and 363 +/- 35 microM trolox equivalents, respectively. Apple consumption caused an acute, transient increase in both plasma FRAP and FRAPAO, with increases after 1 h of 54.6 +/- 8.7 and 61.3 = 17.2 microM trolox equivalents, respectively. This increase in plasma antioxidant capacity was paralleled by a large increase in plasma urate, a metabolic antioxidant, from 271 +/- 39 microM at baseline to 367 +/- 43 microM after 1 h. In contrast, FRAP and FRAPAO time-dependently decreased after bagel consumption, together with urate. Consumption of fructose mimicked the effects of apples with respect to increased FRAP, FRAPAO, and urate, but not ascorbate. Taken together, our data show that the increase in plasma antioxidant capacity in humans after apple consumption is due mainly to the well-known metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids.     

Amino acids and plasma antioxidant capacity

Summary Amino acids antioxidant capacity has been investigated and compared with the chain-breaking antioxidant activity of known compounds as ascorbic acid and Trolox. Basic and acidic amino acids and most of neutral ones showed no antioxidant capacity. On the contrary, tryptophan, tyrosine, cysteine and homocysteine showed antioxidant ability at concentrations which are within the usually reported physiological ranges.These findings are discussed in connection with the antioxidant capacity ascribed to plasma proteins, as human serum albumin.     

New method to study oxidative damage and antioxidants in the human small bowel: effects of iron application

Iron may induce oxidative damage to the intestinal mucosa by its catalyzing role in the formation of highly reactive hydroxyl radicals. This study aimed to determine iron-induced oxidative damage provoked by a single clinical dosage of ferrous sulfate and to elucidate the antioxidant defense mechanisms in the human small intestine in vivo. A double-lumen perfusion tube was positioned orogastrically into a 40-cm segment of the proximal small intestine in six healthy volunteers (25 +/- 5 yr). The segment was perfused with saline and subsequently with saline containing 80 mg iron as ferrous sulfate at a rate of 10 ml/min. Intestinal fluid samples were collected at 15-min intervals. Thiobarbituric acid reactive substances concentrations as an indicator of lipid peroxidation increased significantly from 0.07 microM (range, 0-0.33 microM) during saline perfusion to 3.35 microM (range, 1.19-7.27 microM) during iron perfusion (P < 0.05). Nonprotein antioxidant capacity increased significantly from 474 microM (range, 162-748 microM) to 1,314 microM (range, 674-1,542 microM) (P < 0.05). These data show that a single dosage of ferrous sulfate induces oxidative damage and the subsequent release of an antioxidant in the small intestine in vivo in healthy volunteers.     

Dihydrolipoic acid inhibits 15-lipoxygenase-dependent lipid peroxidation

The potential antioxidant effects of the hydrophobic therapeutic agent lipoic acid (LA) and of its reduced form dihydrolipoic acid (DHLA) on the peroxidation of either linoleic acid or human non-HDL fraction catalyzed by soybean 15-lipoxygenase (SLO) and rabbit reticulocyte 15-lipoxygenase (RR15-LOX) were investigated. DHLA, but not LA, did inhibit SLO-dependent lipid peroxidation, showing an IC(50) of 15 microM with linoleic acid and 5 microM with the non-HDL fraction. In specific experiments performed with linoleic acid, inhibition of SLO activity by DHLA was irreversible and of a complete, noncompetitive type. In comparison with DHLA, the well-known lipoxygenase inhibitor nordihydroguaiaretic acid and the nonspecific iron reductant sodium dithionite inhibited SLO-dependent linoleic acid peroxidation with an IC(50) of 4 and 100 microM, respectively, while the hydrophilic thiol N-acetylcysteine, albeit possessing iron-reducing and radical-scavenging properties, was ineffective. Remarkably, DHLA, but not LA, was also able to inhibit the peroxidation of linoleic acid and of the non-HDL fraction catalyzed by RR15-LOX with an IC(50) of, respectively, 10 and 5 microM. Finally, DHLA, but once again not LA, could readily reduce simple ferric ions and scavenge efficiently the stable free radical 1,1-diphenyl-2-pycrylhydrazyl in ethanol; DHLA was considerably less effective against 2,2'-azobis(2-amidinopropane) dihydrochloride-mediated, peroxyl radical-induced non-HDL peroxidation, showing an IC(50) of 850 microM. Thus, DHLA, at therapeutically relevant concentrations, can counteract 15-lipoxygenase-dependent lipid peroxidation; this antioxidant effect may stem primarily from reduction of the active ferric 15-lipoxygenase form to the inactive ferrous state after DHLA-enzyme hydrophobic interaction and, possibly, from scavenging of fatty acid peroxyl radicals formed during lipoperoxidative processes. Inhibition of 15-lipoxygenase oxidative activity by DHLA could occur in the clinical setting, eventually resulting in specific antioxidant and antiatherogenic effects.     

Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions

Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.     

Dual method for the determination of peroxidase activity and total peroxides-iodide leads to a significant increase of peroxidase activity in human sera

Peroxidases are very important enzymes, e.g., as preventive antioxidants by removing noxious peroxides from the blood. For this reason we evaluated a colorimetric method which detects the activity of endogenous peroxidases by their reaction with hydrogen peroxide, using tetramethylbenzidine as the chromogenic substrate. This assay design can be easily reversed by change of the variable compound to measure also total peroxides in plasma or serum. An increased total antioxidant status was reported previously by the addition of iodide to human serum. In this study iodide activated the endogenous peroxidases significantly in comparison to control sera and isomolar NaCl as well as horseradish peroxidase. Corresponding to the increased peroxidase activity a concomitant decrease of total peroxides occurred in the same samples. This exchangeable assay design is a beneficial opportunity to screen total peroxide levels as well as peroxidase activity in human sera without time-consuming preparations. The method proved to be simple and is favorable due to its specificity, reproducibility, and low costs. Moreover, we were able to find an explanation for the increased total antioxidant status in the presence of iodide, which is presumably an indirect protective effect via an enhanced activity of enzymatic antioxidants, thereby reducing endogenous peroxides.     

Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L

The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K(m) of 35 microM and a V(max) of 55 microM/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K(m) of 2.2mM and a V(max) of 43 microM/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

Antioxidant activity of an aminothiazole compound: possible mechanisms

Oceans are among the richest natural sources of many bioactive compounds. Several of these compounds have shown pharmacological activities for many diseases. Dendrodoine (5-[(3-N-dimethylamino)-1,2,4-thiadiazolyl]-3-indanyl methanone) is an alkaloid extracted from the marine tunicate Dendrodoa grossularia. Aminothiazoles have a wide range of biological activities including anti-tumor and antioxidant properties. The aim of our study was to examine the antioxidant ability of an aminothiazole derivative, dendrodoine analogue (DA) [(4-amino-5-benzoyl-2-(4-methoxy phenylamino) thiazole] which has been chemically synthesized and is similar to dendrodoine. In all the biochemical assays used in our study, corresponding to different levels of protection, DA showed concentration dependent antioxidant ability. DA (3.07 microM) showed an ability to inhibit 2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical formation to the extent of 0.17 microM of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). The ferric complex reducing ability of 3.07 microM DA was equivalent to 110 microM Trolox. 3.07 microM DA gave 84% protection against deoxyribose degradation, a measure of hydroxyl radical scavenging. DA also has an ability to scavenge NO radical, 3.07 microM DA effecting 20% scavenging. Concentration dependent inhibition of lipid peroxidation and protein oxidation induced by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and ascorbate-Fe2+ was observed with low concentrations of DA (1.5-3.07 microM). Mechanistic studies using pulse radiolysis revealed that DA scavenges peroxyl radicals with a bimolecular rate constant of 3 x 10(8)M(-1)s(-1). Moreover, the initially formed nitrogen-centered radical gets transformed into sulfur-centered radical before furnishing any final product. Our results indicated that DA can be a free radical scavenger and potential antioxidant for future application.     

"Antioxidant" (reducing) efficiency of ascorbate in plasma is not affected by concentration

Ascorbate (vitamin C), an important dietary derived antioxidant, reportedly shows decreasing "antioxidant efficiency" with increasing concentrations in indirect radical trapping methods of antioxidant capacity. This study investigated the effect of concentration on antioxidant efficiency of ascorbate using a direct test of antioxidant capacity, the ferric reducing/antioxidant power test (FRAP assay). Results showed that the antioxidant efficiency factor of ascorbate was 2 and was constant over a wide concentration range in both plasma and pure aqueous solution. However, the absolute amount of ascorbate lost per unit of time increased with concentration. Furthermore, ascorbate was less stable in plasma than in aqueous solutions of similar pH and less stable in ethylenediamine tetraacetic acid (EDTA) than in heparinized plasma. Results indicate that previously reported concentration-dependent changes in antioxidant efficiency of ascorbate may have been caused by loss of ascorbate prior to and during testing, and by methodologic characteristics of indirect peroxyl radical trapping tests of antioxidant capacity. Therefore, it is suggested that the premise that the antioxidant efficiency of ascorbate is concentration-dependent is largely methodologically derived and does not reflect the antioxidant behavior of ascorbate per se.     

Effect of alloxan on survival and antioxidant defense of Escherichia coli

The effect of alloxan has been studied on survival and activity of antioxidant and associated enzymes of E. coli wild strains and ones lacked of OxyR and SoxR proteins--regulators of antioxidant defense in E. coli. Bacteria treatment by 500 microM alloxan for 30 min caused an increase of catalase and peroxidase activity in wild and deltasoxRS strains. Catalase activity was not changed in response to alloxan exposure of oxyR deficient strain. It was proposed that under used condition the effect of alloxan on E. coli could be related to the growth of steadystate concentration of hydrogen peroxide. This supposition is also in agreement with the increase of soxRS regulon enzymes activities, because in our previous work it has been shown that H2O2-induced stress increases the activity of some soxRS regulon enzymes.     

Inhibitory effect of linoleyl-hydroxamic acid on the oxidation of linoleic acid by 12-lipoxygenase from porcine leukocytes]

Linoleic acid oxidation by 12-lipoxygenase from porcine leukocytes has been studied as affected by linoleyl-hydroxamic acid. Linoleyl-hydroxamic acid has been found to be an effective inhibitor of porcine leucocyte 12-lipoxygenase. Aerobic preincubation of 12-lipoxygenase with 0.1-6 microM of linoleyl-hydroxamic acid led to a time- and dose-dependent inhibition of the enzyme. The inhibitor's concentration able to induce a 50% loss of the enzyme activity with and without 15-min preincubation were 3.5 and 0.65 microM, respectively. Experimental results obeyed a kinetic scheme, which supposed 2 extra substrate molecules bounding with the enzyme-substrate complex in the presence of linoleyl-hydroxamic acid.     

Induction of apoptosis in a leukemia cell line by triterpene saponins from Albizia adianthifolia

Triterpenoid saponins, which are present in plants and some marine animals, exert various important pharmacological effects. The present study examines the effects of adianthifoliosides A, B, and D (AdA, AdB, and AdD) together with two prosapogenins (Pro1 and Pro2) obtained from Albizia adianthifolia (Mimosaceae) on human leukemia T-cells (Jurkat cells) and on splenocytes. AdA, AdB, and AdD were found to exhibit a cytotoxic effect on Jurkat cells, whereas the prosapogenins were found to exert a lymphoproliferative effect on this cell type. Furthermore, all tested compounds were found to exert a synergistic lymphoproliferative activity with concanavalin A (ConA) on splenocytes. The concentrations where the saponins were found to be cytotoxic on Jurkat cells are far below the concentration of hemolysis. These results indicate that another mechanism than membrane permeabilization formation is responsible of the cell cytotoxicity. Thus, we demonstrated that at 5 microM for AdA and at 1 microM for AdD, these compounds induce apoptosis in Jurkat cells. Early apoptotic events were detected by flow cytometry analysis by using a double annexin-V-FITC and propidium iodide staining. In addition, a disrupted mitochondrial membrane potential was observed in cells treated with AdA, AdB, and AdD. Furthermore, a DNA ladder was observed when Jurkat cells were incubated with 1 microM AdD for 24h. By comparison between the biological activities of the native compounds with the prosapogenins, we show in this work the important role of the acylation and esterification by different moieties at C-21 and C-28 of the aglycone (acacic acid) in the apoptosis-inducing capacity. Particularly, the monoterpene-quinovosyl moiety is shown to be important for the induction of apoptosis.