Electrophysiological responses to seizurogenic compounds dependent on E/I balance in human iPSC-derived cortical neural networks.

Construction of in vitro functional assay systems using human-induced pluripotent stem cells (iPSCs) as indicators for evaluating seizure liability of compounds has been anticipated. Imbalance of excitation/inhibition (E/I) inputs triggers seizure; however, the appropriate ratio of E/I neurons for evaluating seizure liability of compounds in a human iPSC-derived neural network is unknown. Here, five neural networks with varying E/I ratios (88/12, 84/16, 74/26, 58/42, and 48/52) were constructed by altering the ratios of glutamatergic (E) and GABA (I) neurons. The responsiveness of each network against six seizurogenic compounds and two GABA receptor agonists was then examined by using six representative parameters. The 52% GABA neuron network, which had the highest ratio of GABA neurons, showed the most marked response to seizurogenic compounds, however, it suggested the possibility of producing false positives. Moreover, analytical parameters were found to vary with E/I ratio and to differ for seizurogenic compounds with different mechanism of action (MoA) even at the same E/I ratio. Clustering analysis using six parameters showed the balance of 84/16, which is the closest to the biological balance, was the most suitable for detection of concentration-dependent change and classification of the MoA of seizurogenic compounds. These results suggest the importance of using a human-iPSC-derived neural network similar to the E/I balance of the living body in order to improve the prediction accuracy in the in vitro seizure liability assessment.

Detection of synchronized burst firing in cultured human induced pluripotent stem cell-derived neurons using a 4-step method.

Human induced pluripotent stem cell-derived neurons are promising for use in toxicity evaluations in nonclinical studies. The multi-electrode array (MEA) assay is used in such evaluation systems because it can measure the electrophysiological function of a neural network noninvasively and with high throughput. Synchronized burst firing (SBF) is the main analytic parameter of pharmacological effects in MEA data, but an accurate method for detecting SBFs has not been established. In this study, we present a 4-step method that accurately detects a target SBF confirmed by the researcher's interpretation of a raster plot. This method calculates one set parameter per step, in the following order: the inter-spike interval (ISI), the number of spikes in an SBF, the inter-SBF interval, and the number of spikes in an SBF again. We found that the 4-step method is advantageous over the conventional method because it determines the preferable duration of an SBF, accurately distinguishes continuous SBFs, detects weak SBFs, and avoids false detection of SBFs. We found also that pharmacological evaluations involving SBF analysis may differ depending on whether the 4-step or conventional threshold method is used. This 4-step method may contribute to improving the accuracy of drug toxicity and efficacy evaluations using human induced pluripotent stem cell-derived neurons.

Induction of long-term potentiation and depression phenomena in human induced pluripotent stem cell-derived cortical neurons.

Plasticity such as long-term potentiation (LTP) and long-term potentiation depression (LTD) in neuronal networks has been analyzed using in vitro and in vivo techniques in simple animals to understand learning, memory, and development in brain function. Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for understanding the plasticity mechanism in human neuronal networks, thereby elucidating disease mechanisms and drug discoveries. In this study, we attempted the induction of LTP and LTD phenomena in a cultured hiPSC-derived cerebral cortical neuronal network using multi-electrode array (MEA) systems. High-frequency stimulation (HFS) produced a potentiated and depressed transmission in a neuronal circuit for 1 h in the evoked responses by test stimulus. The cross-correlation of responses revealed that spike patterns with specific timing were generated during LTP induction and disappeared during LTD induction and that the hiPSC-derived cortical neuronal network has the potential to repeatedly express the spike pattern with a precise timing change within 0.5 ms. We also detected the phenomenon for late-phase LTP (L-LTP) like plasticity and the effects for synchronized burst firing (SBF) in spontaneous firings by HFS. In conclusion, we detected the LTP and LTD phenomena in a hiPSC-derived neuronal network as the change of spike pattern. The studies of plasticity using hiPSC-derived neurons and a MEA system may be beneficial for clarifying the functions of human neuronal circuits and for applying to drug screening.

Long-term electrophysiological activity and pharmacological response of a human induced pluripotent stem cell-derived neuron and astrocyte co-culture.

Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for drug discovery and cell-based therapy. However, the immaturity of cultured human iPSC-derived neurons and the lack of established functional evaluation methods are problematic. We here used a multi-electrode array (MEA) system to investigate the effects of the co-culture of rat astrocytes with hiPSC-derived neurons on the long-term culture, spontaneous firing activity, and drug responsiveness effects. The co-culture facilitated the long-term culture of hiPSC-derived neurons for >3 months and long-term spontaneous firing activity was also observed. After >3 months of culture, we observed synchronous burst firing activity due to synapse transmission within neuronal networks. Compared with rat neurons, hiPSC-derived neurons required longer time to mature functionally. Furthermore, addition of the synapse antagonists bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione induced significant changes in the firing rate. In conclusion, we used a MEA system to demonstrate that the co-culture of hiPSC-derived neurons with rat astrocytes is an effective method for studying the function of human neuronal cells, which could be used for drug screening.

Raster plots machine learning to predict the seizure liability of drugs and to identify drugs.

In vitro microelectrode array (MEA) assessment using human induced pluripotent stem cell (iPSC)-derived neurons holds promise as a method of seizure and toxicity evaluation. However, there are still issues surrounding the analysis methods used to predict seizure and toxicity liability as well as drug mechanisms of action. In the present study, we developed an artificial intelligence (AI) capable of predicting the seizure liability of drugs and identifying drugs using deep learning based on raster plots of neural network activity. The seizure liability prediction AI had a prediction accuracy of 98.4% for the drugs used to train it, classifying them correctly based on their responses as either seizure-causing compounds or seizure-free compounds. The AI also made concentration-dependent judgments of the seizure liability of drugs that it was not trained on. In addition, the drug identification AI implemented using the leave-one-sample-out scheme could distinguish among 13 seizure-causing compounds as well as seizure-free compound responses, with a mean accuracy of 99.9 ± 0.1% for all drugs. These AI prediction models are able to identify seizure liability concentration-dependence, rank the level of seizure liability based on the seizure liability probability, and identify the mechanism of the action of compounds. This holds promise for the future of in vitro MEA assessment as a powerful, high-accuracy new seizure liability prediction method.

Analysis of signal components < 500 Hz in brain organoids coupled to microelectrode arrays: A reliable test-bed for preclinical seizure liability assessment of drugs and screening of antiepileptic drugs.

Brain organoids with three-dimensional structure and tissue-like function are highly demanded for brain disease research and drug evaluation. However, to our knowledge, methods for measuring and analyzing brain organoid function have not been developed yet. This study focused on the frequency components of an obtained waveform below 500 Hz using planner microelectrode array (MEA) and evaluated the response to the convulsants pentylenetetrazol (PTZ) and strychnine as well as the antiepileptic drugs (AEDs) perampanel and phenytoin. Sudden and persistent seizure-like firing was observed with PTZ administration, displaying a concentration-dependent periodic activity with the frequency component enhanced even in one oscillation characteristic. On the other hand, in the administration of AEDs, the frequency of oscillation decreased in a concentration-dependent manner and the intensity of the frequency component in one oscillation also decreased. Interestingly, at low doses of phenytoin, a group of synchronized bursts was formed, which was different from the response to the perampanel. Frequency components contained information on cerebral organoid function, and MEA was proven useful in predicting the seizure liability of drugs and evaluating the effect of AEDs with a different mechanism of action. In addition, frequency component analysis of brain organoids using MEA is an important analysis method to perform in vitro to in vivo extrapolation in the future, which will help explore the function of the organoid itself, study human brain developments, and treat various brain diseases.

Principal Component Analysis to Distinguish Seizure Liability of Drugs in Human iPS Cell-Derived Neurons.

Screening for drug discovery targeting the central nervous system requires the establishment of efficient and highly accurate toxicity test methods that can reduce costs and time while maintaining high throughput using the function of an in vitro neural network. In particular, an evaluation system using a human-derived neural network is desirable in terms of species difference. Despite the attention, the microelectrode array (MEA) is attracting among the evaluation systems that can measure in vitro neural activity, an effective analysis method for evaluation of toxicity and mechanism of action has not yet been established. Here we established analytical parameters and multivariate analysis method capable of detecting seizure liability of drugs using MEA measurement of human iPS cell-derived neurons. Using the spike time series data of all drugs, we established periodicity as a new analytical parameter. Periodicity has facilitated the detection of responses to seizurogenic drugs, previously difficult to detect with conventional analytical parameters. By constructing a multivariate analytical method that identifies a parameter set that achieves an arbitrary condition, we found that the parameter set comprising total spikes, maximum frequency (MF), inter- MF interval (IMFI), coefficient of variance of IMFI, and periodicity can uniformly detect the seizure liability of seizurogenic drugs with different mechanisms of action. Seizurogenic drugs were suggested to increase the regularity of the network burst in MEA measurements in human iPS cell-derived neurons.

Neuroprotective efficacy of thymoquinone against amyloid beta-induced neurotoxicity in human induced pluripotent stem cell-derived cholinergic neurons.

The natural antioxidant Thymoquinone (TQ) is the most abundant ingredient in the curative plant Nigella sativa seed's oil. An extensive number of studies have revealed that TQ is the most active and most responsible component for the plant's pharmacological properties. It has been documented in several studies that TQ has a wide range of protective activities and many neuropharmacological attributes. Amyloid beta (Aß) is the major role player peptide in the progression of Alzheimer's disease (AD). Our current study has been implemented to explore the protective possibilities of TQ on Aß1-42 -induced neurotoxicity. To test TQ's effect we used cultured human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. The obtained results showed that Aß1-42 caused cell death and apoptosis, which was efficiently attenuated by the co-treatment of TQ. Moreover, TQ restored the decrease in the intracellular antioxidant enzyme glutathione levels and inhibited the generation of reactive oxygen species induced by Aß1-42. Furthermore, using the fluorescent dye FM1-43 we demonstrated that TQ was able to reduce synaptic toxicity caused by Aß1-42. Thus, the findings of our study suggest that TQ holds a neuroprotective potential and could be a promising therapeutic agent to reduce the risk of developing AD and other disorders of the central nervous system.

Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system.

Functional evaluation assays using human induced pluripotent stem cell (hiPSC)-derived neurons can predict the convulsion toxicity of new drugs and the neurological effects of antiepileptic drugs. However, differences in responsiveness depending on convulsant type and antiepileptic drugs, and an evaluation index capable of comparing in vitro responses with in vivo responses are not well known. We observed the difference in synchronized burst patterns in the epileptiform activities induced by pentylentetrazole (PTZ) and 4-aminopryridine (4-AP) with different action mechanisms using multi-electrode arrays (MEAs); we also observed that 100 µM of the antiepileptic drug phenytoin suppressed epileptiform activities induced by PTZ, but increased those induced by 4-AP. To compare in vitro results with in vivo convulsive responses, frequency analysis of below 250 Hz, excluding the spike component, was performed. The in vivo convulsive firing enhancement of the high γ wave and ß wave component were observed remarkably in in vitro hiPSC-derived neurons with astrocytes in co-culture. MEA measurement of hiPSC-derived neurons in co-culture with astrocytes and our analysis methods, including frequency analysis, appear effective for predicting convulsion toxicity, side effects, and their mechanism of action as well as the comparison of convulsions induced in vivo.

Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture.

The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20-30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

Synthesis of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetate, a hypolipidemic agent, and related compounds.

A series of 2-aryl and 2-alkyl derivatives of 5-furyl-4-oxazoleacetic acid and their homologues having alkyl groups at the alpha-position of the acids were synthesized and evaluated for their hypolipidemic activities in Sprague-Dawley rats. On the basis of the structure-activity relationships and subacute toxicities, ethyl 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetate (35) was selected as a candidate compound for development. Compound 35 reduced serum cholesterol and triglyceride levels by 23% and 35%, respectively, at a dose of 0.05% in a diet in normal rats, and it was about 10 times more active in hereditary hyperlipidemic rats (THLR/1) than in normal rats. Compound 35 inhibited platelet aggregation in vitro and also normalized hyperaggregability of hyperlipidemic plasma platelet ex vivo.

Antiplatelet mechanisms of TA-993 and its metabolite MB3 in ADP-induced platelet aggregation.

We investigated the antiplatelet mechanisms of TA-993 [(-)-cis-3-acetoxy-5-(2-(dimethylamino)ethyl)-2, 3-dihydro-8-methyl-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)-one maleate] and its metabolite MB3 (deacetyl and N-monomethyl TA-993) in human platelets stimulated by ADP in vitro. TA-993 and MB3 concentration-dependently inhibited fibrinogen binding to the ADP-stimulated platelets as well as inhibiting platelet aggregation. The antiplatelet effect of MB3 was about 300 times more potent than those of TA-993 and a glycoprotein IIb/IIIa receptor antagonist, Arg-Gly-Asp-Ser (RGDS). Aggregation of ADP-treated fixed platelets caused by the addition of fibrinogen was inhibited by RGDS but not by TA-993 and MB3. TA-993 and MB3 inhibited ADP-induced polymerization of actin filaments. Neither TA-993 nor MB3 affected cyclic AMP and cyclic GMP levels in resting platelets, and nor suppressed the increase in intracellular Ca(2+) concentration induced by ADP. These results suggest that the antiplatelet mechanisms of TA-993 and MB3 may involve inactivation of glycoprotein IIb/IIIa receptors via inhibition of the polymerization of actin.

Effects of the antiplatelet agent TA-993 and its metabolite MB3 on the hemorheological properties of rat and human erythrocytes.

In the present study, we investigated the effects of the antiplatelet agent TA-993 and its metabolite MB3 on the hemorheological properties of rat and human erythrocytes in comparison with ticlopidine and aspirin. TA-993 and MB3 concentration-dependently lowered the viscosity of rat erythrocyte suspensions. TA-993 and MB3 inhibited both the hypotonic hemolysis of human erythrocytes and the mechanical hemolysis of rat erythrocytes induced by turbulent flow. Treatment of rats with TA-993 (10 mg/kg/day po) for 10 days significantly increased blood filterability, but ticlopidine and aspirin did not show this effect. TA-993 and MB3 enhanced the interaction of 1-anilino-8-naphthalene sulfonate (ANS), a hydrophobic probe, with human erythrocyte ghosts and reduced the fluorescence polarization in 1,6-diphenyl 1,3,5-hexatriene (DPH, a fluidity probe)-labeled human erythrocyte ghosts. TA-993 and MB3 induced aggregation of liposome suspensions prepared from acidic phospholipids. These findings suggest that TA-993 and MB3 may affect the erythrocyte membrane by interacting with acidic phospholipids and thus improve the hemorheological properties.

Inhibitory effect of clentiazem (TA-3090) on platelet aggregation--alone and in combination with aspirin or ticlopidine.

Clentiazem (a novel calcium antagonist) and its basic metabolites (MB1-MB7) showed inhibitory effects on collagen-induced platelet aggregation in human platelets. All the basic metabolites (IC50:8-22 micrograms/ml) had much stronger inhibitory effects than clentiazem itself (IC50:53 micrograms/ml), but the acidic metabolites (MA1-MA4) had no inhibitory effects even at 300 micrograms/ml. Other calcium antagonists (diltiazem, verapamil, nicardipine and nimodipine) also showed similar inhibitory effects although nicardipine and nimodipine were less active than the other drugs. The inhibitory effect of clentiazem was enhanced in the presence of aspirin or ticlopidine. Diltiazem and nicardipine also exhibited a similar potentiation of the anti-platelet effect in combination with aspirin or ticlopidine. Clentiazem also inhibited collagen-induced thromboxane B2 production by the platelets, and this inhibition by clentiazem was additively enhanced by the presence of aspirin. When both clentiazem and aspirin were orally administered to rats, platelet aggregation was additively inhibited. These results indicate that a combination therapy with clentiazem plus aspirin or clentiazem plus ticlopidine may be useful for the prevention and/or treatment of thrombotic disorders.

Thymoquinone protects cultured hippocampal and human induced pluripotent stem cells-derived neurons against α-synuclein-induced synapse damage.

The seeds of Nigella sativa are used worldwide to treat various diseases and ailments. Thymoquinone (TQ) that is present in the essential oil of these seeds mediates most of the plant's diverse therapeutic effects. The present study aimed to determine whether TQ protects against α-synuclein (αSN)-induced synaptic toxicity in rat hippocampal and human induced pluripotent stem cell (hiPSC)-derived neurons. Here, we report that αSN decreased the level of synaptophysin, a protein used as an indicator of synaptic density, in cultured hippocampal and hiPSC-derived neurons. However, simultaneous treatment with αSN and TQ protected neurons against αSN-induced synapse damage, as revealed by immunostaining. Moreover, administration of TQ efficiently induced protection in these cells against αSN-induced inhibition of synaptic vesicle recycling in hippocampal and hiPSC-derived neurons as well as against mutated P123H ß-synuclein (ßSN) in hippocampal neurons, as revealed by experiments using the fluorescent dye FM1-43. Using a multielectrode array, we further demonstrated that the treatment of hiPSC-derived neurons with αSN induced a reduction in spontaneous firing activity, and cotreatment with αSN and TQ partially reversed this loss. These results suggest that TQ protects cultured rat primary hippocampal and hiPSC-derived neurons against αSN-induced synaptic toxicity and could be a promising therapeutic agent for patients with Parkinson's disease and dementia with Lewy bodies.

Inhibition of platelet aggregation by diltiazem. Comparison with verapamil and nifedipine and inhibitory potencies of diltiazem metabolites.

The inhibitory effects of three calcium blockers, diltiazem (d-form), verapamil, and nifedipine, on ADP- and collagen-induced platelet aggregation of human and rabbit platelets were compared. The potency of diltiazem was greater than those of verapamil and nifedipine in human platelet-rich plasma. A similar order of the inhibitory potencies was observed in rabbit platelet-rich plasma, but this order was reversed when washed platelets were aggregated in buffered saline. Antiaggregatory potencies of metabolites on diltiazem and their optical isomers were examined in human platelet-rich plasma. All the metabolites except deacetyl-N-demethyl diltiazem showed greater activity than diltiazem in ADP- or collagen-induced platelet aggregation. The potencies of the l-isomers of the deacetyl-N-demethyl metabolites were greater than those of the d-isomers. On the other hand, the inhibitory effect of the d-isomer of deacetyl-O-demethyl diltiazem was greater than that of the l-form at lower concentrations, whereas the relationship was opposite at higher concentrations. There was no marked difference in antiaggregatory potency between the d- and l-forms of diltiazem and N-demethyl diltiazem.

Imidapril, an angiotensin-converting enzyme inhibitor, inhibits thrombosis via reduction in aortic plasminogen activator inhibitor type-1 levels in spontaneously hypertensive rats.

It is known that angiotensin II (Ang II) exerts an antifibrinolytic effect by stimulating synthesis of plasminogen activator inhibitor type-1 (PAI-1), a specific inhibitor of tissue plasminogen activator (t-PA). The aim of this study was to compare the antithrombotic potency of imidapril, an angiotensin-converting enzyme (ACE) inhibitor, and candesartan, an angiotensin II type 1 (AT1) receptor antagonist, in a model of arterial thrombosis in spontaneously hypertensive rats (SHRs). Oral treatment with 5 mg/kg imidapril 1 h before induction of thrombosis resulted in a significant reduction in thrombus weight, whereas candesartan did not affect thrombus weight under the same treatment conditions. Candesartan lowered blood pressure to the same degree as in the imidapril-treated rats. Imidapril not only reduce the serum and aortic ACE activities, but also reduced aortic PAI-1 protein levels, while candesartan had no effect on theses. These results suggest that imidapril, but not the AT1 receptor antagonist, candesartan, enhances fibrinolysis via a reduction of aortic PAI-1 levels by inhibiting ACE and prevents thrombus formation in SHRs.

Inhibitory effect of clentiazem (TA-3090), a new calcium antagonist, on balloon catheter-induced intimal thickening of rabbit aorta.

Male Japanese white rabbits were fed a restricted amount (100 g/head/day) of an atherogenic diet containing 0.2% cholesterol and 6% peanut oil during an 8-week experimental period. Atherosclerotic lesions, characterized by intimal thickening with lipid deposition, were produced by de-endothelialization of the rabbit aorta with a 4 F balloon catheter halfway through the experiment. Clentiazem (TA-3090), a new calcium antagonist, was administered at an oral dose of 30 mg/kg/day for 4 weeks starting on the day of deendothelialization. Clentiazem significantly depressed the intimal thickening without any effect on serum lipid levels. Clentiazem (1, 3, and 10 microM) significantly and dose-dependently inhibited the in vitro proliferation of smooth muscle cells that had been explanted from the neointima of the deendothelialized aorta. At a higher concentration, this drug markedly inhibited collagen-induced aggregation of rabbit platelets. Diltiazem also showed similar effects, but the effects of clentiazem were more potent than those of diltiazem. These results suggest that clentiazem exhibits an antiatherogenic effect, at least partly through prevention of smooth muscle cell proliferation in atheromatous lesions, in addition to its hypotensive action.

An in vitro study of the hydroxyl radical scavenging property of fluvastatin, and HMG-CoA reductase inhibitor.

We investigated the in vitro hydroxyl radical scavenging activity of fluvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor. Fluvastatin showed hydroxyl radical scavenging activity as potent as that of dimethylthiourea and alpha-tocopherol, which are well-known respectively, as a hydroxyl radical scavenger and a natural antioxidant. Since this effect was not observed in other HMG-CoA reductase inhibitors, such as pravastatin and simvastatin, the scavenging effect of fluvastatin on hydroxyl radicals would not be a common property of HMG-CoA reductase inhibitors, but is derived from the unique chemical structure of fluvastatin. The hydroxyl radical scavenging activities of human metabolites of fluvastatin were also determined. All the tested metabolites possessing the fluorophenyl indole moiety showed activity. Among them, the metabolites which possess a phenolic hydroxyl group on the indole moiety showed stronger effects than that of fluvastatin. We suggest that the fluorophenyl indole moiety of fluvastatin is important for manifestation of the activity and that the phenolic hydroxyl group enhances the potency.

In vitro inhibitory effects of the optical isomers and metabolites of fluvastatin on copper ion-induced LDL oxidation.

Fluvastatin is a synthetic hypolipidemic drug which inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. We compared in vitro the antioxidative effects of two enantiomers (3R, 5S and 3S, 5R) of fluvastatin, which is clinically used as a racemic mixture, on copper ion-induced oxidation of human low-density lipoprotein (LDL). Although 3R,5S-enantiomer of fluvastatin has 30-fold stronger inhibitory activity on HMG-CoA reductase than its optical counterpart, the antioxidative effects of these enantiomers on copper ion-induced LDL oxidation were similar. The antioxidative effects of the metabolites of fluvastatin (M2, M3, M4 and M7) on the copper ion-induced LDL oxidation were also investigated. All the metabolites tested showed an inhibitory effect on this system. Among them, the effects of M2 and M3, which have a phenolic hydroxyl group in each indole moiety, were strong and their potencies were 30-50 times greater than that of fluvastatin. We conclude that not only 3R,5S-enantiomer of fluvastatin but also its optical counterpart and the metabolites also have a potential to show the anti-atherosclerotic effect through their antioxidative activities on lipid peroxidation.