In vitro antiproliferative and antiangiogenic effects of synthetic chalcone analogues

As flavonoids, chalcones possess a wide variety of biological activities including anticancer properties. In the present study we have investigated the in vitro antiproliferative and antiangiogenic effects of four synthetic chalcones.E-2-(4′-methoxybenzylidene)-1-benzosuberone (3) was the most active compound with IC₅₀ = 10⁻⁷ mol l⁻¹ in Jurkat cells. In both Jurkat and HeLa chalcone 3-treated cells we found a significant increase in the proportion of cancer cells in the G₂/M phase of the cell cycle as well as an increase in cells having sub-G₀/G₁ DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. These effects were associated with reduced expression of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax.Furthermore, chalcone 3 was selected to evaluate its effect on some angiogenic events. In non-toxic concentrations, chalcone 3 inhibited VEGF-induced migration of human umbilical vein endothelial cells. Moreover, it also decreased secretion of matrix metalloproteinase (mainly MMP-9) and vascular endothelial growth factor (VEGF).In conclusion, the present study has assessed the in vitro antiproliferative/antiangiogenic potential of chalcone 3. This results generate a rationale for in vivo efficacy studies with this compound in preclinical cancer models.     

Synthetic polysulfane derivatives induce cell cycle arrest and apoptotic cell death in human hematopoietic cancer cells

Natural polysulfanes including diallyltrisulfide (DATS) and diallyltetrasulfide (DATTS) from garlic possess antimicrobial, chemopreventive and anticancer properties. However these compounds exhibit chemical instability and reduced solubility, which prevents their potential clinical applicability. We synthesized six DATS and DATTS derivatives, based on the polysulfane motif, expected to exhibit improved physical and chemical properties and verified their biological activity on human leukemia cells.We identified four novel cytotoxic compounds (IC₅₀ values: compound 1, 24.96 ± 12.37 μM; compound 2, 22.82 ± 4.20 μM; compound 3, 3.86 ± 1.64 μM and compound 5, 40.62 ± 10.07 μM, compared to DATTS: IC₅₀: 9.33 ± 3.86 μM). These polysulfanes possess excellent differential toxicity, as they did not affect proliferating mononuclear blood cells from healthy donors.We further demonstrated ability of active compounds to induce apoptosis in leukemia cells by analysis of nuclear fragmentation and of cleavage of effector and executioner caspases. Apoptosis was preceded by accumulation of cells in G2/M phase with a pro-metaphase-like nuclear pattern as well as microtubular alterations. Prolonged and persistent arrest of cancer cells in early mitosis by the benzyl derivative identifies this compound as the most stable and effective one for further mechanistic and in vivo studies.     

Synthesis and toxicity assessment of 3-oxobutanamides against human lymphocytes and isolated mitochondria

To reduce costly late-phase compound scrubbing, there has been an increased focus on assessing compounds within in vitro assays that predict properties of human safety liabilities, before preclinical in vivo studies. The aim of our study was to answer the questions that whether the toxicity risk of a series of 3-oxobutanamide derivatives could be predicted by using of human lymphocytes and their isolated mitochondria. Using biochemical and flow cytometry assessments, we demonstrated that exposure of lymphocytes and isolated mitochondria to five 3-oxobutanamide derivatives (1–5) did not exhibit remarkable toxicity at low concentrations (50–500 μM) but toxicity could be observed at high concentrations (1000 and 2000 μM), particularly for N-(5-(4-bromophenyl)-3-isoxazolyl)-3-oxobutanamide (4) and N-(2-benzothiazolyl)-3-oxo butanamide (5). Compounds 4, 5 and partly N-(5-methyl-3-isoxazol yl)-3-oxo butanamide (1) also showed a marked cellular and mitochondrial toxicity while compound 5 displayed superior toxicity. Compound 5 induced cytotoxicity on human blood lymphocytes which was associated with the generation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) collapse, lysosomal membrane injury, lipid peroxidation and depletion of glutathione. Our results suggested that among assessed compounds, increased toxicity of compound 5 compared to other compounds could be likely attributed to the presence of bromine substituent in 5. Finally our findings proposed that using of antioxidants and mitochondrial/lysosomal protective agents could be beneficial in decreasing the toxicity of 5.     

Synthesis and pharmacological properties of new GABA uptake inhibitors

Backgroundγ-Aminobutanoic acid (GABA) is the principal inhibitory neurotransmitter in the mammalian central nervous system. The identification and subsequent development of the GABA transport inhibitors which enhance the GABA-ergic transmission has shown the important role that GABA transporters play in the control of numerous functions of the nervous system. Compounds which inhibit GABA uptake are used as antiepileptic drugs (tiagabine - a selective GAT1 inhibitor), they are also being investigated for other indications, including treatment of psychosis, general anxiety, sleep disorders, drug addiction or acute and chronic pain.MethodsIn this paper, the synthesis of 2-substituted-4-(1,3-dioxoisoindolin-2-ylo)-butanamides and 2-substituted-4-amino-butanoic acids derivatives is described. These compounds were tested in vitro for their ability to inhibit GABA uptake. The inhibitory potency towards murine plasma membrane GABA transporters (mGAT1-4) was performed as [³H]GABA uptake assay based on stably transfected HEK cells. Compound 18, which demonstrated the highest affinity for mGAT1-4 (pIC50 ranged from 4.42 for mGAT1 to 5.07 for mGAT3), was additionally investigated in several behavioral tests in mice.ResultsCompound 18 increased the locomotor activity (14–38%) and had anxiolytic-like properties in the four-plate test (ED₅₀ = 9.3 mg/kg). It did not show analgesic activity in acute pain model, namely the hot plate test, however, it was antinociceptive in the acetic acid-induced writhing test (ED₅₀ = 15.3 mg/kg) and in the formalin model of tonic pain. In the latter assay, it diminished nocifensive behavior in both phases and in the first (neurogenic) phase of this test the obtained ED50 value (5.3 mg/kg) was similar to morphine (3.0 mg/kg).ConclusionCompound 18 exhibited significant anxiolytic-like properties and was antinociceptive in some models of pain in mice. Moreover, it did not impair animals' motor coordination in the chimney test. Some of the described pharmacological activities of compound 18 can be partly explained based on its affinity for plasma membrane GABA transporters.     

Synthesis and pharmacological evaluation of pyrrolidin-2-one derivatives as antiarrhythmic,antihypertensive and α-adrenolytic agents

A series of novel arylpiperazines bearing a pyrrolidin-2-one fragment were synthesized and evaluated for their binding affinity for α₁– and α₂ -adrenoceptors (ARs) as well as their antiarrhythmic and antihypertensive activities. The highest affinity for the α₁-AR was displayed by 1-{3-[4-(2-chloro-phenyl)-piperazin-1-yl]-propyl}-pyrrolidin-2-one 7, which binds with a pK_i = 7.13. The highest affinity for the α₂-AR was shown by 1-{3-[4-(4-chloro-phenyl)-piperazin-1-yl]-propyl}-pyrrolidin-2-one 18, which binds with a pK_i = 7.29. Among the compounds tested, 1-{3-[4-(2-ethoxy-phenyl)-piperazin-1-yl]-propyl}-pyrrolidin-2-one 13 had the highest prophylactic antiarrhythmic activity in epinephrine-induced arrhythmia in anesthetized rats. Its ED₅₀ value was 1.0 mg/kg intravenously (iv). The compounds with a hydroxy group in the 4-position of the phenyl ring or two substituents such as fluorine atoms in the 2 and 4 positions of the phenyl ring significantly decreased systolic and diastolic pressure in normotensive anesthetized rats at a dose of 2.5 mg/ kg iv, and their hypotensive effects lasted for longer than an hour.     

Isothiazole dioxide derivative 6n inhibits vascular smooth muscle cell proliferation and protein farnesylation

Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4–C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC₅₀ ranging from 8.56 to 1015 μM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC₅₀ = 61.4 μM) to those required to inhibit [³H]-farnesol incorporation into cellular proteins (−44.1% at 100 μM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [³H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.     

Highly potent and selective inhibition of bovine viral diarrhea virus replication by γ-carboline derivatives

Several novel γ-carboline derivatives were identified as selective inhibitors of bovine viral diarrhea virus (BVDV) replication in cell cultures. Among them, 3,4,5-trimethyl-γ-carboline (SK3M4M5M) was the most active against BVDV (Nose strain) in MDBK cells, with a 50% effective concentration of 0.017 ± 0.005 μM and a selectivity index of 435. The compound inhibited viral RNA synthesis in a dose-dependent fashion. In a time of drug-addition experiment during a single viral replication cycle, SK3M4M5M lost its antiviral activity when first added at 8 h or later after infection, which coincides with the onset of viral RNA synthesis. When selected γ-carboline derivatives, including SK3M4M5M, were examined for their inhibitory effect on the mutant strains resistant to some classes of nonnucleoside BVDV RNA-dependent RNA polymerase inhibitors, all of which target the top of the finger domain of the polymerase, the strains displayed cross-resistance to the γ-carboline derivatives. These results indicate that the γ-carboline derivatives may possibly target a hot spot of the RNA-dependent RNA polymerase. Although SK3M4M5M was highly active against BVDV, the compound proved inactive against hepatitis C virus (HCV) in HCV RNA replicon cells.     

Novel quinazoline and acetamide derivatives as safe anti-ulcerogenic agent and anti-ulcerative colitis activity

Two novel quinazoline derivatives named as; 3-[(4-hydroxy-3-methoxy-benzylidene)-amino]-2-p-tolyl-3H-quinazolin-4-one (5) and 2-p-Tolyl-3-[3,4,5-trimethoxy-benzylidene-amino]-3H-quinazolin-4-one (6) in addition to one acetamide derivative named as 2-(2-Hydroxycarbonylphenylamino)-N-(4-aminosulphonylphenyl) 11 were synthesized, and evaluated for their anti-ulcerogenic & Anti-Ulcerative colitis activities.All of the three compounds showed curative activity against acetic acid induced ulcer model at a dose of 50 mg/kg, they produced 65%, 85% & 57.74% curative ratio for compounds 5, 6 & 11 respectively. The effect of the tested compounds 5, 6 & 11 at dose 50?mg/kg were significantly (P < 0.01) more effective than dexamesathone (0.1?mg/kg) in reducing all parameters.Compounds showed curative activity of for peptic ulcer (induced by absolute alcohol (at a dose of 50?mg/kg, it produced Curative of control ulcer 56.00%, 61.70% & 87.1% for compounds 5, 6 & 11 respectively at dose 50?mg/kg, while the standard drug (Omeprazole 20?mg/kg) produced 33.3%. In both tests, the activity of our target compounds were higher than the standard drugs used for treatment of peptic ulcer and ulcerative colitis. No side effects were reported on liver and kidney functions upon prolonged oral administration of this compounds.     

Induction of G2/M arrest,caspase activation and apoptosis by α-santonin derivatives in HL-60 cells

Sesquiterpene lactones (SLs) are natural products with a variety of biological activities. Previously, we demonstrated the cytotoxic effects of three new α-santonin derivatives on different tumor cell lines with low toxic effects upon peripheral human leukocytes. Here, we evaluated the mechanism of action triggered by these derivatives. HL-60 cell cycle determined after 24 h treatment revealed a significant inhibition on cell-cycle progression and leading to an increasing of cells in G₂/M [7.6% and 9.0% for compound 3% and 9.0% and 8.6% for compound 4 (1 and 2 μM, respectively)]. However, after 48 h exposure, all compounds caused G₂/M reduction and a significant DNA fragmentation. Compounds 2, 3 and 4 were able to induce apoptosis on leukemia cells, which was corroborated by phosphatidyserine externalization and activation of caspases-3 and -7 after 24 h exposure. None of the derivatives analyzed caused depolarization of mitochondrial membrane within 24 h of incubation, suggesting the involvement of the extrinsic apoptotic pathway in the death process. The antiproliferative action of these compounds is related to the DNA synthesis inhibition and cell cycle arrest, which probably lead to apoptosis activation. Therefore, these santonin derivatives are promising lead candidates for development of new cytotoxic agents.     

Development of novel N-linked aminopiperidine-based mycobacterial DNA gyrase B inhibitors: Scaffold hopping from known antibacterial leads

DNA gyrase of Mycobacterium tuberculosis (MTB) is a type II topoisomerase that ensures the regulation of DNA topology and has been genetically demonstrated to be a bactericidal drug target. We present the discovery and optimisation of a novel series of mycobacterial DNA gyrase inhibitors with a high degree of specificity towards the mycobacterial ATPase domain. Compound 5-fluoro-1-(2-(4-(4-(trifluoromethyl)benzylamino)piperidin-1-yl)ethyl)indoline-2,3-dione (17) emerged as the most potent lead, exhibiting inhibition of MTB DNA gyrase supercoiling assay with an IC₅₀ (50% inhibitory concentration) of 3.6 ± 0.16 μM, a Mycobacterium smegmatis GyrB IC₅₀ of 10.6 ± 0.6 μM, and MTB minimum inhibitory concentrations of 6.95 μM and 10 μM against drug-sensitive (MTB H37Rv) and extensively drug-resistant strains, respectively. Furthermore, the compounds did not show any signs of cardiotoxicity in zebrafish ether-à-go-go-related gene (zERG), and hence constitute a major breakthrough among the otherwise cardiotoxic N-linked aminopiperidine analogues.     

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

IntroductionP-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine.MethodsMale Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time.ResultsThe Ab-HiLyte P-gp mediated efflux had a K = 1.00 × 10^? 2 min^? 1 and t_1/2 = 68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K = 1.65 × 10^? 3 min^? 1) and the half life is extremely increased (t_1/2 = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K = 1.81 × 10^? 2 min^? 1 and t_1/2 = 38.28 min).DiscussionThe results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.     

Biarylmethoxy isonipecotanilides as potent and selective inhibitors of blood coagulation factor Xa

New chloro-substituted biarylmethoxyphenyl piperidine-4-carboxamides were synthesized and assayed in vitro as inhibitors of the blood coagulation enzymes factor Xa (fXa) and thrombin. An investigation of effects of the amidine and isopropyl groups attached at the piperidine nitrogen and 5-(halogenoaryl)isoxazol-3-yl groups as biaryl substituents led us to identify new compounds which proved to be selective fXa inhibitors, with inhibition constants in the low nanomolar range. The most potent compound 21e, that incorporates 2-Cl-thiophen-5-yl group as the P1 motif and 1-isopropylpiperidine P4 group, inhibited fXa with K_i value of 0.3 nM and very high selectivity over thrombin and some other tested serine proteases, achieving moderate levels of anticoagulant activity in the low micromolar range, as assessed by the prothrombin time clotting assay (PT₂ = 3.30 μM). Based on reliable docking simulations, molecular modeling provided a rationale for interpreting structure–activity relationships. The predicted binding modes highlighted the structural requirements for addressing the subsites S1 and S4 of the fXa enzyme.     

Synthesis and anticonvulsant screening of 1,2,4-triazole derivatives

Background

Currently available antiepileptic drugs offer limited symptomatic treatment and fail to cure more than 30% of the epileptic seizures. (Arylalkyl)azoles are a class of anticonvulsants including nafimidone and loreclezole. Here, we report the design and synthesis of new (arylalkyl)azoles in N-[1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethylidene]hydroxylamine ester structure, their anticonvulsant screening and in silico prediction studies of their pharmacokinetic properties.

Autodisplay of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors

Hyaluronic acid (HA) is the major biopolymer of the extracellular matrix and contributes significantly to cell proliferation and migration. Human hyaluronidase hPH-20 has been identified as a tumor marker for breast and laryngeal cancer. A hPH-20-autotransporter fusion protein for cell surface display was transformed into Escherichia coli BL21 (DE3) and hPH-20 was displayed on the surface of E. coli. Enzymatic activity, however, was not detectable due to competitive inhibition by lipopolysaccharide (LPS). Finally, expression in E. coli F470, a strain missing the O-polysaccharide of LPS, yielded cells with sufficient hyaluronidase activity.6-Palmitoyl-l-ascorbic acid (Vcpal) and two indole-carboxamides, N-(4-fluorobenzyl)-1-benzyl-1H-indole-2-carboxamide (1) and N-(4-chlorobenzyl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide (2), were tested on inhibition of hPH-20. Vcpal with a concentration of 5 μM inhibited hPH-20 to 93% at pH 7, compounds 1 and 2 showed 61% and 21% inhibition at a concentration of 50 μM. At the same inhibitor concentrations the most frequently used bovine testes hyaluronidase (BTH) was inhibited by Vcpal to a similar extent (95%), whereas compound 1 (80%) and compound 2 (66%) showed much differing inhibition. Thus it can be assumed that BTH is not applicable as an alternative to human PH-20. These results indicate that Autodisplay enables the expression of human target enzymes normally forming inclusion bodies in E. coli and accelerates inhibitor testing as shown by the example of human hyaluronidase PH-20.     

Isolates of Alpinia officinarum Hance as COX-2 inhibitors: Evidence from anti-inflammatory,antioxidant and molecular docking studies

BackgroundInflammation triggered by oxidative stress can cause various ailments, such as cancer, rheumatoid arthritis, asthma, diabetes etc. In the last few years, there has been a renewed interest in studying the antioxidant and anti-inflammatory action of plant constituents such as flavonoids and diarylheptanoids.AimTo evaluate the antioxidant, anti-inflammatory activity and the total phenolic content of isolated compounds from Alpinia officinarum rhizomes. Furthermore, molecular docking was performed to study the binding mode of these compounds into the active site of cyclooxygenase-2 (COX-2).MethodsA. officinarum rhizomes were extracted by maceration, using methanol. This extract was further fractionated by partitioning with hexane, chloroform and ethyl acetate and these fractions on further purification resulted in isolation of five pure compounds. Characterization was carried out by using ¹H NMR, ¹³C NMR and MS. They were further evaluated for antioxidant and anti-inflammatory activity using carrageenan-induced paw edema model in rats. Molecular docking study was performed using Glide module integrated in Schrodinger molecular modeling software.ResultsThe compounds were identified as 1,7-diphenylhept-4-en-3-one (1), 5-hydroxy-1,7-diphenyl-3-heptanone (2), 3,5,7-trihydroxyflavone (Galangin, 3), 3,5,7-trihydroxy-4′-methoxyflavone (Kaempferide, 4) and 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone (5). The compound-3 and compound-5 (10 mg/kg) showed significant (p < 0.001) antioxidant and anti-inflammatory potential. Moreover, total phenolic content was detected as 72.96 mg and 51.18 mg gallic acid equivalent respectively. All the five isolates were found to be good binders with COX-2 (average docking score − 9.03).ConclusionsGalangin and 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone exhibited anti-inflammatory and in-vitro antioxidant activity which may be due to presence of phenolic content in it. The molecular docking study revealed that these compounds have affinity towards COX-2 active site which can further be explored as selective COX-2 inhibitors. The results obtained in this work justify the use of A. officinarum in the treatment of inflammatory disorders like rheumatoid arthritis and inflammatory bowel diseases.     

Evaluation of in vitro cytotoxicity of one-dimensional chain [Fe(salen)(L)]n complexes against human cancer cell lines

The 1d-polymeric iron(III) complexes [Fe(salen)(μ-L)]_n (1–6), involving a deprotonated form of the N-donor heterocyclic compounds (L) imidazole (complex 1), 1,2,4-triazole (2), benztriazole (3), 5-methyltetrazole (4), 5-aminotetrazole (5) and 5-phenyltetrazole (6), were studied for their in vitro cytotoxic activity against human cancer cell lines including lung carcinoma (A549), cervix epithelial carcinoma (HeLa), osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma (A2780) and cisplatin-resistant ovarian carcinoma (A2780cis). Cytotoxicity in vitro (IC₅₀ = 0.39–0.48 μM) was achieved for 2–6 against A2780 (IC₅₀ of cisplatin equals 11.5 μM) as well as for 5 and 6 against all the tested cells, with IC₅₀ = 2.5–37.7 μM. The Uv–Vis spectroscopic study showed that the complexes are unstable in organic solvents (e.g. dimethyl sulfoxide, dimethylformamide) containing even trace amounts of water (and thus also in the medium, i.e. 0.1% DMF, v/v, used in the MTT assay), where they partially or completely decompose to the mixtures involving, besides [Fe(salen)(μ-L)]_n itself, also the starting compounds [{Fe(salen)}₂(μ-O)] and appropriate organic compound (HL). In efforts to find how the resulting cytotoxicity of the most active compounds 5 and 6 is influenced by this fact, the in vitro cytotoxicity testing of mixtures of reactants [{Fe(salen)}₂(μ-O)] and HL, as well as the respective reactants, was also performed. It has been found that the cytotoxicity of 5 and 6 against all the tested cell lines is probably caused by a combined effect of the individual components presented within the corresponding mixture in the medium used.     

Phenolic constituents of Pulicaria undulata (L.) C.A. Mey. sub sp. undulata (Asteraceae): Antioxidant protective effects and chemosystematic significances

One new naturally isoflavone compound, 5,7,2′,3′,4′ penta hydroxyl isoflavone-4′-O-β-glucopyranoside (1) was isolated from the aqueous methanol extract (AME) of Pulicaria undulata subsp. undulata, together with seven known compounds: kaempferol (2), kaempferol 3-O-β-glucoside (3), quercetin (4), quercetin 3-O-β-glucoside (5), quercetin 3-O-β-galactoside (6), quercetin 3,7-di OCH₃ (7), and caffeic acid (8). Their structures were established through chemical (acid hydrolysis) and spectral analysis (UV, NMR, and ESIM). The AME and some isolated compounds were evaluated as protective agents. Free radical scavenging using a microscaled 2,2-diphenyl-1-picrylhydrazyl assay was used to assess the direct antioxidant properties that were evaluated by the ability to protect murine Hepa1c1c7 liver cells against damage induced by the organic peroxide tert-butyl hydroperoxide. The neutral red uptake assay (NRU) was used to record the activity. Results of the 2,2-diphenyl-1-picrylhydrazyl assay recorded differential scavenging properties in ascending order: 5,7,2′,3′,4′ penta hydroxyl isoflavone-4′-O-β-glucopyranoside > quercetin > quercetin 3-O-galactoside > caffeic acid > quercetin 3,7-di-OCH₃ > kaempferol with 50% inhibitory concentrations of 3.9 μM, 7.5 μM, 11.4 μM, 12.2 μM, 78.1 μM, and 252.3 μM, respectively. The antioxidative potential reveals the potency of AME, quercetin, and quercetin 3,7-di-OCH₃. The latter compound showed full protection at 100 μM (33 μg/mL) against the induced toxicant effect where the 50% effective concentration was calculated as 33.6 ± 1.7 μM (11.1 μg/mL). In addition to quercetin, which was extensively shown previously as a cytoprotective agent, AME was less potent; it was capable of protecting 75% at 100 μg/mL with 50% effective concentration of 92.3 ± 4 μg/mL. Moreover, the isolated flavonoids were found to be significantly chemosystematic markers.