Antimutagenic, Antiproliferative and Antioxidant Properties of Sea Grape Leaf Extract Fractions (Coccoloba uvifera L.).

BACKGROUND: Cancer is a disease characterized by the invasion and uncontrolled growth of cells. One of the best ways to minimize the harmful effects of mutagens is through the use of natural antimutagens. In this regard, the search for new antimutagens that act in the chemoprevention could represent a promising field in this area. OBJECTIVE: In this study biological potential of 11 fractions from Coccoloba uvifera L. leaf hexane extract was evaluated by several in vitro tests. METHODS: Leaves were lyophilized and hexane extraction was performed. The extract was fractionated by column chromatography with hexane, ethyl acetate, and methanol. The antimutagenic (Ames test), antiproliferative (MTT test), and antioxidant capacity (DPPH, ABTS, and ferrous ion chelation) of the fractions were evaluated. RESULTS: Fractions 4, 6, 8, and 9 have antimutagenic activity (against sodium azide in strain TA100), fraction 11 showed antiproliferative capacity (IC50 of 24 ± 9 µg/mL in cells of HCT 116). The fractions with the highest activity were analyzed by HPLC-MS and lupeol, acacetin, and ß-sitosterol were identified. CONCLUSION: This study demonstrates, for the first time, the bioactivity of C. uvifera leaf as a new source of High Biological Value Compounds (HBVC), which can be of interest to the food and pharmaceutical industries.

A Comparison of Potential Azide Antidotes in a Mouse Model.

Three cobalt-containing macrocyclic compounds previously shown to antagonize cyanide toxicity have been comparatively evaluated for the amelioration of sublethal azide toxicity in juvenile (7-8 weeks) Swiss-Webster mice. The lowest effective doses were determined for hydroxocobalamin, a cobalt porphyrin, and a cobalt-Schiff base macrocycle by giving the antidotes 5 min prior to the toxicant, 27 mg (415 µmol) /kg sodium azide. Both male and female mice were evaluated for their response to the toxicant as well as the antidotes, and no significant differences were noted once weight differences were taken into account. Two of the three compounds significantly decreased the recovery time of azide-intoxicated mice at 10 min after the administration of sodium azide, as determined by a behavioral test (pole climbing). Additionally, azide was determined to cause a several degree drop (∼3 °C) in measured tail temperature, and warming the mice led to a more rapid recovery. The mice were also shown to recover more rapidly when given sodium nitrite, 24 mg (350 µmol)/kg, 5 min after the toxicant; this treatment also suppressed the azide-induced tail temperature decrease. Electron paramagnetic resonance (EPR) measurements of mouse blood treated with sodium azide demonstrated the presence of nitrosylhemoglobin at levels of 10-20 µM which persisted for ∼300 min. The presence of the methemoglobin azide adduct was also detected by EPR at a maximum level of ∼300 µM, but these signals disappeared around 200 min after the administration of azide. The treatment of mice with 15N sodium azide proved that the nitrosylhemoglobin was a product of the administered azide by the appearance of a two-line hyperfine (due to the 15N) in the EPR spectrum of mouse blood.

A Cobalt Schiff-Base Complex as a Putative Therapeutic for Azide Poisoning.

There is presently no antidote available to treat azide poisoning. Here, the Schiff-base compound Co(II)-2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaenyl dibromide (Co(II)N4[11.3.1]) is investigated to determine if it has the capability to antagonize azide toxicity through a decorporation mechanism. The stopped-flow kinetics of azide binding to Co(II)N4[11.3.1] in the absence of oxygen exhibited three experimentally observable phases: I (fast); II (intermediate); and III (slow). The intermediate phase II accounted for ∼70% of the overall absorbance changes, representing the major process observed, with second-order rate constants of 29 (±4) M-1 s-1 at 25 °C and 70 (±10) M-1 s-1 at 37 °C. The data demonstrated pH independence of the reaction around neutrality, suggesting the unprotonated azide anion to be the attacking species. The binding of azide to Co(II)N4[11.3.1] appears to have a complicated mechanism leading to less than ideal antidotal capability; nonetheless, this cobalt complex does protect against azide intoxication. Administration of Co(II)N4[11.3.1] at 5 min post sodium azide injection (ip) to mice resulted in a substantial decrease of righting-recovery times, 12 (±4) min, compared to controls, 40 (±8) min. In addition, only two out of seven mice "knocked down" when the antidote was administered compared to the controls given toxicant only (100% knockdown).

Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia.

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.

Mdivi­1 attenuates sodium azide­induced apoptosis in H9c2 cardiac muscle cells.

The aim of the current study was to investigate the effect of mitochondrial division inhibitor 1 (Mdivi­1) in sodium azide­induced cell death in H9c2 cardiac muscle cells. Mdivi­1 is a key inhibitor of the mitochondrial division protein dynamin­related protein 1 (Drp1). Mdivi­1 was added to H9c2 cells for 3 h, after which, the cells were treated with sodium azide for 24 h. Cell viability was measured by Cell Counting kit­8 assay. DAPI staining was used to observe nuclear morphology changes by microscopy. To further investigate the role of mitochondria in sodium azide­induced cell death, mitochondrial membrane potential (ΔΨm) and the cellular ATP content were determined by JC­1 staining and ATP­dependent bioluminescence assay, respectively. Reactive oxygen species (ROS) production was also assessed by use of the specific probe 2',7'­dichlorodihydrofluorescein diacetate. In addition, the expression of Drp1 and of the apoptosis­related proteins BCL2 apoptosis regulator (Bcl­2), and BCL2 associated X (Bax) was determined by western blotting. The present findings demonstrated that pretreatment with Mdivi­1 attenuated sodium azide­induced H9c2 cell death. Mdivi­1 pretreatment also inhibited the sodium azide­induced downregulation of Bcl­2 expression and upregulation of Bax and Drp1 expression. In addition, the mitochondrion was revealed to be the target organelle of sodium azide­induced toxicity in H9c2 cells. Mdivi­1 pretreatment moderated the dissipation of ΔΨm, preserved the cellular ATP contents and suppressed the production of ROS. The results suggested that the mechanism of sodium azide­induced cell death in H9c2 cells may involve the mitochondria­dependent apoptotic pathway. The present results indicated that Mdivi­1 may have a cardioprotective effect against sodium azide­induced apoptosis in H9c2 cardiac muscle cells.

Neuroprotective effects of hydrogen sulfide on sodium azide­induced autophagic cell death in PC12 cells.

Sodium azide (NaN3) is a chemical of rapidly growing commercial importance. It is very acutely toxic and inhibits cytochrome oxidase (COX) by binding irreversibly to the heme cofactor. A previous study from our group demonstrated that hydrogen sulfide (H2S), the third endogenous gaseous mediator identified, had protective effects against neuronal damage induced by traumatic brain injury (TBI). It is well­known that TBI can reduce the activity of COX and have detrimental effects on the central nervous system metabolism. Therefore, in the present study, it was hypothesized that H2S may provide neuroprotection against NaN3 toxicity. The current results revealed that NaN3 treatment induced non­apoptotic cell death, namely autophagic cell death, in PC12 cells. Expression of the endogenous H2S­producing enzymes, cystathionine­ß­synthase and 3­mercaptopyruvate sulfurtransferase, decreased in a dose­dependent manner following NaN3 treatment. Pretreatment with H2S markedly attenuated the NaN3­induced cell viability loss and autophagic cell death in a dose­dependent manner. The present study suggests that H2S­based strategies may have future potential in the prevention and/or therapy of neuronal damage following NaN3 exposure.

In vitro mutagenic, antimutagenic, and antioxidant activities evaluation and biotransformation of some bioactive 4-substituted 1-(2-methoxyphenyl)piperazine derivatives.

In vitro mutagenic, antimutagenic, and antioxidant potency evaluation and biotransformation of six novel 4-substituted 1-(2-methoxyphenyl)piperazine derivatives demonstrating antidepressant-like activity were investigated. Mutagenic and antimutagenic properties were assessed using the Ames test; free radical scavenging activity was evaluated with 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay and biotransformation was performed with liver microsomes. It was found that all tested compounds are not mutagenic in bacterial strains TA100 and TA1535 and exhibit antimutagenic effects in the Ames test. Noteworthy, compounds possessing propyl linker between phenoxyl and N-(2-methoxyphenyl)piperazine displayed more pronounced antimutagenic properties than derivatives with ethoxyethyl linker. Additionally, compounds 2 and 6 in vitro biotransformation showed that primarily their hydroxylated or O-dealkylated metabolites are formed. Some of the compounds exhibited intrinsic clearance values lower than those reported previously for antidepressant imipramine. To sum up, the results of the present study might represent a valuable step in designing and planning future studies with piperazine derivatives.

Kolaviron was protective against sodium azide (NaN3) induced oxidative stress in the prefrontal cortex.

Kolaviron is a phytochemical isolated from Garcina kola (G. kola); a common oral masticatory agent in Nigeria (West Africa). It is a bioflavonoid used--as an antiviral, anti-inflammatory and antioxidant--in relieving the symptoms of several diseases and infections. In this study we have evaluated the neuroprotective and regenerative effect of kolaviron in neurons of the prefrontal cortex (Pfc) before or after exposure to sodium azide (NaN3) induced oxidative stress. Separate groups of animals were treated as follows; kolaviron (200 mg/Kg) for 21 days; kolaviron (200 mg/Kg for 21 days) followed by NaN3 treatment (20 mg/Kg for 5 days); NaN3 treatment (20 mg/Kg for 5 days) followed by kolaviron (200 mg/Kg for 21 days); 1 ml of corn-oil (21 days-vehicle); NaN3 treatment (20 mg/Kg for 5 days). Exploratory activity associated with Pfc function was assessed in the open field test (OFT) following which the microscopic anatomy of the prefrontal cortex was examined in histology (Haematoxylin and Eosin) and antigen retrieval Immunohistochemistry to show astroglia activation (GFAP), neuronal metabolism (NSE), cytoskeleton (NF) and cell cycle dysregulation (p53). Subsequently, we quantified the level of Glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) in the brain tissue homogenate as a measure of stress-related glucose metabolism. Kolaviron (Kv) and Kolaviron/NaN3 treatment caused no prominent change in astroglia density and size while NaN3 and NaN3/Kv induced astroglia activation and scar formation (astrogliosis) in the Pfc when compared with the control. Similarly, Kolaviron and Kv/NaN3 did not alter NSE expression (glucose metabolism) while NaN3 and NaN3/Kv treatment increased cortical NSE expression; thus indicating stress related metabolism. Further studies on enzymes of glucose metabolism (G6PDH and LDH) showed that NaN3 increased LDH while kolaviron reduced LDH in the brain tissue homogenate (P < 0.001). In addition kolaviron treatment before (P < 0.001) or after (P < 0.05) NaN3 treatment also reduced LDH expression; thus supporting its role in suppression of oxidative stress. Interestingly, NF deposition increased in the Pfc after kolaviron treatment while Kv/NaN3 showed no significant change in NF when compared with the control. In furtherance, NaN3 and NaN3/Kv caused a decrease in NF deposition (degeneration). Ultimately, the protective effect of KV administered prior to NaN3 treatment was confirmed through p53 expression; which was similar to the control. However, NaN3 and NaN3/Kv caused an increase in p53 expression in the Pfc neurons (cell cycle dysregulation). We conclude that kolaviron is not neurotoxic when used at 200 mg/Kg BW. Furthermore, 200 mg/Kg of kolaviron administered prior to NaN3 treatment (Kv/NaN3) was neuroprotective when compared with Kolaviron administered after NaN3 treatment (NaN3/Kv). Some of the observed effects of kolaviron administered before NaN3 treatment includes reduction of astroglia activation, absence of astroglia scars, antioxidation (reduced NSE and LDH), prevention of neurofilament loss and cell cycle regulation.

Protective effects of methanol extracts from Cladonia rangiformis and Umbilicaria vellea against known mutagens sodium azide and 9-aminoacridine.

Lichens and their various extracts have been occasionally used in the treatment of many diseases. Cladonia rangiformis and Umbilicaria vellea are two important species of these lichens and they have several biological activities. In the present study, methanol extracts of these lichens, which are grown in the Eastern Anatolia Region of Turkey, were isolated, and their mutagenic and antimutagenic properties were investigated by using AMES-Salmonella and Zea mays Root Tip Mitotic Index mutagenicity and antimutagenicity assay systems. Known mutagens sodium azide (NaN(3)) and 9-Aminoacridine (9-AA) were used to determine antimutagenic properties of methanol extracts. The results showed that all methanol extracts, investigated in the present study, can be considered genotoxically safe because they do not have mutagenic activity at the tested concentrations. Besides, all of them have antimutagenic activity against 9-AA known as a model intercalator agent in the AMES-Salmonella test system. The inhibition rates obtained from the antimutagenicity assays ranged from 37.07% (C. rangiformis-5 µg/plate) to 54.39% (C. rangiformis-5 µg/plate). Furthermore, all the methanol extracts have significant antimutagenic activity against NaN(3) mutagenicity in Z. mays Root Tip Mitotic Index assay system. These activities are valuable towards an extension of the employ of these drugs as new phytotherapeutic or preservative ingredients.

Extracellular antimutagenic activities of selected probiotic Bifidobacterium and Lactobacillus spp. as a function of growth phase.

The capabilities of selected strains from genera Lactobacillus and Bifidobacterium to produce extracellular bioactive compounds with antimutagenic properties against benzo[a]pyrene (BaP) and sodium azide (SA) were tested as a function of growth phase. The bacterial supernatants from exponential and stationary phases were characterized with different patterns of antimutagenic activity against the two mutagens. All lactobacilli exhibited either no effect or low antimutagenicity against BaP during exponential growth. Higher antimutagenic activities of lactobacilli supernatants were observed in the stationary phase against SA as well. An exception was Lactobacillus sakei 23K which expressed a relatively low percent of inhibition of mutagenesis (PI = 28.14 +/- 7.41) in the exponential phase and no antimutagenic activity in the stationary phase. Of the bifidobacteria, only Bifidobacterium adoleascentis ATCC 15703 exhibited higher antimutagenecity against BaP in the exponential phase. The same bacterial supernatants however, did not possess any antimutagenicity against SA in either the exponential or stationary phases. B. bifidum ATCC 11863 did not express any significant differences in its activity against either BaP or SA in the exponential or stationary phases. Only B. breve ATCC 15700 expressed a high antimutagenic effect against SA in the stationary phase but exhibited no effect during exponential growth. Overall, bacterial antimutagenic responses were associated with growth phase and type of mutagen.

Stearic acid protects primary cultured cortical neurons against oxidative stress.

AIM: To observe the effects of stearic acid against oxidative stress in primary cultured cortical neurons. METHODS: Cortical neurons were exposed to glutamate, hydrogen peroxide (H2O2), or NaN3 insult in the presence or absence of stearic acid. Cell viability of cortical neurons was determined by MTT assay and LDH release. Endogenous antioxidant enzymes activity[superoxide dismutases (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT)] and lipid peroxidation in cultured cortical neurons were evaluated using commercial kits. {3-[1(p-chlorobenzyl)- 5-(isopropyl)-3-t-butylthiondol-2-yl]-2,2-dimethylpropanoic acid, Na} [MK886; 5 micromol/L; a noncompetitive inhibitor of proliferator-activated receptor (PPAR) alpha], bisphenol A diglycidyl ether (BADGE; 100 micromol/L; an antagonist of PPAR gamma), and cycloheximide (CHX; 30 micromol/L, an inhibitor of protein synthesis) were tested for their effects on the neuroprotection afforded by stearic acid. Western blotting was used to determine the PPAR gamma protein level in cortical neurons. RESULTS: Stearic acid dose-dependently protected cortical neurons against glutamate or H2O2 injury and increased glutamate uptake in cultured neurons. This protection was concomitant to the inhibition of lipid peroxidation and to the promotion activity of Cu/Zn SOD and CAT in cultured cortical neurons. Its neuroprotective effects were completely blocked by BADGE and CHX. After incubation with H2O2 for 24 h, the expression of the PPAR gamma protein decreased significantly (P<0.05), and the inhibitory effect of H2O2 on the expression of PPAR gamma can be attenuated by stearic acid. CONCLUSION: Stearic acid can protect cortical neurons against oxidative stress by boosting the internal antioxidant enzymes. Its neuroprotective effect may be mainly mediated by the activation of PPAR gamma and new protein synthesis in cortical neurons.

A notable antimutagenicity of two nonmutagenic novel oxadiazoles in Salmonella mutagenicity assay.

The mutagenic activity of two newly synthesized oxadiazoles: 1,3-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M1) and 1,4-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M2) was studied in Salmonella typhimurium strains TA97, TA100, TA102 and TA1537 in the presence and absence of S9mix. The antimutagenicity of M1 and M2 against H2O2, sodium azide (SA) and 4-nitro-o-phenylene diamine (NPD) using the tester strains TA102, TA100 and TA97, respectively, was also investigated. The two compounds were found to be nonmutagenic using the four tester strains. However, they showed high mutagenic repression activity against hydrogen peroxide (95% and 97% for M1 and M2, respectively, at a concentration of 335 micrograms/plate). Moderate mutagenic repression against NPD (58% and 55% for M1 and M2, respectively, at a concentration of 167.5 micrograms/plate) and low mutagenic repression against SA (21% and 33% for M1 and M2 respectively, at a concentration of 335 micrograms/plate) was detected. The obtained results are very encouraging to test the above mentioned compounds as anticarcinogens.

Protective effect of benidipine against sodium azide-induced cell death in cultured neonatal rat cardiac myocytes.

We investigated the effect of benidipine, a calcium antagonist, against sodium azide (NaN(3))-induced cell death in cultured neonatal rat cardiac myocytes with increase of LDH release, depletion of cellular ATP contents, and collapse of mitochondrial membrane potential (DeltaPsi) as indicators. Cells were treated with 1 mmol/L NaN(3) for 18 h. Benidipine concentration-dependently inhibited NaN(3)-induced cell death. The protective effect of benidipine was compared with those of amlodipine, nifedipine, candesartan, and captopril. Calcium antagonists exhibited a protective effect and the IC(50) values of benidipine, amlodipine, and nifedipine were 0.65, 90, and 65 nmol/L, respectively. NaN(3)-induced cell death was inhibited completely with the calpain inhibitor. It was considered that the sustained elevation of [Ca(2+)](i) might be implicated in NaN(3)-induced cell death. Benidipine, moreover, concentration-dependently preserved cellular ATP contents and maintained DeltaPsi the extent of the control level. In conclusion, benidipine exhibited the protective effect at an approximately 100-fold lower concentration than those of amlodipine and nifedipine in the NaN(3)-induced cardiac cell death model. It was considered that both the inhibition of Ca(2+) influx and the preservation of cellular ATP contents might play an important role in the protective effect of benidipine.

Methylene blue restores spatial memory retention impaired by an inhibitor of cytochrome oxidase in rats.

Cytochrome oxidase is the mitochondrial enzyme that catalyzes the utilization of oxygen for the electron transport chain during cellular respiration. Chronic subcutaneous infusion of sodium azide, an inhibitor of cytochrome oxidase, produced a spatial memory retention deficit in rats in a holeboard maze. Methylene blue, which has been shown to increase oxygen consumption in vitro, was used to restore mitochondrial electron transport in order to facilitate memory consolidation. Administration of 1 mg/kg methylene blue after training, during the memory consolidation period, completely restored the memory retention impaired by the inhibitor of cytochrome oxidase. This suggests that methylene blue may compensate for impaired mitochondrial respiration and improve spatial memory retention. Memory retention deficits found in some neurodegenerative diseases may be improved by drugs targeting impaired mitochondrial respiration.

Anti-mutagenic activity of Phyllanthus amarus Schum & Thonn in vitro as well as in vivo.

Methanolic extract of Phyllanthus amarus was tested for its anti-mutagenic activity in Salmonella typhimurium strains TA1535, TA100, and TA102 (Ames test). P. amarus extract was able to inhibit the activation and mutagenicity of 2-acetaminofluorene (2-AAF) and aflatoxinB(1) at concentrations of 0.25-2 mg/plate. It was also found to inhibit mutagenicity induced by direct acting mutagens sodium azide (NaN(3)), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and 4-nitro-0-phenylenediamine (NPD), at concentrations of 1 mg to 0.25 mg/plate. Urinary mutagenicity produced in rats by benzo[a] pyrene was found to be significantly inhibited by the oral administration of Phyllanthus extract. These results indicate significant anti-mutagenicity of the extract in vitro as well as in vivo.

Characterization of an inducible citrate uptake system in Penicillium simplicissimum.

When citrate was used as a sole source of carbon, citrate uptake by Penicillium simplicissimum increased 267-fold (if glucose-grown mycelium was adapted to citrate) or 1400-fold (if the fungus was grown on citrate) compared to glucose-grown mycelium. Inhibition of macromolecular synthesis prevented this stimulation of citrate uptake. Citrate uptake by glucose-grown mycelium was low (0.0015 nmol min(-1) (mg DW)(-1)) and most probably due to diffusion of undissociated citric acid. Citrate-adapted mycelium had a K(M) of 65 micromol l(-1) and a V(max) of 0.34 nmol min(-1) (mg DW)(-1). In citrate-grown mycelium K(M) was 318 micromol l(-1) and V(max) was 8.5 nmol min(-1) (mg DW)(-1). Citrate uptake was inhibited by sodium azide and uncouplers (TCS, 3,3',4',5-tetrachlorosalicylanilide; FCCP, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone). Because of this we postulate that the induced citrate uptake must be an active transport process. The pH optimum of citrate uptake was between pH 6 and 7. EDTA and Mg2+, Mn2+, Cu2+, Zn2+, Fe2+, Ca2+ only weakly influenced the induced citrate uptake. The properties of citrate uptake by Aspergillus niger and P. simplicissimum are compared.

Antimutagenic potential of extracts isolated from Terminalia arjuna.

Terminalia arjuna is an important medicinal plants widely used in the preparation of Ayurvedic formulations used against several ailments. The present investigation was aimed at the fractionation of crude extracts from the bark of T. arjuna in order to isolate and purify the antimutagenic factors present. The antimutagenicity assay was performed to check the modulatory effect of these fractions against NPD, sodium azide, and 2AF, using the Ames Salmonella his+ reversion assay. Most of the phenolic fractions exhibited mutagen specificity against direct-acting mutagens, being effective in suppressing the frameshift mutagen NPD but failing to inhibit sodium azide (base pair substitution)-induced his+ revertants. ET-1 fraction triterpenoid diglycoside showed a marked effect against sodium azide but was ineffective against NPD. In the case of the indirect-acting mutagen 2AF, all the fractions were found to be quite potent in modulating its mutagenicity in both TA98 and TA100 tester strains of Salmonella typhimurium. The results indicate that the bark of T. arjuna harbors constituents with promising antimutagenic/anticarcinogenic potential that should be investigated further.