Effect of Achyranthes aspera Root and Terminalia arjuna Bark on Aerotolerant Responses in Streptococcus mutans

In the present study, the effect of hydroalcoholic extract of Achyranthes aspera root (AAR) and Terminalia arjuna bark (TAB) on the aerotolerant capacities of Streptococcus mutans was examined. AAR and TAB extracts were found to inhibit the prime antioxidant enzymes likemanganese superoxide dismutase (MnSOD), NADHoxidases, and glutathione peroxidase (GPx), altering the aerotolerant responses in S. mutans. Consequently, S. mutans are unable to withstand the oxidative stress and thus are made more susceptible to the antimicrobial activity.


Introduction
Streptococcus mutans, the main pathogen involved in the development of dental caries, is an aerotolerant microorganism.S. mutans cannot synthesize heme and therefore lacks catalase and cytochrome oxidases required for energylinked oxygen metabolism.However, S. mutans can grow in the presence of oxygen and even consume molecular oxygen through the action of nicotinamide adenine dinucleotide (NADH) oxidase and manganese superoxide dismutase (MnSOD) [1,2].ese oxidative enzymes prevent the bacterial cell from oxidative stress, thereby preventing the generation of highly toxic radicals and conferring oxygen tolerance [3,4].In view of the defense against oxygen toxicity, the lack of catalase in lactic acid bacteria is inconsistent with their aerotolerance [5].us, aerotolerance provides a mechanism for S. mutans to protect them from oxidative damage [6].
Superoxide dismutase is the primary line of defense against the toxicity of oxygen radicals to oxygen and hydroperoxide, through the involvement of metal prosthetic group.MnSOD is found in protecting S. mutans against exogenous superoxide [7].erefore, modulation of superoxide anion generation is an early response as part of the defensive process against pathogen invasion.Superoxide promotes hydroxyl-radical formation and consequent DNA damage in cells of all types [8].NADH oxidases have been produced as a response of S. mutans to oxidative stress.erefore, reductions of NADH oxidases directly sensitize the S. mutans to oxidative stress and no longer give protection.Glutathione peroxidase (GPx) catalyzes the reduction of hydroperoxides at the expense of GSH.It catalyses the reduction of H 2 O 2 and organic hydroperoxides including those derived from unsaturated lipids to alcohol.It also protects the biomembrane from oxidative attack.Induction of protective systems aer exposure to free radicals or ROS may be an important adaptive response to nonlethal insults by these reactive species.e main objective of our study was to examine the effect of Achyranthes aspera root (AAR) and Terminalia arjuna bark (TAB) on the aerotolerant properties in S. mutans.e two plant materials were mentioned in siddha literature to give relief from tooth ache.Based on Siddhar's claims, anticariogenic activity of AAR and TAB extracts could be well pronounced by interfering with the defense system of S. mutans and making them more susceptible for the drug action.A preliminary phytochemical evaluation of the AAR and TAB extracts was performed.

Bacterial Strain and Growth Conditions.
Streptococcus mutans A 32-2 strain (serotype c) is used in our study.S. mutans A 32-2 strain is obtained as gi from Dr. Richard L.Gregory, Ph.D., Professor of Oral biology and Pathology, School of Dentistry and medicine, Indiana University, USA.
S. mutans was maintained in our laboratory with weekly subculture on Todd Hewitt agar or Brain heart infusion agar or Trypticase soy agar plates, all supplemented with 1% glucose and long-term storage in the speci�ed medium at −70 ∘ C in 50% glycerol solution.S. mutans A 32-2 was incubated at 37 ∘ C at 10% CO 2 in Candle jar.

Collection of Plant Materials & Extract
Preparation.e roots of Achyranthes aspera and Terminalia arjuna bark were collected from the Tamil University campus, anjavur, India.e collected roots and barks were shade dried and powdered.e dried powders of AAR and TAB were separately extracted with 50% ethanol at 60 ∘ C by continuous hot percolation using Soxhlet apparatus and used for the study.

Preliminary Phytochemical
Testing.500 mg of the dried ethanolic extract was reconstituted in 10 mL of ethanol and it was subjected to preliminary phytochemical testing for the presence of alkaloids, �avonoids, tannins, saponins, terpenoids and other different chemicals groups of compounds by standard methods [9,10].e color intensity or the precipitate formation was used as analytical responses to the tests.

Preparation of Bacterial Cell Lysate.
Overnight culture of S. mutans A 32-2 grown in Todd-Hewitt broth was harvested and incubated with 100 mg/L and 500 mg/L of AAR and TAB extracts, respectively.Cells devoid of extracts served as control.e samples were incubated at 37 ∘ C for 3 hours.Aer incubation, the cells were removed, homogenized, and centrifuged to remove the cell debris.e cell lysate was used as the enzyme source.

Role of AAR and TAB on Superoxide Dismutase in S.
mutans Assay of Superoxide Dismutase (SOD).Superoxide dismutase activity was determined by the procedure of Kakkar et al. [11] with slight modi�cations.
Bacterial cell lysate was used as the enzyme source.Cell lysate included both the treated (AAR/TAB) and untreated samples.
0.5 mL of sample was diluted to 1 mL with water.en 2.5 mL of ice-cooled ethanol and 1.5 mL ice-cooled chloroform were added.is mixture was shaken for 1 minute at 4 ∘ C and then centrifuged.e enzyme activity in the supernatant was determined.e assay mixture contained 1.2 mL of sodium pyrophosphate buffer (0.025 M, pH 8.3); 0.01 mL of phenazine methosulphate, 0.3 mL of nitro blue tetrazolium (NBT); 0.2 mL of NADH (780 M), appropriately diluted enzyme preparation and water in a total volume of 3 mL.Reaction was initiated by the addition of NADH.Aer incubation at 30 ∘ C for 90 seconds, the reaction was stopped by the addition of 1 mL glacial acetic acid.e reaction mixture was stirred vigorously and shaken with 4 mL of n-butanol.e intensity of the chromogen in the nbutanol layer was measured at 560 nm against butanol blank.
A system devoid of enzyme served as control.Enzyme activity was expressed as 50% of NBT reduction/min/mg protein.[12] with slight modi�cation.

Role of AAR and TAB on NADH Oxidase in S. mutans NADH Oxidase Assay. NADH oxidase was assayed by the method of Steyn-Parve and Benert
NADH oxidase activity was assayed at 30 ∘ C by monitoring the oxidation of NADH in the reaction mixture (1 mL) at absorbance, 340 nm.e reaction mixture contained 40 mM potassium phosphate buffer (PPB), 0.2 mM EDTA, 0.17 mM-NADH, and cell lysate containing enzyme.One unit of NADH oxidase was de�ned as the amount of enzyme (mg protein) that catalyzed the oxidation of 1.0 mol NADH min −1 .
e reaction mixture consisted of 0.2 mL each of 0.8 mM EDTA, 10 mM sodium azide, 2.5 mM H 2 O 2 , 0.4 mL of phosphate buffer (0.32 M; pH 7.0), and 0.1 mL of sample was incubated at 37 ∘ C at different time intervals.e reaction was arrested by the addition of 0.5 mL of 10% TCA and the tubes were centrifuged at 2000 rpm.To 0.5 mL of supernatant, 4 mL of 0.3 M disodium hydrogen phosphate and 0.5 mL DTNB (40 mg of DTNB in 100 mL of 1% sodium citrate) were added and the color developed was read at 420 nm immediately.e activity of GPX was expressed as moles of glutathioneoxidized/min/mg protein.

Protein Estimation.
Total proteins were assayed by the method of Lowry et al. [14].

Statistical Analysis.
All the experiments were done in triplicates and the results were presented as the mean ± SD.One-way analysis of variance (ANOVA) followed by the Tukey's test for multiple comparison values of   1 was considered to be signi�cant.Statistical Package for Social Studies (SPSS) 7.5 version was used for this analysis.

Results and Discussion
In the present study, preliminary phytochemical testing shows (Table 1) the presence of high amount of glycosides, alkaloids, tannins, phenolics and other all the principal secondary metabolites in hydroalcoholic extracts of AAR and TAB.e result obtained in the present experiment is well supported by previous studies.Phytochemical studies on Achyranthes aspera revealed the presence of glycosides of oleanolic acid, peroxidase, saponins, indole acetic acid, polyphenol, ecdysome, and �avanoids in various parts of the plant.Betaine and achyranthine are the principal alkaloids identi�ed from the whole plant [15].e various constituents of the dried bark extract of Terminalia arjuna (IPC-53) include acids such as arjunic acid, gallic acid, ellagic acid, arjunin and terminic acid, glycosides such as arjunetin, arjunosides I�IV, the �avone arjunolone, tannins, oligomeric proanthocyanidins (OPCs), coloring matter, and essential oils and minerals such as calcium, magnesium, zinc, and copper [16].
In our experiment, Table 2 showed the activity of MnSOD in control and AAR/TAB treated cells.AAR at 100 mg/L and 500 mg/L concentration showed signi�cant reduction in the MnSOD activity when compared to the control cells.In case of TAB treated cells, TAB at 100 mg/L concentration showed signi�cant reduction, while at 500 mg/L concentration showed enhanced MnSOD activity when compared to the control cells.erefore, TAB at 500 mg/L concentration is not able to cause oxygen toxicity.
Interestingly, the lowest concentrations of TAB (100 mg/L) resulted in decreases in MnSOD activity in S. mutans.An understanding of the optimal concentrations of TAB extract may require careful titration to determine predictable and dependable therapeutic strategies against adverse effects.Previous report suggests that T. arjuna extract-induced oxidative stress is upstream of signaling events that might alter the pro-and antiapoptotic balance in HepG2 cells.Phenolic compounds are generally known to show not only their antioxidant effects but also prooxidant actions under in vitro conditions.Hence, it is possible that treatment of HepG2 cells with T. arjuna extract can deplete the GSH levels and promote oxidation induction, which switches the mode of death via apoptosis.erefore, the cytotoxic action of this drug may be attributed to its prooxidant action on the cells.is may be able to account for the discrepancy between in vitro cytotoxicity and in vivo antitumor activities of T. arjuna extract [17].
It has been reported that reduction in SOD activity increases the sensitivity of S. mutans to oxygen tolerance, through the enhancement of Fenton.It increases the amount of hydroxyl radical leading to cell death [18].Since SOD is proven to play role in aerotolerance in S. mutans [19], decrease in SOD activity by AAR and TAB might bene�t them as anticaries.
Iron ions stimulate the generation of highly reactive and toxic oxygen species such as hydroxyl radicals.In vitro experiments have shown that Fe 2+ catalyzes non-enzymatic hydroxyl radical synthesis from H 2 O 2 via the Fenton reaction.But, H 2 O 2 remained intact in the absence of iron ions at physiological pH [20].erefore, tight regulation of the intracellular free iron ion concentration is believed to be signi�cant factor in organism survival under aerobic conditions.One of the important characteristics that distinguish S. mutans from other oral streptococci is its ability to ferment mannitol and sorbitol [21].According to Yamamoto et al., A new antioxidant gene, Dpr, in the chromosomal DNA of S. mutans was identi�ed [5].Dpr was found to have ironbinding ability.us, a possible protective role of Dpr might be to sequester iron, thereby protecting cells from peroxides and conferring oxygen tolerance.Dpr did not directly react with oxygen and reactive oxygen species.e iron-binding ability of Dpr might indirectly contribute to oxygen tolerance in S. mutans.
Although Streptococci have a preference for anaerobiosis, O 2 affected the growth on mannitol with a variation dependent on strain [3].e growth response to O 2 was correlated with the ability of strains to induce NADH oxidase and superoxide dismutase (SOD) under aerobic conditions [4].ese �ndings suggested that NADH oxidase plays an important role in the regulation of aerobic metabolism through the regulation of NAD from the additional NADH derived from the oxidation of mannitol-1-PO 4 to fructose-6-PO 4 .
In our study, the NADH oxidase activity of control and AAR/TAB treated cells was estimated under aerobic condition, as evidenced in Table 3. AAR and TAB treated cells at concentration of 100 mg/L and 500 mg/L showed signi�cant reduction in the NADH oxidase activity compared to the control cells.TAB at 100 mg/L concentration showed maximum inhibition of 74.77%.AAR and TAB imposes an oxygen dependent toxicity, apparently both by inhibition of growth and by loss of viability.Decrease in NADH oxidases in treated cells relates to the susceptibility of the organism to oxidative challenge.
Reduced transition metal cations commonly enhance oxidative damage to cells caused by hydroperoxides formed as a result of oxygen metabolism or added externally.As expected, the cations Fe 2+ and Cu + enhanced killing of Streptococcus mutans GS-5 by hydroperoxides [22].Iron and copper present in AAR and TAB substantiate their role on increased oxidative damages caused due to the reduction in the antioxidant enzymes like SOD and NADH oxidases in S. mutans.
Polyphenols as catechin act on different bacterial strains belonging to different species (Escherichia coli, Bordetella bronchiseptica, Serratia marcescens, Klebsiella pneumonaie, Salmonella choleraesuis, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis) by generating hydrogen peroxide [23] and by altering the permeability of the microbial membrane [24].Polyphenolic compounds which have anticariogenic property [25] also reported to exhibit prooxidant properties under appropriate conditions [26].us, AAR and TAB, rich in polyphenols might act as pro oxidant, providing free radicals at higher rate during the treatment of S. mutans cells with AAR and TAB.As a consequence, SOD and NADH oxides, the major antioxidant enzymes present in S. mutans, could no longer able to compete and scavenge the overcrowding free radical formation and ultimately lead to the deterioration of the SOD and NADH activities.
Table 4 showed the GPx activity of the control and AAR/TAB-treated cells (100 mg/L and 500 mg/L).e GPx activity was signi�cantly reduced in AAR-and TAB-treated cells when compared to the control cells.e reduction in GPx activity in the both extracts may be attributed to the presence of terpenoids.e present study shows the presence of terpenoids in AAR and TAB extracts.e mechanism of action of terpenes is speculated to involve membrane disruption by the lipophilic compounds.Accordingly, Mendoza et al. [27] found that increasing the hydrophilicity of kaurene diterpenoids by addition of a methyl group drastically reduced their antimicrobial activity.Food scientists have found the terpenoids present in essential oils of plants to be useful in the control of Listeria monocytogenes [28].
Another possible mechanism of GPx inhibitory activity might be favored by �avonoids present in AAR and TAB.Activity of �avonoids are probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls, as described above for quinones.More lipophilic �avonoids may also disrupt microbial membranes [29].Both terpenoids, and �avonoids present in AAR and TAB were found to disturb the membrane of the bacteria.According to the �nding of omas, GSH, the substrate for GPX activity, was normally imported from external source into the S. mutans cells [30].us, when the bacterial cell membrane is being disturbed by �avoniods and terpinoids present in AAR and TAB, S. mutans no longer able to import GSH and ultimately, no substrate is available for carrying out GPx activity.AAR and TAB were found to reduce the enzyme activity, thereby reducing the tolerance response.is results in the accumulation of potent cytotoxic agents like free radicals, peroxides in S. mutans that cause lethality.
Ellagic acid and quercetin were found were to be potent inhibitors of GPx [31].Our results show the presence of polyphenolic compounds in hydroalcoholic extract of AAR and TAB and these results are consistent with earlier studies that reported the presence of ellagic acid TAB [32].
e GSH-dependent reduction system is responsible for maintaining a reduced environment and plays an important role in survival in the presence of oxidative stress in Escherichia coli and Saccharomyces cerevisiae [33].Sherrill and Fahey found that cellular GSH protected S. mutans ATCC 33402 against growth inhibition by the thiol oxidizing agent diamide [34].Now, presence of AAR/TAB makes S. mutans incapable of being protected from thiol oxidizing agents.erefore, reduction in the glutathione-related enzymes ultimately makes S. mutans unable to withstand the oxidative stress and, hence, made it more liable to the antimicrobial activity.

Conclusion
e anticariogenic activity of AAR and TAB extracts is well pronounced by interfering with the defense system of S. mutans and, thus, making the organism more susceptible for the drug action.Our results suggest that the inability of S. mutans to consume oxygen has a negative impact on its capability to infect and cause disease.e disruption of the oxygen metabolizing pathways by AAR and TAB extracts may lead to novel drug targets and a way to control the growth of the organism and diminish the incidence of dental caries.e extracts can be subjected to isolation of the active compound to carry out further pharmacological evaluation and molecular mechanisms of their actions against S. mutans.is will surely complement to the previously known therapeutic values of AAR and TAB and improve the popularization of the plants.