Aging, characterized by progressive alterations in cells and tissues of the organic structure, is associated with fading of cellular map [ 1 ] . Overproduction of Reactive O species ( ROS ) including free groups such as superoxide ( O2•? ) , hydroxyl extremist ( •OH ) , peroxyl extremist ( RO2•? ) every bit good as nonradical species such as H peroxide ( H2O2 ) are accepted as the chief modulators of aging [ 2 ] . Under oxidative emphasis status, H2O2 diffuses into lysosomes ( PH ~ 4 – 5 ) and reacts with Fe metal ions released by metalloprotein debasement ( Fenton chemical science ) . This leads to the formation of extremely reactive O species such as hydroxyl groups ( •OH ) which later oxidize critical supermolecules such as lipoids, proteins and nucleic acids. The oxidised merchandises would so respond with each other taking to the formation of sums known as lipofuscin pigments or age pigments ( Fig. 1 ) [ 3,4 ] .
Lipofuscin is therefore, an intracellular indigestible amber autofluorescent stuff composed chiefly of oxidized protein ( 30-58 % ) and lipid ( 19-51 % ) bunchs. It is extremely immune to proteolytic debasement and accumulates largely in station mitotic cells such as nerve cells [ 5-7 ] . The intracellular rate of lipofuscin formation is negatively correlated with the staying life span of cells and additions with age [ 7-8 ] . Sing the elevated degree of polyunsaturated fatty acids in encephalon, lipofuscin pigments largely accumulate within this tissue by age [ 9 ] .
However, cells have several antioxidant defence mechanisms to forestall and/or to rarefy the destructive effects of ROS. These defence mechanisms include antioxidative enzymes, such as superoxide dismutase ( SOD ) , catalase ( CAT ) , glutathione peroxidase ( GPx ) and little molecules such as glutathione and vitamins C and E [ 10 ] . The efficiency of the antioxidant defence system is weaken under oxidative emphasis conditions. Therefore, to endorse up the system under these conditions, it might be good to utilize exogenic natural or man-made antioxidants.
Recently, much attending has been devoted to the phenolic antioxidants of medicative and dietetic workss [ 11-14 ] . Yakuchinone B ( 1- [ 4′- hydroxy-3′-methoxyphenyl ] -7-phenylhept-1-en-3-one ) has been characterized in Alpinia oxyphylla from zingiberaceae household [ 15 ] . This conjugated 1, 4- enones belongs to chalcone compounds with known diverse biological activities such as anti-inflammatory [ 16 ] , antiproliferative [ 17 ] , antiviral [ 18 ] and anti-neurodegenerative [ 19 ] . To acquire a better apprehension on the structural entities required for free extremist scavenging, we evaluated the protective effects of yakuchinone B derived functions ( JC1-JC6, table 1 ) against H2O2-induced harm on SK-N-MC cells in footings of cell viability, intracellular ROS content, MDA and lipofuscin degrees and besides on the CAT and SOD activities. Our consequences indicated that yakuchinone B derivatives particularly JC4, JC5 and JC6 decreased the extent of programmed cell death and ROS degrees and increased the activities of SOD and CAT relative to H2O2-treated cells. Antioxidant activity of yakuchinone B derivatives seems to be related to their molecular construction: the presence of a hydroxyl group on pealing A ( JC1-JC6 ) , presence of pealing B ( JC3, JC4, JC5 and JC6 ) , presence an alkyl group in pealing B ( JC4, JC5 and JC6 ) and the junction and resonance effects on rings A and B seemed to be indispensable for JCs activities.
2. Materials and methods
The cell civilization medium ( RPMI-1640 ) , foetal bovine serum ( FBS ) and penicillin-streptomycin were purchased from Gibco BRL ( Life Technology, Paisley, Scotland ) . Cell line was obtained from Pasteur Institute of Iran ( Tehran, Iran ) . The civilization home bases were obtained from Nunc ( Brand merchandises, Denmark ) . Nicotinamide adenine dinucleotide reduced ( NADH ) , phenazine methosulphate ( PMS ) , glutaraldehyde, nitroblue tetrazolium ( NBT ) , H2O2 and 2-thiobarbituric acid ( TBA ) were obtained from Merck ( Germany ) . Ethidium bromide, acridine orange and Triton x-100 were purchased from Pharmacia LKB Biotechnology ( Sweden ) . MTT [ 3- ( 4, 5-dimethyl tiazol-2-yl ) -2, 5-diphenyl tetrazolium bromide ] and phenylmethylsulphonyl fluoride ( PMSF ) were from Sigma Chem. Co ( Germany ) . 2? , 7?-dichlorofluorescein diacetate ( DCFH-DA ) was obtained from Molecular Probe ( Eugene, Oregon, USA ) . Ethylenediaminetetraacetic acid ( EDTA ) was from Aldrich. Lead citrate, Na-cacodylate and uranyl ethanoate were purchased from PELCO ( California, USA ) . Osmium tetroxide ( OsO4 ) was obtained from TAAB Laboratories Equipment Ltd ( Aldermaston, UK ) . Epoxy ( Araldite ) was from AGAR scientific LTD. Benzylideneacetophenone derived functions ( JC1-JC6 ) were a generous gift from Dr. Seikwan Oh ( Ewha Womans University of Korea ) . JC1-JC6 were dissolved in a minimal sum of dimethyl sulfoxide ( DMSO ) and so diluted with the civilization medium to acquire the coveted concentration. The concentration of DMSO in the civilization medium has been kept lower than 0.1 % and the control cells has been treated with the vehicle incorporating the same sum of DMSO.
2.2. Cell civilization and experimental intervention
Human SK-N-MC neuroblastoma cells, obtained from Pasteur Institute ( Tehran, Iran ) , were cultured at a denseness of 5-104/ml RPMI 1640 medium supplemented with FBS ( 10 % , v/v ) , streptomycin ( 100 ?g/ml ) and penicillin ( 100 U/ml ) and kept at 37 & A ; deg ; C in a 5 % CO2 humidified atmosphere. Drug interventions were normally done 24 H after seeding the cells. To bring on the oxidative emphasis, H2O2 was newly prepared from 8.5 millimeter stock solution prior to each experiment. SK-N-MC cells were incubated with JC1-JC6 for 3 H before exposure to 300 ?M H2O2.
2.3. Cytotoxicity rating of JC1-JC6 in SK-N-MC cells
Cell viability was estimated utilizing the MTT check. This method is dependent on the transition of xanthous tetrazolium bromide to its violet formazan derivative by mitochondrial succinate dehydrogenase of the feasible cells [ 20 ] . The cells were seeded in 96-well home bases at a concentration of 5-104 cells/ml for 24 H, and so treated with JC1-JC6 at different concentrations ( 10, 20, 40, 60 and 80 µM ) . After 24 H, MTT stock solution ( 10 µl ; 5 mg/ml ) was applied to each well. After 4 H of incubation, the home bases were centrifuged for 15 min at 2500 revolutions per minute and the supernatants were aspirated. The formazan crystals in each well were dissolved in 200 µl of DMSO, and the optical density was measured via ELISA technique at a wavelength of 570 nanometers. Consequences were expressed in per centum of MTT decrease relation to the control cell samples, assuming that the optical density of the control cells was 100 % .
2.4. Cytoprotective consequence of JC1-JC6 in H2O2-treated SK-N-MC cells
To measure the cytoprotective effects of JC1-JC6 against H2O2 harm, SK-N-MC cells were plated at a denseness of 5-104 cells/ml for 24 h. The cells were treated with 20 µM of JC1-JC6. Three hours subsequently, 300 µM H2O2 was added to the home base followed by incubation for an extra 24 H and 48 h. Cell viability was estimated utilizing the MTT check and it was expressed in per centum of survival relation to the control cell samples.
2.5. Fluorescence microscopy and sensing of apoptotic cells
The SK-N-MC cells were seeded in 24-well home bases. After 24 H, the cells were pretreated with 20 µM of each drugs and so were treated with 300 ?M H2O2 for a clip class of 24 h. Apoptotic cells were characterized morphologically by staining the cells with acridine orange/ethidium bromide ( AO/EtBr ) followed by fluorescence microscopy review [ 21 ] . After intervention, cells were washed twice with PBS and adjusted to a cell denseness of 1-106 cells/ml of PBS and stained with AO/EtBr solution ( 1:1 v/v ) in a concluding concentration of 100 µg/ml. The atomic morphology was evaluated by Axoscope 2 plus fluorescence microscope from Zeiss ( Germany ) . The cells with condensed or disconnected karyons were counted as apoptotic cells. All experiments were repeated three times. In each tally, the entire Numberss of cells along with entire figure of apoptotic cells were determined in 10 different microscopic Fieldss. The extent of programmed cell death was so expressed as a per centum of the entire cell count.
2.6. Measurement of Intracellular ROS
The ROS coevals was monitored utilizing 2 ‘ , 7’- dichlorofluorescein diacetate ( DCFH-DA ) which readily diffuses into cells [ 22 ] .Within the cells, this nonfluorescent dye reacts with intracellular ROS and is converted into dichlorofluorescein ( DCF ) which is a fluorophor. SK-N-MC cells were cultured in a 12-well home base with 1ml of medium for 24 h. Then, cells were pretreated with drugs and so exposed to 300 µM H2O2. After 24 h incubation, cells were rinsed with serum-free RPMI medium and so, DCFH-DA ( 10 µM ) was added to the cells followed by incubation at 37-C for 1 h. After incubation, cells were washed twice with PBS and the fluorescence strength was monitored utilizing a Varian-spectrofluorometer, theoretical account Cary Eclipse with excitement and emanation wavelengths of 485nm and 530 nanometer, severally.
2.7. Catalase activity assay
The CAT activity was measured by the method of Aebi [ 23 ] in which the rate of decomposition of H2O2 was determined spectrophotometrically at 240 nanometer. The enzyme activity was expressed as -10?1 k/mg protein, where K represents the rate invariable of the first order reaction of catalase. Protein concentration was determined by the method of Lowry et Al [ 24 ] .
2.8. Superoxide dismutase activity assay
The SOD activity was determined harmonizing to the method of Kakkar et Al [ 25 ] , based on the extent suppression of amino bluish tetrazolium formazan formation in the mixture of nicotinamide A dinucleotide, phenasine methosulphate and nitroblue tetrazolium ( NADH-PMS-NBT ) . One unit of enzyme activity was defined as the sum of enzyme which caused 50 % suppression of NBT reduction/mg protein.
2.9. Determination of lipid peroxidation
Thiobarbituric acid reactive substances ( TBARS ) , as indexs of lipid peroxidation, were assayed as described in the literature [ 26 ] . Briefly, after exposure of SK-N-MC cells to JC1-JC6 for 3 H, cells were exposed to 300 µM H2O2 for 48 h. MDA degrees were measured by the dual warming method [ 27 ] . The method is based on spectrophotometeric measuring of the violet colour generated by the reaction of thiobarbituric acid ( TBA ) with MDA. Briefly, the cells were assorted with 0.5 milliliters of tricholoroacetic acid ( TCA, 10 % , w/v ) solution followed by boiling in a H2O bath for 15 min. After chilling to room temperature, the samples were centrifuged at 3000 revolutions per minute for 10 min and 0.5 milliliter of each sample supernatant was transferred to a trial tubing incorporating 0.25 milliliter of TBA solution ( 0.67 % , w/v ) . Each tubing was so placed in a boiling H2O bath for 15 min. After chilling to room temperature, the optical density was measured at 532 nanometer with regard to the clean solution. The concentration of MDA was calculated based on the optical density coefficient of the TBA-MDA composite ( I = 1.56 – 105 cm?1 M?1 ) and it was expressed as nmol/mg protein [ 28 ] .
2.10. Acid phosphatase check
The Na ethanoate buffer ( 0.1 M, pH 5 ; 100 ?l ) , incorporating 0.1 % ( vol/vol ) Triton X-100 and 10 mM p-nitrophenyl phosphate, was added to each well. The home bases were placed in the brooder at 37 & A ; deg ; C for 3 h. The reaction was stopped by the add-on of 10 ?l of NaOH ( 100 millimeter, pH=10.5 ) to each well, and the Wellss were measured via ELISA technique at a wavelength of 405 nm [ 29 ] .
2.11. Evaluation of intracellular lipofuscin pigments
Extraction of intracellular lipofuscin was achieved undermentioned lysis of each cell sample harmonizing to Emig and co-workers procedure with little alteration [ 30 ] . The cells ( 5-104 cells/well ) were seeded in 24-well home bases for 24 H prior to pretreatments. After pretreatment with 20 µM of each derived function for 3 H, each cell sample was treated with 300 µM H2O2 for 24 H, 48 H and 72 h. The affiliated cells in each well were trypsinized with trypsin- EDTA solution followed by cell numbering utilizing a hemocytometer. Each home base was so centrifugated and the cell pellet was washed with PBS, and the cell content was lysed with lysis buffer incorporating 1 % Triton x-100, 1mM EDTA and 1mM PMSF. Each cell lysate was harvested and its fluorescence strength was monitored on a varian-spectrofluometer, theoretical account cary Eclipse with excitement and emanation wavelengthes of 310 and 620 nanometer, severally [ 31 ] . All experiments were repeated three times and the fluorescence strengths of the samples were so normalized for equal cell Numberss.
2.12. Transmission negatron microscopy ( TEM )
SK-N-MC cells were cultured in a cell civilization flask for 24 h. Then, cells were pretreated with JC4 and after 3 H, the cells were exposed to 300 µM H2O2. After 24 h incubation, the cells were fixed in 2.5 % glutaraldehyde ( 0.1 M Na-cacodylate, pH 7.2, 4 & A ; deg ; C ) , postfixed in 1 % OsO4 ( H2O ) and embedded in a 2 % agar gel ( to ease farther processing ) followed with desiccation with ethyl alcohol ( 50-100 % ) and so embedded in epoxy rosin ( Araldite ) . Finally, thin subdivisions were cut with a diamond knife, stained with 2 % lead citrate and 2 % uranyl ethanoate ( UAc ) , and examined by a transmittal negatron microscope ( Leo 906, Germany ) .
2.13. Statistical analyses
Datas are expressed as average ±SD of three independent experiments and statistically analyzed utilizing Student ‘s t-test.Values of P & A ; lt ; 0.05 were considered important.
3.1. Effect ( s ) on cell viability
The toxicities of JC1-JC6 compounds were evaluated based on the viability of SK-N-MC cells exposed to variable concentrations of the parallels utilizing MTT check. Table 2 indicates that benzylideneacetophenone parallels ( JC1-JC6 ) have slight cytotoxic effects on the cells following a 24 H rating clip. Based on Table 2, the full probes with the derived functions were achieved at doses loss than 20 µM to avoid the cytotoxic effects of the drugs. We evaluated the cytoprotective consequence of JC1-JC6 against harm induced in cells by H2O2. For this end, we pretreated SK-N-MC cells for 3 H with 20 µM of JC1-JC6. Then, the prtreated cells were exposed to 300 µM H2O2 for 24 H and 48 h. As shown in Fig. 2, intervention with H2O2 reduced cell viability by about 40 and 55 % , comparative to H2O2-untreated cells, after 24 and 48 H, severally. However, pretreatment of cells with 20 µM of JC1-JC6 reduced the detrimental effects of H2O2 by about 11, 15, 18, 24, 24 and 22 % after 24 H and by about 4, 19, 23, 29, 25 and 27 % after 48 H, severally. Sing these observations, it can be concluded that JC4, JC5 and JC6 at 20 µM concentration scavenging ROS every bit strongly as catechin at 20 µM.
3.2. Inhibitory effects on programmed cell death
As shown in Fig. 3A, the neurite length, as a marker of neural aging among H2O2-treated cells, has markedly increased. This is in contrast to the cells which have been pre-treated with JC parallels. For the drug-pretreated cells, homogeneousness in forms and sizes were clearly apparent and the cell withdrawals from the civilization home bases were significantly lower than the H2O2-treated control cells. To farther measure the repressive effects of benzylideneacetophenone parallels ( JC1-JC6 ) against H2O2 toxicity, we studied the extent of cell decease utilizing AO/EtBr dual staining technique [ 21 ] . In this attack, the non-apoptotic control cells appeared uniformly green ; the apoptotic cells showed orangish points in their nuclei corresponding to atomic DNA atomization. The extent of apoptotic cell decease for untreated cells was lower than 5 % . The figure of apoptotic cells increased by 45 % upon exposure to H2O2 for 24 h. However, intervention with H2O2 in the presence of JC1-JC6 decreased the extent of apoptotic cells by 4.5, 8.3, 14.5, 17.4, 21.0 and 16.3 % , severally ( Fig 3B ) .
3.3. Deactivation of reactive O species
Exposure of the cells to 300 ?M H2O2 caused 165 % addition in ROS content relation to H2O2-untreated control cells. Pretreatment of the cells with JC1-JC6 at concentrations of 20 ?M diminished the degrees of ROS by 38, 14, 50, 77, 56 and 71 % , severally compared to cells exposed merely to H2O2 ( Fig. 4A ) . The intracellular ROS degree did non varied among cells treated entirely with each of the drugs ( JC1-JC6, 20 ?M, informations non shown ) . Based on our informations, JC3, JC4, JC5 and JC6 can surely be classified as free-radical scavengers.
3.4. Effectss on indogenous antioxidant enzymes
To analyze whether the consequence of yakuchinone B derived functions ( JC1-JC6 ) is related to the change of intracellular antioxidant position, the activities of CAT and SOD, as the most antiphonal antioxidant enzymes of the biological system, were determined at 20 µM of each drug ( JC1-JC6 ) among the H2O2-treated cells. As shown in Fig. 4B, 300 ?M H2O2 reduced both CAT and SOD activities by 55 and 32 % , severally. However, pre-treatment of the cells with 20 µM of JC1-JC6 increased the CAT activity by 5.3, 10.5, 11.8, 32.6, 18.9 and 32.4 % and the dismutase activity by 2.3, 7.2, 8.2, 14.2, 10.0 and 17.1 % , severally comparative to cells treated entirely with H2O2.
3.5. Inhibition of lipid peroxidation
Biological membranes and intracellular constituents, which are rich in polyunsaturated fatty acids, can be easy affected by free groups through peroxidation. The extent of lipid peroxidation significantly increased among the cells exposed to 300 µM H2O2 for 24 H as measured in footings of MDA ( Fig. 5A ) . In SK-N-MC cells treated with H2O2 for 24 H, the degree of MDA was 0.65 nmol/mg protein, which was about 4.3- crease higher than that of untreated cells ( 0.15 nmol/mg protein ) . As shown in Fig 5A, JC4, JC5 and JC6 efficaciously inhibited the formation of MDA. The MDA degrees in cells pretreated with JC1-JC6 were 0.52, 0.50, 0.54, 0.34, 0.43 and 0.47 nmol/mg protein, severally. In other words, JC1-JC6 have quenched the formation of MDA by 20.4, 22.7, 16.8, 47.9, 34.2 and 28.1 % comparative to H2O2-treated cells.
3.6. Effectss on the acerb phosphatase activity
Acid phosphatase is a stable lysosomal enzyme. Accretion of lipofuscin in lysosome Acts of the Apostless as a trap for acerb phosphatase taking to its inactivation. Our consequences showed a important lessening ( by 38 % ) in acerb phosphatase activity among the H2O2-treated cells. However, pretreatment of the cells with each of the drugs ( JC1-JC6 ) increased the acerb phosphatase activity by 1, 8, 12, 20, 19 and 13 % , severally comparative to H2O2-trested cells ( Fig 5B ) .
3.7. Influence on lipofuscin formation
Exposure of the cells to 300 ?M H2O2 for 24 H, 48h and 72 H caused 85, 158 and 208 % addition in the intracellular degree of lipofuscin pigments comparative to H2O2-untreated control cells, severally. Pretreatment of the cells with each of the derived functions at a concentration of 20 ?M, diminished the formation of lipofuscin pigments by 11, 13, 40, 72, 58 and 61 % after 24 H of exposure ( Fig 6 ) . Sing these informations, JC drugs can surely cut down lipofuscin formation due to their free-radical scavenging capablenesss as one of their path of action.
3.8. Transmission electron microscopy findings
TEM micrographs of SK-N-MC control cells were devoid of no lipofuscin granules ( Fig 7A ) , After H2O2 intervention, lipofuscin granules with uniformly dense and approximately spherical forms appeared in the cell cytol. The sizes and distribution of these fluorescent inclusions were consistent with sizes and distributions of the lipid droplets and/or chondriosomes ( Fig 7B ) . Pretreatment of the cells with the most active derived function ( JC4 ) resulted in lower lipofuscin content within the cells, as shown in Fig 7C.
It is by now good accepted that free groups play critical functions in induction and patterned advance of age-related complications [ 32 ] . Hydrogen peroxide, as a pro-oxidant, is capable of incursion into lysosomes, where it interacts with redox sensitive passage metals via fenton reaction and produces hydroxyl groups [ 33 ] . Hydroxyl groups, in bend, originate lipid peroxidation with MDA production which will steer the cross-linking of supermolecules such as proteins and the lipid peroxidation merchandises taking to the formation of lipofuscin sums [ 4 ] . Therefore, lipofuscin accretion is linked to oxidative emphasis and/or inefficient anti-oxidant defence system. Even liposomal Fe overload and mitochondrial disfunction are considered to play functions in lipofuscin accretion within the cells [ 34 ] . Based on these facts, it is logical to foretell that anti-oxidant-based therapeutical attacks would rarefy lipofuscin accretion via disturbance of free extremist concatenation reactions [ 3,35 ] .
In the present probe, we attempted to correlate the antioxidant belongings of several yakuchinone B parallels ( JC1-JC6 ) to the intracellular construct up of lipofuscin sums. Our cumulative consequences indicated that the detrimental effects of H2O2 on the cells viabilities, morphology and programmed cell death were all reversed by pretreating the cells with each of the parallels. For illustration, the viabilities of the drug-pretreated cells increased by11, 15, 18, 24, 24 and 22 % after 24 H and by about 4, 19, 23, 29, 24 and 27 % after 48 H for JC1-JC6, severally comparative to H2O2-treated cells ( Fig 2 ) . The extent of programmed cell death among the drug-pretreated cells decreased by 4.5, 8.3, 14.5, 17.4, 21.0 and 16.3 % compared to H2O2-treated cells ( Fig 3B ) . Similar to normal cells, the drug-pretreated cells were largely attached to while the H2O2-treated cells were largely detached from the civilization home base surface. Based on Fig 4A, H2O2 enhanced the intracellular degrees of ROS and this was accompanied with important depression of CAT and SOD activities as shown in Fig 4B. However, pre-treatments of the cells with a individual dosage of each of the parallels ( JC1-JC6 ) decreased the intracellular degree of ROS and restored the bulk of cellular CAT and SOD activities, as it is apparent from Fig 4A and 4B, severally. Superoxide dismutase ( SOD ) catalyses the dismutation of superoxide anion into O and H2O2 and CAT catalyses the transition of H2O2 to H2O and molecular O. Repression of SOD and CAT activities by H2O2 ( Fig 4B ) was accompanied with enhanced intracellular degree of lipofuscin as indicated in Fig 6. As mentioned before, lysosome is engaged in lipofuscinogenesis. Accretion of age pigments within the lysosome might interfere with the activities of lysosomal enzymes [ 36 ] . Acid phosphatase is such an enzyme whose activity, upon exposure to H2O2, was significantly declined. However, pre-treatment of the cells with JC3, JC4, JC5 and JC6, followed by exposure to H2O2, led to a marked addition in the acerb phosphatase activity ( Fig 5B ) .
Transmission electron microscope is an appropriate technique to document the intracellular events under non-physiological conditions. By this technique, the lipofuscin pigments appear as irregularly shaped dark multitudes largely in cytol. Harmonizing to TEM consequences in Fig7, JC4 has strongly attenuated the accretion of age pigments.
Our cumulative informations clearly documented the yakuchinone B derived functions as free extremist scavengers with the following order of activity: JC4 & A ; gt ; JC6 & A ; gt ; JC5? JC3 & A ; gt ; JC2 & A ; gt ; JC1. Previous surveies have shown that the phenolic hydroxyl group on the vaniline ring ( pealing A, table 1 ) is required for the antioxidant activity of each of the derived functions [ 3, 37 ] . The stronger free extremist scavenging activity of JC4, JC5 and JC6, comparative to JC1, JC2 and JC3, might be attributed to two structural characteristics: the presence of another aromatic ring ( pealing B, table 1 ) and the presence of at least one methylene group between the carbonyl group and the aromatic ring B. These structural elements would add up to higher free extremist scavenging activity due to the formation of the undermentioned intermediates ( I and II ) for JC4, JC5 and JC6 as extra paths for neutralisation of free groups such as •OH.The other parallels ( JC1, JC2 and JC3 ) can non organize either of these intermediates and therefore, they will be capable of neutralizing the free groups merely through pealing A. This is in contrast to compounds JC4, JC5 and JC6 which would neutralize the free groups through both rings A and B. Therefore, they have higher anti-oxidant activities [ 38 ] .
In drumhead, the consequences of this probe, besides of reconfirming the free-radical hypothesis of aging, clearly indicate that the molecular complications of aging such as intramolecular accretion of lipofuscin pigments, could be attenuated by equilibrating the intracellular degrees of free groups to their normal physiological degrees. This might be achieved through delicate disposal of appropriate exogenic antioxidants.
The writers appreciate the fiscal support of this probe by the Research Council of University of Tehran. In add-on, the writers thank Mr. N. Pirhajati for his sort aid in TEM sample readyings.