Author: Priyarani, M
Date published: April 1, 2010
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Antioxidants are molecules that can neutralize free radicals by accepting or donating an electron to eliminate the unpaired condition. They are used by the food industry to delay the oxidation process. The oxidation of lipids in food is responsible for the formation of off-flavors and undesirable chemical compounds which may be detrimental to health. The addition of antioxidants to food products becomes popular as a powerful means for extending the shelflife of products and decreasing the nutritional losses by preventing or slowing the oxidation process . The antioxidants constitute a range of substances that play a role in protecting biological systems against the deleterious effects of oxidation processes on macromolecules such as proteins, lipids, carbohydrates and DNA . Research in recent years has shown the implication of oxidative and free radical mediated reactions in degenerative processes related to aging  and diseases such as cancer, coronary heart disease and neurodegenerative disorders such as Alzheimer 's disease etc . However, the commonly added synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) were restricted by legislative rules because of doubts over their toxic and carcinogenic effects. Therefore, a considerable interest in the food industries has developed to find natural antioxidants to replace the synthetic ones. Further, growing interest of consumer preferences towards natural antioxidants, there is more impetus to explore natural sources of antioxidants. Epidemological studies suggest that persons with high dietary antioxidant intake are less likely to develop cancer, particularly lung cancer but unfortunately benefits appear to be relatively small.
Awide variety of anti-cancer drugs exhibit cytotoxic effect by interfering with cell-cycle kinetics. These drugs are effective against cells that are proliferating and produce cytotoxic effect either by damaging the DNA during the S-phase of the cell cycle or by blocking the formation of the mitotic spindle in Mphase . However, most of the cytotoxic drugs exhibit serious side effects . Hence, there is a need for drugs that are equally efficacious but have lesser side effects.
Cinnamon (Cinnamomum verum) is an evergreen tree 10-15 m tall, belonging to the family Lauraceae and is native to Sri Lanka and South India. Cinnamon leaves and barks are used as spices and have many applications in perfumery, flavouring and pharmaceutical industries. Cinnamaldehyde, one of the components in the bark has been found to posses significant antitumor, cytotoxic , antiallergic, antiulcerogenic, antipyretic, anesthetic  and antimutagenic properties . The objective of the present study was to evaluate the antioxidant and cytotoxic potential of Cinnamomum zeylanicum verum fresh and dry bark extracts by different in vitro methods.
MATERIALS AND METHODS
Plant material:Cinnamomum zeylanicum verum stem bark, was obtained from a local garden in Trivandrum, Kerala, during January 2009.
Chemicals: 2,2'-azinobis-(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS), 2,2 Diphenyl -1-picrylhydrazyl (DPPH), thiobarbituricacid (TBA), gallic acid, catechin, trolox etc were purchased from Sigma chemical Pvt Ltd, Mumbai. Ascorbic acid was purchased from Sarabhai chemicals, Baroda. Hydrogen peroxide (H^sub 2^O^sub 2^), potassium persulphate, Tween-20 etc were purchased from SD fine chemicals Pvt Ltd Mumbai. 2-deoxy-2-ribose and ferrous chloridewere purchased from Fluka chemical Pvt Ltd. Ethylene diamine tetra acetic acid (EDTA), Trichloroacetic acid (TCA), ferrozine, Folin-Ciocalteu reagent, linoleic acid etc were purchased fromSisco research laboratories Pvt Ltd, Mumbai. Butylated hydroxytoluene (BHT), Potassium Ferricynanide, FeCl^sub 3^, potassium phosphate buffer were purchased from Central drug house limited, NewDelhi. RPMI-1640- R-1383Medium,MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide] from Sigma- Aldrich, Belgium. All the other chemicals employed were of standard analytical grade.
Preparation of cinnamon bark extracts: 100 g of coarsely powdered fresh stem bark was extracted using Soxhlet apparatus with acetone and 100 g of powdered dried bark was extracted using Soxhlet apparatus with methanol for 6 hours. The solvent was evaporated using rotary evaporator under reduced pressure. Acetone and methanol extracted viscous oleoresins of Cinnamomum zeylanicum from fresh (CZAE) and dry (CZME) stem bark yielded 29.45 % and 21.06 % respectively (on dry weight basis).
DPPH free radical scavenging assay:The antioxidant activity was determined using DPPH, a stable free radical.Antioxidant effect onDPPH radical was estimated according to the procedure explained by Brand-Williams .A methanolic stock solution of the acetone and methanol extracts were prepared. 0.1 ml of the sample solution at different concentrations ranging from20-60 µg/ml for acetone extract and 10-50 µg/ml for methanol extracts were added to 1.4 ml of freshly prepared DPPH. .The samples were kept at room temperature in the dark for 30 minutes. After 30 minutes absorbance was measured at 517 nmusing a Shimadzu UV1601 UVVIS spectrophotometer. Gallic acid was used as the standard. The radical scavenging capacity was calculated by the equation, .Scavenging capacity (%) = (1-A^sub sample^/A^sub control^) x 100, - where Asample is the absorbance of samples and Acontrol is the absorbance of control (contains all the reagents except samples). All determinations were performed in triplicate. The percentage inhibition ofDPPHradical was plotted against the sample or standard concentration to obtain the amount of antioxidant necessary to decrease the initial concentration of DPPH. to 50% (IC^sub 50^). A lower IC^sub 50^ value indicates greater antioxidant activity.
ABTS radical cation decolorisation assay: ABTS decolorisation assay was carried out by the method of Re et al.  in which ABTS^sup +^. was generated by oxidation of ABTS with potassium persulfate. The ABTS radical cation (ABTS+.) was produced by reacting 7 mMstock solution ofABTS with 2.45mM potassium persulphate (final concentration) and allowing the mixture to stand in the dark for 6 h at room temperature before use. The ABTS^sup +^. solution was diluted to an absorbance of 0.7 ± 0.05 at 734 nm. Appropriate volumes were taken from stock solution of acetone extract (2 - 12 µg/mL) and methanol extract (1-10 µg/mL) of bark for measurements. Absorbance was measured at 7 min after the initial mixing of different concentrations of the sample with 800 µL ofABTS+. solution. Trolox was used as the standard. Experiments were carried out in triplicate and mean values were taken. The radical scavenging capacity was calculated by the equation, -Scavenging capacity (%) = (1-A^sub sample^/A^sub control^) x 100 -where A^sub sample^ is the absorbance of samples and Acontrol is the absorbance of control (contains all the reagents except samples). All determinations were performed in triplicate.
Metal chelating activity: The chelation of ferrous ions by the bark extracts was estimated by the method of Dinis et al  with slight modification and compared with EDTA. Various concentrations of acetone extract (100-500 µg/ml) and methanol extract (100-500 µg/mL) of bark were prepared and to that added a mixture of 0.1 ml of 2 mM FeCl^sub 2^.7H^sub 2^O and 0.2 ml 5 mM ferrozine. Themixture was shaken vigorously and left standing at room temperature for 10 min. A control solution was prepared without adding sample. EDTA was used as the standard. A sample blank was also prepared without adding ferrozine. Absorbance of the red coloured solution was then measured spectrophotometrically at 562 nm. All test samples were run in triplicate and averaged. Readings of control, sample blank and standard were measured. The percentage inhibition of ferrozine-Fe^sup 2+^ complex formation was calculated using the formula, - % inhibition = [1- A^sub sample^/A^sub control^] x 100- where Asample is the absorbance of tested sample and A^sub control^ is the absorbance of control
Hydroxyl radical (OH*) scavenging activity: The hydroxyl radical scavenging activity was measured by the deoxyribose method  with slight modifications. Various concentrations of acetone extract (0.2-1 mg/ml) and methanol extract (0.2-1 mg/ml) of bark were mixed with adequate amount of potassium buffer (pH 7.4) to make the solution as 150 ml. Then added 100 ml freshly prepared FeCl^sub 3^ (200 µM), 50 ml H^sub 2^O^sub 2^ (1 mM), 100 ml EDTA (1.04 mM), 50 ml deoxy ribose (28 mM) and freshly prepared 50 ml ascorbic acid (1 mM) and themixture was incubated at 37 °C for one hour. Then 500 µl 2 % (w/v) trichloroacetic acid and 500 µl 1% (w/v) thiobarbituric acid were added and the mixture was heated in a water bath at 100 °C for 15 minutes. Absorbance of resulting solution was measured at 532 nm. A blank was prepared without sample and catechin was used as the standard. The scavenging effect of hydroxyl radical (%) was calculated using the following,- Scavenging effect (%) = (1-A^sub sample^/ A^sub control^) x 100 -where Asample is the absorbance in the presence of the tested samples and Acontrol is the absorbance of the control (contained all the reaction reagents except the tested samples).
Antioxidant Activity in linoleic acid emulsion system: The antioxidant activity of the cinnamon extracts and BHT was determined by the thiocyanate method of Duh et al. . 100 µg/ml concentrations of the acetone and methanol bark extracts and 100 µg/ml BHT were mixed with linoleic acid emulsion in potassium phosphate buffer (0.02 M, pH 7.0). Linoleic acid emulsion was prepared by mixing and homogenizing the 155 µl linoleic acid, 175 µg Tween- 20 as emulsifier and 50 ml 0.02 M phosphate buffer. The reaction mixture was incubated at 37 ± 0.5 °C. Aliquots of 0.1 ml were taken at various intervals during incubation from the mixture. The degree of oxidation was measured by sequentially adding ethanol (5 ml, 75%v/v), ammoniumthiocyanate (0.1 ml, 30% w/v), and ferrous chloride (0.1 ml, 0.02 M in 3.5% HCl w/v) to sample solution (0.1 ml) and then reading the absorbance at 500 nm. Solutions without added extracts were used as blank samples. The degree of oxidation was measured every 24 hrs and the data are the averages of triplicate analyses. The inhibition of lipid peroxidation in percent was calculated by the following equation: -LPI (%) = [100 - (A^sub 1^/A^sub 0^) x 100], where A^sub 1^ was the absorbance at 500 nm in the presence of sample and A^sub 0^ was the absorbance of control.
Reductive potential: The reducing power of the extracts was determined by the method of Oyaizu  with slight modifications. One ml of sample solutions of different concentrations of acetone (10- 50 µg/ml) and methanol (10-50 µg/ml) extracts of bark were mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and potassiumferricyanide (1%, 2.5 ml). The mixture was incubated at 50 °C for 20 minutes. Afterwards, 2.5 ml of trichloroacetic acid (TCA, 10 %) was added to the mixture. The mixture was shaken well and 2.5 ml fromthis solution was mixed with 2.5 ml distilled water and 0.5 ml FeCl^sub 3^ (0.1 %) and absorbance measured at 700 nm. A blank was prepared without sample. Gallic acid was used as the standard. Higher absorbance of the reaction mixture indicated greater reductive potential.Agraph was plotted using absorbance against concentration.
Total phenolics: Total phenolic content of the extracts were measured by the method described by Singleton . An aliquot of methanol containing different concentrations of acetone and methanol extracts ranging from 30-50 µg/ml was added to 5 ml of Folin-Ciocalteu reagent, waited for 8 minutes and added 4 ml of 7.5% sodium carbonate and kept in dark for 2 hrs and absorbance was measured at 765 nm against blank. Readings were taken in triplicate. The total phenolic content was expressed as Gallic acid equivalents (GAE) / gram of the plant material, using a standard curve generated with gallic acid.
MTT Assay: This experiment was carried out by the modified method of Abate et al. . The MCF- 7 cells were seeded (5 × 10^sup 4^ cells / well) into a 96 well plate and pre-incubated for 24 hrs at 37 °C in 5% CO[ub]2^ incubator. The samples with different concentrations were added to wells in the constant volume 100 µl and cells were incubated at 37 °C for 48 hrs in 5% CO^sub 2^. The plates were checked every 12 hours under microscope to avoid contamination. Following the removal of the medium from the wells, 100 µl of MTT solutions were added. After 3 hrs of incubation at 37 °C, the MTT was completely removed and 100 µl of formazone solubilizing solution (0%SDS in 50% DMF) was added to dissolve the formazan crystals. The absorbancewas read at 550 nmin ELISA reader. The percentage of viable cells was calculated by using the formula,
where A^sub sample^ is the aborbance of sample and A^sub control^ is the absorbance of control. A graph was plotted with percentage viability against sample concentration to obtain the amount necessary to decrease the initial concentration to 50% (LD^sub 50^). A lower LD^sub 50^ value indicates higher cytotoxicity.
Statistical analysis: The experimental results were expressed as means ± SD of three parallel measurements. The results were processed using Microsoft Excel 2007 and the data were subjected to one way analysis of variance (ANOVA) and the significance of differences between sample means were calculated.
RESULTS AND DISCUSSION
DPPH radical scavenging activity: DPPH is a stable free radical compound which has been widely used to test the free radical scavenging activity of the sample. The DPPH. scavenging activity of the samples was compared with the standard gallic acid (IC^sub 50^ 0.483 µg/ml). The acetone extract had an IC^sub 50^ value of 6.76 µg/ml and methanol extract had shown better activity with an IC50 value of 5.046 µg/ml. Increase in concentration increases the DPPH. scavenging activity and further increase in the concentration showed less increment in the activity, this shows that the samples attained their maximum activity with the radicals present. Figure 1 shows the percentageDPPH radical scavenging activity against concentration.
ABTS radical cation scavenging activity: The relative antioxidant activity to scavenge the ABTS^sup +^. radicals had been compared with the standard trolox with an IC^sub 50^ value of 1.72 µg/ml. Acetone and methanol extract showed an IC^sub 50^ value of 6.96 and 4.007 µg/ml respectively. Figure 2 depicts the steady state increase in the ABTS+. radical scavenging capacity of cinnamon extracts. Higher concentration of the extracts was more effective in quenching free radicals in the system .
Metal chelating activity: Chelation of metal ions by food components, particularly antioxidants, reduces the pro-oxidative effect of these ions and raises considerably the energy of activation of the initiation reactions. The efficiency of samples in comparison with EDTA and in reducing the prooxidant effect of iron ions by chelation was studied. Ferrozine can quantitatively formcomplexeswith Fe^sup 2+^. In the presence of other chelating agents, the complex formation was disrupted with the result that the red color of the complex was decreased. As shown in figure 3, the formation of the ferrozine Fe^sup 2+^ complex was not complete in the presence of samples, indicated that it can chelate iron. The absorbance of ferrozine Fe^sup 2+^ complex decreased linearly in a dose dependent manner. The chelating efficiency of samples was much lower than EDTA. The latter showed maximum activity (81.26 %) at 25 µg/ml. Acetone extract showed 70.95 % metal chelating activity at 500 µg/ml concentration and methanol extract showed 74.14 % metal chelating activity at 500 µg/ml concentration.
Hydroxyl radical scavenging activity: The highly reactive OH. can cause oxidative damage to DNA, lipids and proteins . In hydroxyl radical scavenging test, OH. radicals were generated by reaction of ferric-EDTA together with H^sub 2^O^sub 2^ and ascorbic acid to attack the substrate deoxyribose. The resulting products of the radical attack forma pink chromogen when heated with TBA in acid solution . When extracts were incubated with this reaction mixture they were able to interfere with free radical reaction and could prevent damage to the sugar. The standard catechin showed high scavenging activity with an IC^sub 50^ value of 11.28 µg/ml whereas acetone and methanol extracts showed IC^sub 50^ value of 0.54 mg/ml and 0.48 mg/ml respectively. The samples exhibited hydroxyl radical scavenging activity in a dose dependent manner in the reaction mixture as shown in figure 4.
Total antioxidant determination in linoleic acid emulsion system: Lipid peroxidation leads to rapid development of rancid and stale fragrance and is considered as a primary mechanism of quality deterioration in lipid foods and oils .Acetone and methanol extracts exhibited effective and powerful antioxidant activity at 100 µg/ml concentration. The effect of extracts and BHT on peroxidation of linoleic acid emulsion is represented in figure 5. It shows the antioxidant activity of BHT, acetone extract and methanol extract in the linoleic acid emulsion system using the thiocyanatemethod at various intervals. The percentage inhibition of peroxidation in linoleic acid system by 100 µg/ml of BHT, acetone and methanol extract was found to be 79.1%, 70.04% and 72.87% respectively at 48 h.
Reducing power: The reducing power of extracts may be due to the di andmonohydroxyl substitutions in the aromatic ring, which possess potent hydrogen donating abilities. The reducing properties are generally associated with the presence of reductones , which have been shown to exert antioxidant activity by breaking the free radical chain by donating hydrogen atoms. The total reducing power of the samples and gallic acidwere carried out. The samples showed very high activity comparable to gallic acid. The latter showed 0.849 absorbance at a concentration of 50 µg/ml. Acetone extract showed an absorbance of 0.136 at 50 µg/ml andmethanol extract showed an absorbance of 0.156 at 50 µg/ml. The reducing power of samples is depicted in figure 6.
Total phenolic content: The phenolic compounds may contribute directly to antioxidative action . Total phenolic content in acetone and methanol extracts was determined by the Folin-Ciocalteu method which is considered the best method for total phenolic content determination . The total phenolic contents (TPC) of acetone and methanol extracts were 30.52 µg gallic acid equivalents/g of plant material and 32.31 µg gallic acid equivalents/g of plant material respectively. Antioxidative action of plant materials isdue to the presence of phenolics.
MTT Assay: Mitochondrial enzyme in live cells, succinate-dehydrogenase cleaves the tetrazoliumring, converts MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells . Figure 7 shows the cytotoxic activities of acetone and methanol extracts of bark with the MCF-7 cells. The result showed that at low concentration it exerts cytotoxicity. Both the extracts showed a dose dependent inhibitory effect on the growth of MCF 7 cells. Acetone extract showed an LD^sub 50^ value of 19.74 µg/ml and methanol extract showed an LD^sub 50^ value of 14.98 µg/ml. Thus methanol extract showed higher cytotoxic activity than acetone extract against breast cancer cell lines.
The authors are thankful to Dr. K.G. Raghu (Scientist, Biochemistry and Cell culture Laboratory, Agroprocessing and Natural Products division, NIIST, Thiruvananthapuram) for providing necessary facilities for doing the work.
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PRIYARANI, M., VENKATESAN, J., BINILRAJ, S. S., SASIDHARAN, I. AND PADMAKUMRIAMMA, K. P.*
Agro Processing and Natural Products Division, National Institute for Interdisciplinary Science and
Technology (CSIR), Thiruvananthapuram 695019. E. mail: firstname.lastname@example.org
Received: December 1, 2009; Accepted: January 19, 2010