ANTIOXIDANT AND CYTOTOXIC POTENTIAL OF ACETONE AND METHANOLIC EXTRACTS OF FRESH AND DRY BARKS OF CINNAMOMUM ZEYLANICUM VERUM : IN VITRO STUDY

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1 Journal of Cell and Tissue Research Vol. (1) () ISSN: (Available online at Original Article ANTIOXIDANT AND CYTOTOXIC POTENTIAL OF ACETONE AND METHANOLIC EXTRACTS OF FRESH AND DRY BARKS OF CINNAMOMUM ZEYLANICUM VERUM : IN VITRO STUDY PRIYA RANI, M., VENKATESAN, J., BINILRAJ, S. S., SASIDHARAN, I.? AND PADMAKUMRI AMMA, K. P. Agro Processing and Natural Products Division, National Institute for Interdisciplinary Science and Technology (CSIR), Thiruvananthapuram E. mail: kppad@yahoo.co.in Received: December 1, 09; Accepted: January 19, Abstract: In the present study, acetone and methanol extracts of fresh and dry bark of Cinnamomum zeylanicum verum, were studied for their antioxidant activity viz, DPPH, ABTS and hydroxyl radical scavenging activities. Experiments were also conducted to evaluate the total phenolic content, the metal chelation capacity and the reducing power of extracts. The lipid peroxidation capacity of extracts was recorded using a linoleic acid emulsion system, which showed excellent results. Cytotoxic potential of extracts was evaluated using MCF 7 cells. Methanol extract of dry bark showed the highest antioxidant and cytotoxic potential. Key words: Cinnamomum zeylanicum bark, antioxidant, cytotoxic potential INTRODUCTION 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 [1]. 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 [2]. Research in recent years has shown the implication of oxidative and free radical mediated reactions in degenerative processes related to aging [3] and diseases such as cancer, coronary heart disease and neurodegenerative disorders such as Alzheimer s disease etc [4]. 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. A wide 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 M- phase [5]. However, most of the cytotoxic drugs exhibit serious side effects [6]. Hence, there is a need 2131

2 J. Cell Tissue Research for drugs that are equally efficacious but have lesser side effects. Cinnamon (Cinnamomum verum) is an evergreen tree -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 [7], antiallergic, antiulcerogenic, antipyretic, anesthetic [8] and antimutagenic properties [9]. 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 09. 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 2 O 2 ), potassium persulphate, Tween- etc were purchased from SD fine chemicals Pvt Ltd Mumbai. 2-deoxy-2-ribose and ferrous chloride were purchased from Fluka chemical Pvt Ltd. Ethylene diamine tetra acetic acid (EDTA), Trichloroacetic acid (TCA), ferrozine, Folin-Ciocalteu reagent, linoleic acid etc were purchased from Sisco research laboratories Pvt Ltd, Mumbai. Butylated hydroxytoluene (BHT), Potassium Ferricynanide, FeCl 3, potassium phosphate buffer were purchased from Central drug house limited, New Delhi. RPMI-16- R-1383 Medium, 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: 0 g of coarsely powdered fresh stem bark was extracted using Soxhlet apparatus with acetone and 0 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 % and % respectively (on dry weight basis). DPPH free radical scavenging assay:the antioxidant activity was determined using DPPH, a stable free radical. Antioxidant effect on DPPH 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 from - µg/ml for acetone extract and - 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 minutes. After minutes absorbance was measured at 517 nm using a Shimadzu UV 11 UV- VIS spectrophotometer. Gallic acid was used as the standard. The radical scavenging capacity was calculated by the equation, Scavenging capacity (%) = (1-A sample /A control ) x 0, where A sample is the absorbance of samples and A control is the absorbance of control (contains all the reagents except samples). All determinations were performed in triplicate. The percentage inhibition of DPPH radical was plotted against the sample or standard concentration to obtain the amount of antioxidant necessary to decrease the initial concentration of DPPH to % (IC ). A lower IC value indicates greater antioxidant activity. ABTS radical cation decolorisation assay: ABTS decolorisation assay was carried out by the method of Re et al. [11] in which ABTS +. was generated by oxidation of ABTS with potassium persulfate. The ABTS radical cation (ABTS +. ) was produced by reacting 7 mm stock solution of ABTS with 2.45 mm potassium persulphate (final concentration) and allowing the mixture to stand in the dark for 6 h at room temperature before use. The ABTS +. 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- μg/ml) of bark for measurements. Absorbance was measured at 7 min after the initial mixing of different concentrations of the sample with 0 µl of ABTS +. 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, 2132

3 Scavenging capacity (%) = (1-A sample /A control ) x 0 where A sample is the absorbance of samples and A control 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 [12] with slight modification and compared with EDTA. Various concentrations of acetone extract (0-0 μg/ml) and methanol extract (0-0 μg/ml ) of bark were prepared and to that added a mixture of 0.1 ml of 2 mm FeCl 2.7H 2 O and 0.2 ml 5 mm ferrozine. The mixture was shaken vigorously and left standing at room temperature for 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 2+ complex formation was calculated using the formula, % inhibition = [1- A sample /A control ] x 0 where A sample is the absorbance of tested sample and A control is the absorbance of control Hydroxyl radical (OH. ) scavenging activity: The hydroxyl radical scavenging activity was measured by the deoxyribose method [13] 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 1 µl. Then added 0 µl freshly prepared FeCl 3 (0 µm), µl H 2 O 2 (1 mm), 0 µl EDTA (1.04 mm), µl deoxy ribose (28 mm) and freshly prepared µl ascorbic acid (1 mm) and the mixture was incubated at 37 o C for one hour. Then 0 µl 2 % (w/v) trichloroacetic acid and 0 µl 1% (w/v) thiobarbituric acid were added and the mixture was heated in a water bath at 0 o 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 sample / A control ) x 0 where A sample is the absorbance in the presence of the tested samples and A control is the absorbance of the control (contained all the reaction reagents except the tested samples). Priyarani et al 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. [14]. 0 μg/ml concentrations of the acetone and methanol bark extracts and 0 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- as emulsifier and 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), ammonium thiocyanate (0.1 ml, % 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 0 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 (%) = [0 (A 1 /A 0 ) x 0], where A 1 was the absorbance at 0 nm in the presence of sample and A 0 was the absorbance of control. Reductive potential: The reducing power of the extracts was determined by the method of Oyaizu [15] with slight modifications. One ml of sample solutions of different concentrations of acetone (- μg/ml) and methanol (- μg/ml) extracts of bark were mixed with 2.5 ml phosphate buffer (0.2 M, ph 6.6) and potassium ferricyanide (1%, 2.5 ml). The mixture was incubated at o C for minutes. Afterwards, 2.5 ml of trichloroacetic acid (TCA, %) was added to the mixture. The mixture was shaken well and 2.5 ml from this solution was mixed with 2.5 ml distilled water and 0.5 ml FeCl 3 (0.1 %) and absorbance measured at 0 nm. A blank was prepared without sample. Gallic acid was used as the standard. Higher absorbance of the reaction mixture indicated greater reductive potential. A graph was plotted using absorbance against concentration. Total phenolics: Total phenolic content of the extracts were measured by the method described by Singleton [16]. An aliquot of methanol containing different concentrations of acetone and methanol extracts ranging from - 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

4 J. Cell Tissue Research % RSA 1 0 GA CZME CZAE Fig.1: DPPH radical scavenging activity % RSA 1 TLX CZME CZAE Fig. 2: ABTS radical scavenging activity CZME CZAE % inhibition Fig. 3: Metal chelating activity

5 Priyarani et al. 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. [17]. The MCF- 7 cells were seeded (5 4 cells / well) into a 96 well plate and pre-incubated for 24 hrs at 37 C in 5% CO 2 incubator. The samples with different concentrations were added to wells in the constant volume 0 µl and cells were incubated at 37 C for 48 hrs in 5% CO 2. The plates were checked every 12 hours under microscope to avoid contamination. Following the removal of the medium from the wells, 0 µl of MTT solutions were added. After 3 hrs of incubation at 37 C, the MTT was completely removed and 0 µl of formazone solubilizing solution (0% SDS in % DMF) was added to dissolve the formazan crystals. The absorbance was read at 5 nm in ELISA reader. The percentage of viable cells was calculated by using the formula, % viability = A sample X 0 A control where A sample is the aborbance of sample and A 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 % (LD ). A lower LD 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 07 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 µg/ml). The acetone extract had an IC value of 6.76 µg/ml and methanol extract had shown better activity with an IC value of 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 percentage DPPH radical scavenging activity against concentration. ABTS radical cation scavenging activity: The relative antioxidant activity to scavenge the ABTS +. radicals had been compared with the standard trolox with an IC value of 1.72 µg/ml. Acetone and methanol extract showed an IC value of 6.96 and 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 [18]. 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 form complexes with Fe 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 2+ complex was not complete in the presence of samples, indicated that it can chelate iron. The absorbance of ferrozine Fe 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.95 % metal chelating activity at 0 µg/ml concentration and methanol extract showed % metal chelating activity at 0 µg/ml concentration. Hydroxyl radical scavenging activity: The highly reactive OH. can cause oxidative damage to DNA, lipids and proteins [19]. In hydroxyl radical scavenging test, OH. radicals were generated by reaction of ferric-edta together with H 2 O 2 and ascorbic acid to attack the substrate deoxyribose. The resulting products of the radical attack form a pink chromogen when heated with TBA in acid solution []. When extracts were incubated with this reaction mixture 2135

6 J. Cell Tissue Research 0 CAT CZME CZAE % RSA Fig. 4: Hydroxyl radical scavenging activity CZME CZAE % inhibition Fig. 5: Total antioxidant activity in linoleic acid emulsion system Absorbance at 0 nm GA CZME CZAE Fig. 6: Reducing power

7 Priyarani et al. CZME CZAE % Viability Fig. 7: MTT assay 0 0 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 value of µg/ml whereas acetone and methanol extracts showed IC 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 [21]. Acetone and methanol extracts exhibited effective and powerful antioxidant activity at 0 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 thiocyanate method at various intervals. The percentage inhibition of peroxidation in linoleic acid system by 0 µg/ml of BHT, acetone and methanol extract was found to be 79.1%,.04% and 72.87% respectively at 48 h. Reducing power: The reducing power of extracts may be due to the di and monohydroxyl substitutions in the aromatic ring, which possess potent hydrogen donating abilities. The reducing properties are generally associated with the presence of reductones [22], 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 acid were carried out. The samples showed very high activity comparable to gallic acid. The latter showed absorbance at a concentration of μg/ml. Acetone extract showed an absorbance of at µg/ml and methanol extract showed an absorbance of at µg/ml. The reducing power of samples is depicted in figure 6. Total phenolic content: The phenolic compounds may contribute directly to antioxidative action [23]. 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 [24]. The total phenolic contents (TPC) of acetone and methanol extracts were.52 μg gallic acid equivalents/g of plant material and 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 tetrazolium ring, converts MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells [25]. 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 value of μg/ml and methanol extract showed an LD value of μg/ml. Thus methanol extract showed higher cytotoxic activity than acetone extract against 2137

8 J. Cell Tissue Research breast cancer cell lines. ACKNOWLEDGEMENTS 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. Abbreviations used: DPPH = 2, 2 Diphenyl -1- picrylhydrazyl; ABTS = 2, 2 -azinobis-(3- ethylbenzothiazoline-6-sulfonic acid); MTT = 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; GA = Gallic acid; TLX = Trolox; EDTA = Ethylene diammine tetraacetic acid; CAT = Catechin; BHT = Butylated hydroxyl toluene; CZME = Cinnamomum zeylanicum verum methanol extract; CZAE = Cinnamomum zeylanicum verum acetone extract. [16] Singleton, V.L. and Rossi, J.A.: Am. J. Enol. Viticul., 16: (1965). [17] Abate, G., Mshana, R.N. and Miorner, H.: Inter. J. Tubercul. Lung Disease, 2:11-16 (1998). [18] Liangli, Y., Scott, H., Jonathan, P., Mary, H., John, W. and Ming, Q.: J. Agri. Food Chem., : (02). [19] Spencer, J.P.E., Jenner, A., Aruoma, O.I., Evans, P.J., Kaur, H. and Dexter, D.T.: FEBS Letters, 353: (1994). [] Shimada, K, Fujikawa, K, Yahara K.T. and Nekamura.: J. Agri. Food Chem., : (1992). [21] Guntensperger, B., Hammerli-meier, D. E. and Escher, F.E.: J. Food Sci., 63: (1998). [22] Pin-Der-Duh, X.: J. Am. Oil Chem. Soc.., 75: (1998). [23] Duh, P.D., Tu, Y.Y. and Yen, G.C.: Lebnesmittel Wissenschaft Technol., 32: (1999). [24] Engelhardt, U.: Critical Rev. Food Sci. Nutr., 41: (01). [25] Mossmann, T.: J. Immunol. Meth., 65: (1983). REFERENCES [1] Tsuda, T., Ohshima, K., Kawakishi, S. and Osawa,T.: J. Agri. Food Chem., 42: (1994). [2] Halliwell, B. and Gutteridge, J.M.C.: Free Rad. in Bio. And Med., Second edi., Clareodon Press Oxford, pp (1989). [3] Ames, B.N., Shigena, M.K. and Hegen, T.M.: Proceed. Natural Acad. Sci., 90: (1993). [4] Ames, B.N.: Science, 221: (1983). [5] Gali-Muhtasib, H. and Bakkar, N.: Curr. Cancer Drug Targets. 2: (02). [6] Powis, G.: Drug Metab. Rev., 14: (1983). [7] Kwon, B.M., Lee, S.H., Choi, S.U., Park, S.H., Lee, C.O. and Cho, Y.K.: Arch. Pharmacal. Res., 21: (1998). [8] Chopra, R.N., Nayer, S.L. and Chopra, I.C.: Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, (19). [9] Sharma, N., Trikha, T., Athar, M. and Raisuddin, S.: Mut. Res., 4 & 481: (01). [] Brand-Williams, W., Cuvelier, M.E. and Berset, C.: Lebensmittl-Wissenschaft Technologic, 28: 25- (1995). [11] Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice Evans, C.: Free Rad. Bio. and Med., 26(9/): (1999). [12] Dinis, T.C.P., Madeira, V.M.C. and Almeida, L.M.: Arch. Biochem. Biophy., 315: (1994). [13] Halliwell, B., Gutteridge, J.M.C. and Aruoma, O.I.: Analytical Biochem., 165: (1987). [14] Duh, P.D., Yen, W.J., Du, P.C. and Yen, G.C.: J. Am. Oil Chem. Soc., 74: (1997). [15] Oyaizu, M.: Japan J. Nutr., 44: (1986). 2138