COMPARATIVE INVESTIGATION ON RADICAL SCAVENGING ACTIVITY AND PROTECTIVE PROPERTIES OF NATURAL ISOLATED AND SYNTHETIC ANTIOXIDANTS Y. Karamalakova 1,2, J. Sharma 2, R.K. Sharma 2, V. Gadjeva 1, R. Kumar 2 and A. Zheleva 1 1 Trakia University, Medical Faculty, Department Chemistry and Biochemistry, Stara Zagora, Bulgaria 2 Institute of Nuclear Medicine and Allied Sciences, Delhi, India Coresspondence to: Yanka Karamalakova E-mail: ykaramalakova@abv.bg ABSTRACT The purpose of this study is to evaluate and compare the free radical scavenging activity and protective properties of the natural product SQGD-IBG-21 isolated from a radioresistant bacterium Bacillus sp. INM-1 and synthetic nitroxyl free radical containing antioxidant 1-ethyl-1-nitroso-3-[4-(2,2,6,6-tetramethylpiperidine- 1-oxyl)]-urea (SLENU). It was found a higher reducing power potential for SQGD (1.336±0.03357 U abs ) comparing to that of SLENU (0.196±0.002273U abs ); a higher nitric oxide radical scavenging activity 35.645 ±1,122% for SQGD in comparison with SLENU (19.964±2.233%). Total antioxidant capacity of SQGD was found to be 75±0.06%, while for SLENU it was only 22±0.03%. Maximum protection to the liposomes calculated in % inhibitory activity of two agents was found to be 50.04±0.037% for the natural agent and 27.54±0.33% for nitroxyl labeled agent. By direct EPR spectroscopy stable radical structures were recorded in the solutions of both studied antioxidants: an o-semiquinone radical structure was recorded in aqueous solution of SQGD and nitroxyl free radical struture in the ethanol solution of SLENU, respectively. It should be mentioned that the natural antioxidant possesses higher protective properties in comparison with the synthetic antioxidant. In conclusion, because of well expressed free radical scavenging and antioxidant activities of both studied agents they might be used in the combination anticancer chemotherapy for reduction the toxicity caused by anticancer drugs and/or radiation therapy. Keywords: scavenging activity, protective properties, EPR, antioxidants, anticancer agents Introduction Natural isolated products from microbial or herbal materials and synthetic agents are evaluated for pharmacological activities and application in medicine for the treatment of different diseases and cancer therapy (17). The experience in the free radical biology and cancer therapy shows that the normal cells are characterized with low levels of ROS/RNS and constant levels of some scavenging activities. In human body and cells the total ROS/RNS levels are determinated by the levels of: superoxide radicals, nitric oxide radicals, endproducts of lipid peroxidation, liposomal preparation, electron donation potential, enzymes, extracting an electron from natural antioxidants and from synthetic agents (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). Antioxidant prophylaxis and therapy is an effective modern method for preventing abnormal production of ROS and limit their harmful effects on the body. In recent years, increasing studies are performed on the effects of the treatment of many diseases with combinations of synthetic medicines and other radical scavengers. Use of appropriate natural and synthetic antioxidant substances reduces the level of lipid peroxidation and increases the activity of antioxidant enzyme defense system and ultimately reduces radiotherapy- and chemotherapy- induced oxidative 175 stress and negative consequences (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). The purpose of this study was to evaluate and compare the free radical scavenging activity and protective properties of the natural product SQGD isolated from a radioresistant bacterium Bacillus sp. INM-1 and the synthetic antioxidant SELNU containing nitroxyl free radical structure. Radical scavenging activity, membrane lipids protective properties and presence of stable free radical structures in SQGD and SLENU were investigated using in vitro spectrophotometrical methods and direct Electron Paramagnetic Resonance (EPR) technique. Material and Methods Chemicals The natural product SQGD was isolated from a radio-resistant bacterium Bacillus sp. (crystalline form) was provided for the present study by INMAS, India (8). Nitroxyl labeled agent SLENU analogue of the anticancer drug Lomustine (CCNU), was synthesized as previously described (3). 2,2-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), soya-lecithin, 2,2-dipheniyl-1- picrylhydrazyl (DPPH), sodium nitroprusside, naphthylethylenediamine and phenilbuthylsufoxide (PBS) were purchased from Sigma Chemicals (St. Louis, MO, USA). Chloroform, hexane, hydrogen peroxide, ethanol, o-phosphoric acid were purchased from Qualigen, India. Deionized and
distillated water was used for all experiments. Other chemicals used were analytical or HPLC grade. Measurement of reducing power The reducing powers of the natural agent SQGD and synthetic agent SLENU were determined according to the method described by Oyaizu (12) with slight modification. The absorbance was recorder at 700 nm as reducing power using a spectrophotometer (Power Wave, Biotek, USA). The antioxidant activity of two agents were estimated and compared. Nitric oxide scavenging activity assay Nitric oxide radical scavenging activity of natural agent SQGD and synthetic agent SLENU were determined according to the method reported by Green et al. (5). Sodium nitroprusside in aqueous solution at physiological ph (7.4) spontaneously generates nitric oxide which interacts with oxygen and determined by the use of Griess reagent. The nitric oxide scavenging activity was analyzed and compared as a percent of decreasing in the absorbance of the complex formed by diazotization of nitride with sulfanilamide and subsequent coupling with naphthylethylendiamide at 546 nm. Total antioxidant capacity estimation To determine and compare the total antioxidant potential of the two reagents, modified ABTS (2,2- azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay was performed to Re et al., 1999 (10). The ABTS radical formed was incubated with any of the studied agents in a concentration of 0-500µg/ml for 5 min and the absorbance was measured at 734 nm. Membrane lipids protecting abilities of SQGD and SLENU The analysis of membrane protecting ability (peroxidation of membrane lipids) of SQGD and SLENU was performed using artificial membrane system (liposome) as reported by New (11). The resultant mixtures (liposome + SQGD and liposome + SLENU) were heated at 40 0 C for 4 h in a water bath and the absorption of the pink-coloured complexes was read at 535 nm. Electron Paramagnetic Resonance measurements For all EPR measurements an X-band EMXmicro, EPR spectrometer (Bruker, Germany) equipped with standard Resonator was used. Quartz capillaries were used as sample tubes. The capillary tubes were sealed and placed inside a standard EPR quartz tube (i.d. 3mm) that was placed in the EPR cavity. All EPR experiments were performed at room temperature (18-23 0 C) and relative humidity 40%. Spectral processing (g-value calculation) was performed using Bruker WIN-EPR and SimFonia software. 176 Direct EPR spectroscopy in solutions of SQGD and SLENU EPR spectrum of the aqueous solution of SQGD was recorded at the following EPR spectrometer settings: center field 3514 G, sweep width 200 G, modulation amplitude 10.00 G, microwave power 6.494 mw, gain 1x10 5, time constant 163.840 ms, sweep time 16.384 s, 10 scans per sample. EPR spectrum of the ethanol solution of SLENU was recorded at the following EPR settings: center field 3515.73 G, sweep width 100.00 G, modulation amplitude 1.00 G, microwave power 6.24 mw, gain 2x10 4, time constant 10.24 ms, sweep time 60 s, 1 scans per sample. Statistical analysis Statistical analysis was performed with Statistica 6.1, StaSoft, Inc. and results were expressed as means ± standard error (SE). Statistical significance was determined by the Student s t-test. A value of p<0.05 was considered statistically significant. Results and Discussion Reactive oxygen/nitrogen species (ROS/RNS) have a fundamental part in maintaining metabolic homeostasis in the human body. The imbalance of production and generation of free radicals or ROS relative to inactivation caused by outside irritants or effects of chemotherapy, radiation-therapy stress leads to oxidative stress. Radical-mediated oxidative stress have been implicated different disease, including tumors and cancer like lung cancer, leukemia, breast cancer, ovary, rectum cancers, etc. (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). The effects of ROS/RNS can be connected and overcome by using external supplementation of antioxidants with natural or synthetic origin. The reducing ability (reduction of Fe 3+ complex into the Fe 2+ ) of the compounds was monitored by measuring the formation of Fe 2+ at 700 nm. Maximum reducing power for SQGD (1.366±0.03357) was observed at the highest concentration (1000 µg/mi) as compared to the maximum activity of SLENU (0.196±0.002273) at the same concentration. Therefore, the study indicated significantly increased reducing activity of the both compounds linked to the increased concentration. Comparing to the synthetic agent, SQGD exhibits six times higher protective efficacy at in vitro models (Fig. 1A). Nitric oxide, stable free radical, contributed to the toxic reactions with body-biomolecules and for pathogenesis of some inflammatory diseases (10). Results of the study (Fig. 1B) indicated a significant increase in percent inhibition (35% and 19%) of the scavenging nitric oxide activity by SQGD and SLENU, respectively. The total antioxidant capacity (ABTS radicals generation) by SQGD and SLENU were estimated and compared. At all studied concentrations of both
agents significantly higher antioxidant activities were found for SQGD comparing to those of SLENU. Natural agent showed 50% inhibition of ABTS radicals at a concentration of0.50 µg/ml, while this inhibition for the nitroxyl-labeled nitrosourea was 22%. It was also established that to the highest studied concentration the percent of inhibition continuously increased for SQGD (75±0.06%, at 500 µg/mi), while for SLENU it showed only 3% at 500 µg/mi (Fig. 2A). A. B. Fig. 1. The evaluations of reducing power (absorbance- 700 nm) (A) and nitric oxide radical scavenging inhibition (B) of SQGD and SLENU A. B. Fig. 2. The effects of the concentration of the SQGD and SLENU on the inhibition of ABTS radicals (A) and analysis of the membrane protecting ability of SQGD and SLENU utilizing in membrane systems (B) The analysis of membrane protecting ability (peroxidation of membrane lipids) of SQGD and SLENU were utilized an artificial membrane system (liposome). The most effective dose for percentage inhibition of radicals, as compared to the two drugs (50.04±0.037% for SQGD and 27.54±0.33% for SLENU), was found to be the highest tested concentration (1000 µg/mi). Result of the study indicated almost two times higher antilipid peroxydation activity of the natural product as compared with synthetic agent (Fig. 2B). After inhibition lipid peroxide radicals, can be presumed that both agents exhibit an anti-inflammatory effect indirectly, which is demonstrated their protective 177 properties (8). EPR spectroscopy is a highly sensitive technique utilized for investigation of free radical scavenging capacity and radical structures in synthetic and naturally isolated antioxidants (9, 10, 11, 12, 13). Formerly, by in vitro EPR spin trapping technique we reported a well expressed superoxide anion scavenging activity for SLENU due to the presence of the nitroxyl radical moiety in its structure (4).As is seen on (Fig. 3A) in the solution of SLENU the typical triplet EPR signal of the nitroxyl free radical was registered, while in the aqueous solution of SQGD an EPR signal with a single line was recorded (Fig. 3B). Because of the stability and calculated g value (2.0044 ± 0.0002)
the radical registered in SQGD was ascribed to a semiquinone structure. At this stage of the study antioxidant properties of SQGD and SLENU demonstrated by all methods used we could explain by the presence of stable free radical structures. A. B. Fig. 3. EPR spectra of the solutions of SLENU (A) and SQGD (B) radicals Conclusions Because of their free radical scavenging abilities SQGD and SLENU are characterized as potential radio- and hepatoprotectors and might be used in the combination anticancer chemotherapy for reduction the toxicity caused by anticancer drugs and/or radiation therapy. Acknowledgments This study was supported by grants of Ministry of Education, Youth and Science Indo-Bulgarian collaborative project (Bin-7/2008). REFERENCES 1. Arora R. (2008) CABI Publishing, Wallingford, Oxon, UK, 23, 1496. 2. Bernardo dos Santos A., Silva D.H.S., Bolzani V.S., Santos L.A., Schmidt T.M., Baffa O. (2009) J. Braz. Chem. Soc., 20(8), 1483-1492. 3. Gadjeva V. (2002) Eur. J. Med. Chem., 37, 295-300. 178 4. Gadjeva V., Ichimori K., Nakazawa H., Raikov Z. (1994) Free Rad. Res., 21(3), 177-186. 5. Green L.C., Wagner D.A., Glogowski J., Skipper P.L., Wishnok J.S., Tannenbaum S.R. (1982) Anal. Biochem., 126, 131-138. 6. Halliwell B. and Gutteridge J.M.C. (1990) Methods Enzymol., 186, 1-85. 7. Karawita R., Siriwardhana N., Lee Ki-Wan. (2005) Eur. Food Res. Technol., 220, 363-371. 8. Kumar R., Bansal D.D., Patel D.D., Mishra S., Karamalakova Y., Zheleva A., Gadjeva V., Sharma R.K. (2011) Mol. Cell Biochem., 349(1-2), 57-67. 9. Matthaus B. (2002) J. Agrec. Food Chem., 50, 3444-3452. 10. Moncada S., Palmer R.M., Higgs E.A. (1991) Pharmacol. Rev., 43, 109-142. 11. New RRC (1990) In: New RRC (D. Rickwood, B.D. Hames, Eds.), IRL Press, Oxford, UK, 7, 154-161. 12. Oyaziu M. (1986) Nippon Shokuhin Kogyo Gakkai-Shi., 35, 771-775.
13. Polovka M., Brezova V., Stasko A. (2003) Biophys. Chem., 106, 39-56. 14. Re R., Pellegrini N., Proteggente A., Pannala A., Rice-Evans C. (1999) Free Radical Bio. Med., 26, 1231-1237. 15. Roos D., Puntel R.L., Santos M.M., Souza D., Farina F., Nogueira C., Aschner M., Burger M., Barbosa N., Rocha J. (2009) Toxicology in Vitro., 23, 302-307. 16. Sen S., Chakraborty R., Sridhar C., De Biplab R. (2010) Int. J. of PS R and Research., 3(1), 91-100. 17. Trachootham D., Alexandre J., Huang P. (2009) Nat. Rev. Drug Discovery, 8, 579-605. 18. Zhelev Z., Bakalova R., Aoki I., Gadjeva V., Kanno I. (2010) Mol. BioSyst., 6, 2386-2388. 179
33