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S. Jipa a,b , T. Zaharescu a , W. Kappel a , A. F. Danet c , A. C. Popa c ,

EFFECT OF GAMMA-IRRADIATION ON THE ANTIOXIDANT CAPACITY OF ROSEMARY (Rosmarinus officinalis) AND SAGE (Salvia officinalis) EXTRACTS. S. Jipa a,b , T. Zaharescu a , W. Kappel a , A. F. Danet c , A. C. Popa c , T. Setnescu a,b , A. Mantsch a , M. E. Lungulescu a , M. Bumbac b ,

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S. Jipa a,b , T. Zaharescu a , W. Kappel a , A. F. Danet c , A. C. Popa c ,

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  1. EFFECT OF GAMMA-IRRADIATION ON THE ANTIOXIDANT CAPACITY OF ROSEMARY (Rosmarinus officinalis) AND SAGE (Salvia officinalis) EXTRACTS S. Jipaa,b, T. Zaharescua, W. Kappela, A. F. Danetc, A. C. Popac, T. Setnescua,b, A. Mantscha, M. E. Lungulescua, M. Bumbacb, C. Dumitrescub, L. M. Gorghiub a INCDIE ICPE CA, 313 Splaiul Unirii, Bucharest b Valahia University of Targoviste, Faculty of Sciences and Arts, 18-22 Unirii Av., Targoviste 130082 C University of Bucharest, Faculty of Chemistry, 4012 Elisabeta Bd., Bucharest 030018

  2. Radiation produces highly reactive and dangerous molecular species (free radicals) in cells and tissues. As a result of these interactions, the lipid peroxidation chain reactions may induce mutations and structural damage; • Antioxidants are known for their ability to scavenge free radicals and protect living being from radio-oxidation damage: State of art in the field Nowadays, there is a strong interest in the insulating antioxidants from natural sources to be used in this field

  3. State of art in the field • The use of chemicals to protect against the effects of nuclear radiation was attempted in World War II as safeguard humans against the militry use of atomic weapons (was tested the effect of amino-acid cysteine in rats exposed to lethal doses of X-rays • Plants and herbs as radioprotectors were tested to recovery workers from the Chernobyl accident (Gingko biloba: oral dose of 40 mg/day of plant was given 3 times daily for 2 months). Plants such as Panax ginseng, Mentha piperita, Zingiber officinale have been reported to provide protection against sickness induced by radiation

  4. Radioprotector is a chemical compound able to reduce the biological degradation produced by ionizing radiation on the normal tissue The ideal radioprotector agent would fulfill several criteria: (a) it must provide significant protection against the effects of radiation (even at low doses); (b) it must have a general protective effect on the majority of organs; (c) it must have an acceptable route of administation (oral or injection); (d) it must be an acceptable toxicity profile; (e) it must be compatible with other drugs. State of art in the field • The radioprotective efficiency of plant extracts is as a result of their • amount in a large number of antioxidants (phenolic, therpenic and flavonoidic • compounds). The plant preparation is very effective and non-toxic in • radioprotection relative to synthetic compounds. • Rosemary and sage are reputed to be the highest sources of potential • antioxidants consisting in phenolic acids (e.g. caffeic, chlorogenic, carnosic • acid, etc), phenolic diterpenes (e.g. carnosol, rosmanol, carnosic acid, etc.) • and flavonoids (apigenin, luteolin,naringenin, etc).

  5. The structures of some compounds of large interest are presented in figure 1. State of art in the field Phenolic acids Caffeic acid Chlorogenic acid Rosmarinic acid Phenolic diterpenes Carnosol Rosmanol Carnosic acid Flavonoids Naringenin Apigenin Luteolin Fig. 1 – Chemical structure of some phenolics in rosemary and sage

  6. Goal of work The purpose of this study is the exploring of antioxidants in rosemary and sage extracts before and after g-irradiation. The changes resulting from the gamma-radiation dose absorption as well as from the subsequent storage were monitored.

  7. The analyzed dry species were: rosemary (Rosmarinus officinalis) and sage (Salvia officinalis) . The samples were grounded and homogenized immediately before extraction (fig. 2) Experimental Fig. 2 Plant comminuting

  8. The plant material was refluxed in 0.5 liter of ethanol at 75 – 780C for 2 hrs in an extraction apparatus. The ethanolic extract was precipitated by non-solvent method, filtered (fig. 3) and, finally, additived (0.25 %) the pure paraffin. Experimental Fig. 3 Steps of precipitation

  9. Experimental The dried plant (10 g) and the extracting solvent (ethanol) were placed in an separating funnel (250 mL); the ratio of plant material and extracting solvent was 1 : 10 w/v. Maceration was performed for 5 days at room temperature, by permanent shaking. The liquid extract was separated from the plant material by filtration and the solvent was evaporated under vacuum

  10. Experimental rosemary application analysis sage Fig. 4 Steps of maceration

  11. We studied antioxidant activity for extracts obtained by different methods Experimental Table 1. List of extracts analyzed for antioxidant activity R = rosemary; S = sage

  12. The UV-VIS spectra were recorded on SPECTROMETER JASCO V 570 (fig. 5). Experimental Fig. 5 • 210 – 220 nm correspond to carnosol • 230 nm correspond to carnosic acid • 283 nm correspond to the presence of carnosol • 330 nm correspond to the presence of rosmarinic acid Carnosol and carnosic acid have been suggested to account for over 90% of the antioxidant properties of rosemary extract

  13. The FTIR spectra were recorded on SPECTROMETER JASCO 4000 (fig. 6). Experimental Fig. 6 • absorbtion at 3400 cm-1 indicate the phenolic OH group • absorbtion at 2900 – 2600 cm-1 (C – H) • absorbtions at 1680 cm-1 and 1210 cm-1 are characteristic bands of lactone - C – O abs. • II • O • absorbtions 1600 ÷ 1500 cm-1 (aromatic ring)

  14. The fluorescence spectra were recorded on SPECTROFLUORIMETER JASCO FP 6300 (fig. 7). Experimental Fig. 7 Spectrofluorimetry

  15. The chemiluminescence spectra were recorded with OL-94 (fig. 8) and LUMIPOL-3 (fig. 9). Experimental Fig. 8. Chemiluminograph OL-94 Fig. 9. Chemiluminescence instrument LUMIPOL-3

  16. Experimental The meanings of kinetic parameters which are discussed in this work are presented in fig. 10 Fig. 10 Typical CL kinetic parameters (isothermal method)

  17. The total antioxidant activity of the irradiated extracts was • measured by CL reaction of luminol with H2O2 in the presence • of Co (II) ions and EDTA. • Irradiations were carried out with the g-radiation of a 137Cs • GAMMATOR M-38-2 installation (fig. 11). Experimental Fig. 11 g-Irradiator GAMMATOR M 38-2137Cs Isodose irradiation chamber

  18. Figures 12 and 13 present the chemiluminescence spectra (168°C, air) of paraffin stabilized (0.25% w/w) with rosemary extracts by different sources. Extracts can be divided in two groups. Results and discussion As longer is the induction time as stronger is the antioxidant activity Fig. 13. Samples processed at room temperature Fig. 12. Samples processed at higher temperatures The samples extracted at room temperature (maceration) present longer induction times. Conclusion: extraction temperature decreases antioxidant activity. It is known that antioxidant activity depends, first, on genetic and growth conditions, such as the quality of the original plant, its geographical origin and the climatic conditions , storage, processing and second, on the extraction process.

  19. Results and discussion Fig. 14 shows the chemiluminescence spectra (1680C, air)of irradiated rosemary extract at various concentrations in paraffin.

  20. Results and discussion As it was expected, the rosemary extract stopped paraffin degradation at 1680C by trapping radical formed in this system (table 2). This isproved by the increased values of time patameters (ti, t1/2 or tmax) and by diminution of voxmax and Imax, in comparison with theblank sample. Table 2. Values of kinetic CL parameters (1680C, air) of 30 kGy irradiated rosemary extract in paraffin

  21. The rosemary extract equals in antioxidant efficiency butylated hydroxytoluene (BHT) as it may be seen from fig. 15. Results and discussion Fig. 15. CL curves for paraffin with various additives Fig. 16. CL curves for paraffin with preirradiated additives 1: free of additive 2: TOPANOL OC (BHT) 3: Unirradiated rosemary 4: IONOX-100 5: Rosemarin 2.5 KGy after storage 30 days

  22. Results and discussion • The antioxidant activity in rosemary extract has been attributed to above mentioned phenolic diterpenes (carnosol, rosmanol, etc) and also to some phenolic acids (caffeic acid and others) as well as to some flavonoids. The most important antioxidant seems to be carnosic acid and its derivatives.

  23. Results and discussion 2.5 KGy 5 KGy Figs. 17 present the effect of storage time on CL induction time for 2.5 and 5 kGy g-irradiated rosemary extracts.

  24. Results and discussion Fig. 18. Variation in the oxidation induction time for the thermal oxidation of paraffin modified with sage extract previously irradiated at 2.5 and 5 kGy. CL measurements were promptly performed.

  25. The maximum increase of total antioxidative capacity (TAC) was observed at dose of 5 kGy, as it can be seen in fig.19 Results and discussion Fig.19

  26. The oxidation cascade is very interesting from the point of view of the powerful antioxidant activity of rosemary and sage extracts, because new antioxidative compounds such as carnosol is produced during cascade. Results and discussion Carnosol also extracts a free radical becoming rosmanol. Rosmanol, continues the free radical scavenging until gladosol is created and continues the scavenging process (fig. 20). The presence of such compounds in the extracts of rosemary and sage could explain the increase in antioxidant activity observed after exposures at gamma radiation. Fig. 20

  27. Results and discussion • Caffeic acid present in rosemary and sage extracts could react with peroxy radicals by reaction: • The polyhydroxy substituted flavonoids present the highest antioxidant activity whyle the most favorable structural characteristics appear to be the o-di-OH-substitution on the B-ring. Free radicals can abstract the two hydroxyl hydrogens of B-ring, producing the corresponding inactive quinones.

  28. Results and discussion • The active constituents of rosemary like carnosol, carnosic acid, caffeic acid, rosmarinic acid etc have been subjected to pharmacological investigation. Carnosic acid and carnosol were found to exhibit anticarcinogenic activity. Natural polyphenols found in rosemary and sage have not only antioxidant activity but also anticarcinogenic properties. • Treatment of animals with rosemary extract (1000 mg/Kg body) prior to irradiation was found to delay the onset of lethal effects and reduces symptoms od radiation sickness. In the same time a significant decrease of lipid oxidation level was observed.

  29. Chemiluminescence has proved its versatility in fast and accurate assessment degradation studies; • Rosemary and sage extracts show a remarkable activity in thermal stabilisation of organic substrates; • Rosemary and sage extracts could offer protection against the effects of ionizing radiation because of their ability to scavenge free radicals; • Irradiation of rosemary and sage extracts demonstrated that some compounds appeared during “cascade” are responsible for enhancing of the antioxidant activity after the carnosic acid depletion; • More studies are necessary to investigate the exact mechanism of action and clinical applicability of rosemary and sage as radioprotectors. Conclusions

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