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Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi

Year 2019, , 708 - 716, 01.06.2019
https://doi.org/10.21597/jist.475524

Abstract

Bu çalışmada, sıçan böbrek dokusunda karbontetraklorür’ün neden olduğu hasara karşı kuersetin’in etkisi araştırıldı. Çalışmada ağırlıkları yaklaşık (200-300) gr ağırlığında 28 adet yetişkin wistar albino sıçan kullanıldı ve dört gruba ayrıldı. 3 günlük uygulamayı takiben sıçanlar anestezi altına alınarak böbrek dokuları alındı. Böbrek dokusunda SOD, CAT, GPx enzim aktiviteleri ve (8-OHdG) düzeyi değerlendirildi. SOD, CAT, GPx enzim aktiviteleri değerlendirildiğinde, kontrol grubuna oranla CCl4 uygulanan grupta enzim aktivitesinde azalış, CCl4 grubuna oranla CCl4 + kuersetin uygulaması yapılan grupta enzim aktivite düzeyinde artış olduğu belirlendi. Çalışmada, karbontetraklorüre maruz kalan sıçanların böbrek dokusunda 8-OHdG düzeyi değerlendirildiğinde; kontrole oranla karbontetraklorür uygulanan grupta artma, yine kontrole oranla karbontetraklorür + kuersetin uygulanan grupta ise artma, karbontetraklorür grubuna oranla ise karbontetraklorür + kuersetin uygulanan grupta düşme olduğu gözlendi. Kuersetinin, CCl4 tarafından indüklenen böbrek doku hasarı üzerine koruyucu etkisinin olduğu kanaatine varıldı.

References

  • Abarikwu SO, 2014. Protective Effect of Quercetin on Atrazine-Induced Oxidative Stress in the Liver, Kidney, Brain, and Heart of Adult Wistar Rats. Toxicol Int. May-Aug, 21(2): 148–155.
  • Abdel-Raheem IT, Abdel-Ghany AA, Mohamed GA, 2009. Protective effect of quercetin against gentamicin-induced nephrotoxicity in rats. Biological and Pharmaceutical Bulletin, 32(1), 61-67.
  • Abdurrauf Y, Aksakal M, 2007. Ratların Karaciğer ve Testis Dokusundaki Antioksidan Aktivite Üzerine Nar Suyunun Etkisi.F.Ü. Sağ. Bil. Derg, 21(6): 253 -256.
  • Adel Abdel-Moneim A, Ahmed OM, Fahim HI, Zaky MY, 2016. Ameliorative Effects of Quercetin and Naringenin on Diethylnitrosamine/2-acetyl amino flourene-Induced Nephrotoxicity in Male Wistar Rats. American Journal of Biochemistry, 6(5): 113-121.
  • Aebi H, 1984. Catalase in vitro assay methods. Methods Enzymol, 105:121-126.
  • Aldemir M, Okulu E, Kösemehmetoğlu K, Ener K, Topal F, Evirgen O, Gürleyik E, Avcı A,2014. Evaluation of the protective effect of quercetin against cisplatin‐induced renal and testis tissue damage and sperm parameters in rats. Andrologia, 46(10), 1089-1097.
  • Alidadi H, Khorsandi L, Shirani M, 2018. Effects of Quercetin on Tubular Cell Apoptosis and Kidney Damage in Rats Induced by Titanium Dioxide Nanoparticles. Malaysian Journal of Medical Sciences, 25(2), 72-81.
  • Almaghrabi OA, 2015. Molecular and biochemical investigations on the effect of quercetin on oxidative stress induced by cisplatin in rat kidney. Saudi journal of biological sciences, 22(2), 227-231.
  • Akkoyun HT, Bengu AS, Ulucan A, Bayramoglu-Akkoyun M, Arihan O, 2018.Protectıve Effect Of Ellagıc Acıd Agaınst Carbon Tetrachlorıde (CCl4) Induced Oxıdatıve Braın Injury In Rats.Fresenius Environmental Bulletin, 27(5): 3148-3155
  • Akşit H, Akşit D, Bildik A, Kara H, Yavuz Ö, Seyrek K, 1989. Deneysel Karaciğer İntoksikasyonunda N-Asetil Sistein’in Glutatyon Metabolizması Ve Lipid Peroksidasyonuna Etkileri. Ankara Üniv Vet Fak Derg, 62:( 1-5).
  • Arslan A, Demir H, Ozbay MF, Arslan H, 2014. Evaluation of lipid peroxidation and some antioxidant activities in patients with primary and metastatic liver cancer. Journal of Cancer Therapy, 5(2):192.
  • Bansal AK, Bansal M, Soni G, Bhatnagar D, 2005. Protective role of Vitamin E pre-treatment on N-nitrosodiethylamine induced oxidative stress in rat liver. Chemico-biological interactions, 156(2-3): 101-111.
  • Betteridge DJ, 2000. What is oxidative stress? Metabolism, 49(2): 3-8.
  • Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O, 2012. Oxidative stress and antioxidant defense. World Allergy Organization Journal, 5(1):9.
  • Bradford MM, 1976. A rapid and sensitive method fort he quantation of microgram quantaties of protein utiliging the principle of protein-dye. Binding AnalyticalBiochemıstry, 72:248.
  • Dashti H, Jeppsson B, Hagerstrand I, Hultberg B, Srinivas U, Abdulla M, Bengmark S,1989. Thioacetamide and carbon tetrachloride-induced liver cirrhosis. Eur Surg Res, 21:83–91.
  • Donald EP, Valentina WN, 1967. Studies on the quantitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory & Clinical Medicine, 70:158-169.
  • Elbe H, Dogan Z, Taslidere E, Cetin A, Turkoz Y. 2016. Beneficial effects of quercetin on renal injury and oxidative stress caused by ciprofloxacin in rats: A histological and biochemical study. Human & experimental toxicology, 35(3), 276-281.
  • Erboga M, Aktas C, Erboga ZF, Donmez YB, Gurel A, 2015. Quercetin ameliorates methotrexate-induced renal damage, apoptosis and oxidative stress in rats. Renal failure, 37(9), 1492-1497.
  • Escobar JA, Rubio MA, Lissi EA, 1996. SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic Biol Med, 20: 285–290.
  • Herbet M, Gawrońska-Grzywacz M, Jagiełło-Wójtowicz E, 2015. Evaluatıon Of Selected Bıochemıcal Parameters Of Oxıdatıve Stress In Rats Pretreated Wıth Rosuvastatın And Fluoxetıne. Acta Poloniae Pharmaceutica, 72(2): 261-265.
  • Islam MA, Al Mamun MA, Faruk M, Islam MTU, Rahman MM, Alam MN,Alam MA, 2017. Astaxanthin ameliorates hepatic damage and oxidative stress in carbon tetrachloride-administered rats. Pharmacognosy research, 9(1):84.
  • Junnila M, Rahko T, Sukura A, Lindberg LA, 2000. Reduction of carbon tetrachloride-induced hepatotoxic effects by oral administration of betaine in male Han-Wistar rats: a morphometric histological study. Veterinary pathology, 37(3), 231-238.
  • Khan MR, Ahmed D, 2009. Protective effects of Digera muricata (L.) Mart. on testis against oxidative stress of carbon tetrachloride in rat. Food and Chemical Toxicology, 47(6):1393-1399.
  • Liu CM, Ma JQ, Sun YZ, 2010. Quercetin protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead. Environmental toxicology and pharmacology, 30(3): 264-271.
  • Ma JQ, Li Z, Xie WR, Liu CM, Liu SS, 2015. Quercetin protects mouse liver against CCl4-induced inflammation by the TLR2/4 and MAPK/NF-κB pathway. International immunopharmacology, 28(1): 531-539.
  • Makni M, Chtourou Y, Garoui EM, Boudawara T, Fetoui H, 2012. Carbon tetrachloride-induced nephrotoxicity and DNA damage in rats: protective role of vanillin. Human & experimental toxicology, 31(8): 844-852.
  • Moneim AEA, El-Khadragy MF, 2013. The potential effects of pomegranate (Punica granatum) juice on carbon tetrachloride-induced nephrotoxicity in rats. Journal of physiology and biochemistry, 69(3): 359-370.
  • Muthukumaran S, Sudheer AR., Nalini N, Menon VP, 2008. Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes. Redox Report, 13(5): 217-224.
  • Nomoto M, Yamaguchi R, Kawamura M, Kohno K, Kasai H, 1999. Analysis of 8-hydroxyguanine in rat kidney genomic DNA after administration of a renal carcinogen, ferric nitrilotriacetate. Carcinogenesis, 20(5):837-841.
  • Ozturk F, Ucar M, Ozturk IC, Vardi N, Batcioglu K, 2003. Carbon tetrachloride-induced nephrotoxicity and protective effect of betaine in Sprague-Dawley rats. Urology, 62(2): 353-356.
  • Özyurt H, Çevik Ö, Özgen Z, Özden AS, Çadırcı S, Elmas MA, Şener G, 2014. Quercetin protects radiation-induced DNA damage and apoptosis in kidney and bladder tissues of rats. Free radical research, 48(10): 1247-1255.
  • Renugadevi J, Prabu SM, 2010. Quercetin protects against oxidative stress-related renal dysfunction by cadmium in rats. Experimental and Toxicologic Pathology, 62(5): 471-481.
  • Sedky A, Mahboub F, Elsawy H, Eid R, 2017. Protective Potential of Quercetin on Cd-Induced Hepatorenal Damage. Polish Journal of Environmental Studies, 26(5).
  • Sun Yi, Oberley LW, Ying Li. 1988. A simple method for clinical assay of superoxide dismutase. Clinical Chemistry, 3413:497-500.
  • Tirkey N, Pilkhwal S, Kuhad A, Chopra K, 2005. Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbon tetrachloride in rat liver and kidney. BMC pharmacology, 5(1): 2.
  • Yousef MI, Omar SA, El-Guendi M I, Abdelmegid LA, 2010. Potential protective effects of quercetin and curcumin on paracetamol-induced histological changes, oxidative stress, impaired liver and kidney functions and haematotoxicity in rat. Food and Chemical Toxicology, 48(11), 3246-3261.
  • Yuan Y, Ma S, Qi Y, Wei X, Cai H, Dong L, Guo Q, 2016. Quercetin inhibited cadmium-induced autophagy in the mouse kidney via inhibition of oxidative stress. Journal of toxicologic pathology, 29(4): 247-252.
  • Zhang JQ, Shi L, Xu XN, Huang SC, Lu B, Ji LL, Wang ZT, 2014. Therapeutic detoxification of quercetin against carbon tetrachloride-induced acute liver injury in mice and its mechanism. Journal of Zhejiang University Science B, 15(12): 1039-1047.

The Protective Effect of Quercetin Against to Renal Tissue Damage Induced Carbontetrachloride in Rat

Year 2019, , 708 - 716, 01.06.2019
https://doi.org/10.21597/jist.475524

Abstract

In this study, the protective effect of quercetin against to the renal tissue damage caused by carbontetrachloride in rat was investigated. It was used 28 adults wistar albino rats weighing approximately 200-300 gr and were divided into four groups. Following 3 days of intraperitoneal application, rats were anesthetized and kidney tissues were taken.As biochemical parameters, SOD, CAT, GPx activities and (8-OHdG) levels were evaluated in renal tissue. When SOD, CAT, GPx enzyme activity was evaluated, decrease in enzyme activity in CCl4 group class with control group and increase in enzyme activity in CCl4 + quercetin group class withCCl4 group was determined.In this study, when 8-OHdG level of kidney tissue exposed to carbontetrachloride was evaluated; In the group treated with carbontetrachloride compared to the control, it was observed that the increase in the group of carbontetrachloride + quercetin compared to the control and the decrease in the group of carbontetrachloride + quercetin compared to the carbontetrachloride group. It was concluded that quercetin had protective effect on CCl4-induced kidney tissue damage.

References

  • Abarikwu SO, 2014. Protective Effect of Quercetin on Atrazine-Induced Oxidative Stress in the Liver, Kidney, Brain, and Heart of Adult Wistar Rats. Toxicol Int. May-Aug, 21(2): 148–155.
  • Abdel-Raheem IT, Abdel-Ghany AA, Mohamed GA, 2009. Protective effect of quercetin against gentamicin-induced nephrotoxicity in rats. Biological and Pharmaceutical Bulletin, 32(1), 61-67.
  • Abdurrauf Y, Aksakal M, 2007. Ratların Karaciğer ve Testis Dokusundaki Antioksidan Aktivite Üzerine Nar Suyunun Etkisi.F.Ü. Sağ. Bil. Derg, 21(6): 253 -256.
  • Adel Abdel-Moneim A, Ahmed OM, Fahim HI, Zaky MY, 2016. Ameliorative Effects of Quercetin and Naringenin on Diethylnitrosamine/2-acetyl amino flourene-Induced Nephrotoxicity in Male Wistar Rats. American Journal of Biochemistry, 6(5): 113-121.
  • Aebi H, 1984. Catalase in vitro assay methods. Methods Enzymol, 105:121-126.
  • Aldemir M, Okulu E, Kösemehmetoğlu K, Ener K, Topal F, Evirgen O, Gürleyik E, Avcı A,2014. Evaluation of the protective effect of quercetin against cisplatin‐induced renal and testis tissue damage and sperm parameters in rats. Andrologia, 46(10), 1089-1097.
  • Alidadi H, Khorsandi L, Shirani M, 2018. Effects of Quercetin on Tubular Cell Apoptosis and Kidney Damage in Rats Induced by Titanium Dioxide Nanoparticles. Malaysian Journal of Medical Sciences, 25(2), 72-81.
  • Almaghrabi OA, 2015. Molecular and biochemical investigations on the effect of quercetin on oxidative stress induced by cisplatin in rat kidney. Saudi journal of biological sciences, 22(2), 227-231.
  • Akkoyun HT, Bengu AS, Ulucan A, Bayramoglu-Akkoyun M, Arihan O, 2018.Protectıve Effect Of Ellagıc Acıd Agaınst Carbon Tetrachlorıde (CCl4) Induced Oxıdatıve Braın Injury In Rats.Fresenius Environmental Bulletin, 27(5): 3148-3155
  • Akşit H, Akşit D, Bildik A, Kara H, Yavuz Ö, Seyrek K, 1989. Deneysel Karaciğer İntoksikasyonunda N-Asetil Sistein’in Glutatyon Metabolizması Ve Lipid Peroksidasyonuna Etkileri. Ankara Üniv Vet Fak Derg, 62:( 1-5).
  • Arslan A, Demir H, Ozbay MF, Arslan H, 2014. Evaluation of lipid peroxidation and some antioxidant activities in patients with primary and metastatic liver cancer. Journal of Cancer Therapy, 5(2):192.
  • Bansal AK, Bansal M, Soni G, Bhatnagar D, 2005. Protective role of Vitamin E pre-treatment on N-nitrosodiethylamine induced oxidative stress in rat liver. Chemico-biological interactions, 156(2-3): 101-111.
  • Betteridge DJ, 2000. What is oxidative stress? Metabolism, 49(2): 3-8.
  • Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O, 2012. Oxidative stress and antioxidant defense. World Allergy Organization Journal, 5(1):9.
  • Bradford MM, 1976. A rapid and sensitive method fort he quantation of microgram quantaties of protein utiliging the principle of protein-dye. Binding AnalyticalBiochemıstry, 72:248.
  • Dashti H, Jeppsson B, Hagerstrand I, Hultberg B, Srinivas U, Abdulla M, Bengmark S,1989. Thioacetamide and carbon tetrachloride-induced liver cirrhosis. Eur Surg Res, 21:83–91.
  • Donald EP, Valentina WN, 1967. Studies on the quantitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory & Clinical Medicine, 70:158-169.
  • Elbe H, Dogan Z, Taslidere E, Cetin A, Turkoz Y. 2016. Beneficial effects of quercetin on renal injury and oxidative stress caused by ciprofloxacin in rats: A histological and biochemical study. Human & experimental toxicology, 35(3), 276-281.
  • Erboga M, Aktas C, Erboga ZF, Donmez YB, Gurel A, 2015. Quercetin ameliorates methotrexate-induced renal damage, apoptosis and oxidative stress in rats. Renal failure, 37(9), 1492-1497.
  • Escobar JA, Rubio MA, Lissi EA, 1996. SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic Biol Med, 20: 285–290.
  • Herbet M, Gawrońska-Grzywacz M, Jagiełło-Wójtowicz E, 2015. Evaluatıon Of Selected Bıochemıcal Parameters Of Oxıdatıve Stress In Rats Pretreated Wıth Rosuvastatın And Fluoxetıne. Acta Poloniae Pharmaceutica, 72(2): 261-265.
  • Islam MA, Al Mamun MA, Faruk M, Islam MTU, Rahman MM, Alam MN,Alam MA, 2017. Astaxanthin ameliorates hepatic damage and oxidative stress in carbon tetrachloride-administered rats. Pharmacognosy research, 9(1):84.
  • Junnila M, Rahko T, Sukura A, Lindberg LA, 2000. Reduction of carbon tetrachloride-induced hepatotoxic effects by oral administration of betaine in male Han-Wistar rats: a morphometric histological study. Veterinary pathology, 37(3), 231-238.
  • Khan MR, Ahmed D, 2009. Protective effects of Digera muricata (L.) Mart. on testis against oxidative stress of carbon tetrachloride in rat. Food and Chemical Toxicology, 47(6):1393-1399.
  • Liu CM, Ma JQ, Sun YZ, 2010. Quercetin protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead. Environmental toxicology and pharmacology, 30(3): 264-271.
  • Ma JQ, Li Z, Xie WR, Liu CM, Liu SS, 2015. Quercetin protects mouse liver against CCl4-induced inflammation by the TLR2/4 and MAPK/NF-κB pathway. International immunopharmacology, 28(1): 531-539.
  • Makni M, Chtourou Y, Garoui EM, Boudawara T, Fetoui H, 2012. Carbon tetrachloride-induced nephrotoxicity and DNA damage in rats: protective role of vanillin. Human & experimental toxicology, 31(8): 844-852.
  • Moneim AEA, El-Khadragy MF, 2013. The potential effects of pomegranate (Punica granatum) juice on carbon tetrachloride-induced nephrotoxicity in rats. Journal of physiology and biochemistry, 69(3): 359-370.
  • Muthukumaran S, Sudheer AR., Nalini N, Menon VP, 2008. Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes. Redox Report, 13(5): 217-224.
  • Nomoto M, Yamaguchi R, Kawamura M, Kohno K, Kasai H, 1999. Analysis of 8-hydroxyguanine in rat kidney genomic DNA after administration of a renal carcinogen, ferric nitrilotriacetate. Carcinogenesis, 20(5):837-841.
  • Ozturk F, Ucar M, Ozturk IC, Vardi N, Batcioglu K, 2003. Carbon tetrachloride-induced nephrotoxicity and protective effect of betaine in Sprague-Dawley rats. Urology, 62(2): 353-356.
  • Özyurt H, Çevik Ö, Özgen Z, Özden AS, Çadırcı S, Elmas MA, Şener G, 2014. Quercetin protects radiation-induced DNA damage and apoptosis in kidney and bladder tissues of rats. Free radical research, 48(10): 1247-1255.
  • Renugadevi J, Prabu SM, 2010. Quercetin protects against oxidative stress-related renal dysfunction by cadmium in rats. Experimental and Toxicologic Pathology, 62(5): 471-481.
  • Sedky A, Mahboub F, Elsawy H, Eid R, 2017. Protective Potential of Quercetin on Cd-Induced Hepatorenal Damage. Polish Journal of Environmental Studies, 26(5).
  • Sun Yi, Oberley LW, Ying Li. 1988. A simple method for clinical assay of superoxide dismutase. Clinical Chemistry, 3413:497-500.
  • Tirkey N, Pilkhwal S, Kuhad A, Chopra K, 2005. Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbon tetrachloride in rat liver and kidney. BMC pharmacology, 5(1): 2.
  • Yousef MI, Omar SA, El-Guendi M I, Abdelmegid LA, 2010. Potential protective effects of quercetin and curcumin on paracetamol-induced histological changes, oxidative stress, impaired liver and kidney functions and haematotoxicity in rat. Food and Chemical Toxicology, 48(11), 3246-3261.
  • Yuan Y, Ma S, Qi Y, Wei X, Cai H, Dong L, Guo Q, 2016. Quercetin inhibited cadmium-induced autophagy in the mouse kidney via inhibition of oxidative stress. Journal of toxicologic pathology, 29(4): 247-252.
  • Zhang JQ, Shi L, Xu XN, Huang SC, Lu B, Ji LL, Wang ZT, 2014. Therapeutic detoxification of quercetin against carbon tetrachloride-induced acute liver injury in mice and its mechanism. Journal of Zhejiang University Science B, 15(12): 1039-1047.
There are 39 citations in total.

Details

Primary Language Turkish
Subjects Structural Biology
Journal Section Biyoloji / Biology
Authors

Hurrem Turan Akkoyun 0000-0002-4547-8003

Publication Date June 1, 2019
Submission Date October 28, 2018
Acceptance Date January 29, 2019
Published in Issue Year 2019

Cite

APA Turan Akkoyun, H. (2019). Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi. Journal of the Institute of Science and Technology, 9(2), 708-716. https://doi.org/10.21597/jist.475524
AMA Turan Akkoyun H. Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi. J. Inst. Sci. and Tech. June 2019;9(2):708-716. doi:10.21597/jist.475524
Chicago Turan Akkoyun, Hurrem. “Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi”. Journal of the Institute of Science and Technology 9, no. 2 (June 2019): 708-16. https://doi.org/10.21597/jist.475524.
EndNote Turan Akkoyun H (June 1, 2019) Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi. Journal of the Institute of Science and Technology 9 2 708–716.
IEEE H. Turan Akkoyun, “Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi”, J. Inst. Sci. and Tech., vol. 9, no. 2, pp. 708–716, 2019, doi: 10.21597/jist.475524.
ISNAD Turan Akkoyun, Hurrem. “Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi”. Journal of the Institute of Science and Technology 9/2 (June 2019), 708-716. https://doi.org/10.21597/jist.475524.
JAMA Turan Akkoyun H. Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi. J. Inst. Sci. and Tech. 2019;9:708–716.
MLA Turan Akkoyun, Hurrem. “Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi”. Journal of the Institute of Science and Technology, vol. 9, no. 2, 2019, pp. 708-16, doi:10.21597/jist.475524.
Vancouver Turan Akkoyun H. Karbontetraklorür’ün Neden Olduğu Sıçan Böbrek Dokusu Hasarlarına Karşı Kuersetin’in Koruyucu Etkisi. J. Inst. Sci. and Tech. 2019;9(2):708-16.