Research Article
Year 2022,
Volume: 25 Issue: 3, 439 - 448, 30.06.2022
Abstract
Bu çalışmada geleneksel olarak yara tedavisinde kullanılan Potentilla recta L. (Rosaceae) 'nin yara iyileştirme mekanizmalarını değerlendirmek için, ciltteki patojen mikroorganizmalara karşı etkinliği ve antioksidan aktivitesi araştırılmıştır. Antioksidan aktivite 1,1-Difenil-2-pikrilhidrazil (DPPH•) ve 2,2-Azinobis (3-etilbenzotiyazolin-6-sülfonat) (ABTS•+) radikal temizleme, β-karoten ağartmanın inhibisyonu, 2-deoksiriboz ve sığır beyninden türetilmiş fosfolipidlerin hidroksil radikal aracılı degradasyonuna karşı koruma etkileri deneyleri ile 0.001 - 2 mg mL-1 arasında değişen farklı konsantrasyonlarda belirlenmiştir. Antimikrobiyal aktivite, mikrodilüsyon yöntemi ile Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa ve Candida albicans üzerinde değerlendirilmiştir. Ayrıca fare fibroblast hücreleri (L929) üzerindeki canlılığı artırıcı etkiler MTT (3- (4,5-dimetiltiyazol-2-il)-2,5-difeniltetrazolyum bromür) yöntemi ile değerlendirilmiştir. Ekstreler için temel parametreler: DPPH • IC50: 0.19 mg mL-1, TEAC: 0.76 mmol L-1 Trolox, redüksiyon: 0.87 mmol g-1 AsscE ve lipid peroksidasyonun korunması IC50: 0.07 mg mL-1 olarak belirlenmiştir. 125 mg mL-1 konsantrasyonda % 79 inhibisyonla S. epidermidis üzerinde güçlü bir etki gözlenirken 250 mg mL-1 konsantrasyonunun altında L929 hücrelerine toksisite gözlenmemiştir. Sonuçlar, antimikrobiyal aktivite ile birlikte güçlü antioksidan aktivitenin geleneksel kullanımı desteklediğini kanıtlamıştır.
Supporting Institution
Erciyes Üniversitesi
Project Number
TCD-2015-5609
References
- Amri B, Martino E, Vitulo F, Corana F, Bettaieb-Ben Kaâb L, Rui M, Rossi D, Mori M, Rossi S, Collina S 2017. Marrubium Vulgare L. Leave Extract: Phytochemical Composition, Antioxidant and Wound Healing Properties. Molecules 22:1851.
- Aruoma OI, Spencer JP, Warren D, Jenner P, Butler J, Halliwell B 1997. Characterization of Food Antioxidants, Illustrated Using Commercial Garlic and Ginger Preparations. Food Chem 60:149-156.
- Baytop T 1999. Turkiye’de Bitkiler ile Tedavi: Geçmipte ve Bugün-ilaveli ikinci baski Therapy with Medicinal Plants in Turkey: Past and Presnt extended 2nd Edn, Nobel Tip Kitaberleri Ltd, Turkey: Çapa-Istanbul 375sy.
- Bolla SR, Al-subaie AM, Al-Jindan RY, Balakrishna JP, Ravi PK, Veeraraghavan VP, Pillai AA, Gollapalli SSR, Joseph JP, Surapaneni KM 2019. In vitro wound healing potency of methanolic leaf extract of Aristolochia saccata is possibly mediated by its stimulatory effect on collagen-1 expression. Heliyon 5(5):e01648.
- Carter P 1971. Spectrophotometric Determination of Serum Iron at The Submicrogram Level With A New Reagent (Ferrozine). Anal.Biochem 40:450-458.
- Edwards JV, Howley P, Cohen IK 2004. In Vitro Inhibition of Human Neutrophil Elastase by Oleic Acid Albumin Formulations From Derivatized Cotton Wound Dressings. Int. J. Pharm 284:1-12.
- Folta KM, Gardiner SE 2009. Genetics and Genomics of Rosaceae. Springer-Verlag New York 6:315-335.
- Gangwar M, Gautam MK, Ghildiyal S, Nath G, Goel RK 2015. Mallotus philippinensis Muell. Arg Fruit Glandular Hairs Extract Promotes Wound Healing on Different Wound Model in Rats. BMC Complement Altern Med 15:123.
- Grochowski DM, Uysal S, Aktümsek A, Granica S, Zengin G, Ceylan R, Locatell M, Tomczyk M 2017. In Vitro Enzyme Inhibitory Properties, Antioxidant Activities, and Phytochemical Profile of Potentilla thuringiaca. Phytochem. Lett 20:365-372.
- Güner A, Aslan S 2012. Türkiye Bitkileri Listesi:(Damarlı Bitkiler). Nezahat Gökyiǧit Botanik Bahçesi Yayınları İstanbul 803 sy.
- Halliwell B, Gutteridge JM, Aruoma OI 1987. The Deoxyribose Method:A Simple “Test-tube” Assay For Determination of Rate Constants For Reactions of Hydroxyl Radicals. Anal.Biochem 165:215-219.
- Kang Y, Park Y, Lee G 1996. The Nitrite Scavenging and Electron Donating Activity of Phenol Compounds. Korean J Food Sci Technol 28:232-239.
- Karatoprak GŞ, Yücel Ç, Göger F, Sobarzo-Sánchez E, Akkol EK 2020. Potential Antioxidant and Enzyme Inhibitory Effects of Nanoliposomal Formulation Prepared from Salvia aramiensis Rech. f. Extract. Antioxidants 9:293.
- Kundu A, Ghosh A, Singh NK, Singh GK, Seth A, Maurya SK, Hemalatha S, Laloo D 2016. Wound Healing Activity of The Ethanol Root Extract and Polyphenolic Rich Fraction From Potentilla fulgens. Pharm.Biol 54:2383-2393.
- Leopoldini M, Marino T, Russo N, Toscano M 2004. Antioxidant Properties of Phenolic Compounds: H-Atom Versus Electron Transfer Mechanism. J. Phys. Chem. A 108:4916-4922. Liyana‐Pathirana CM, Shahidi F 2006. Antioxidant Properties of Commercial Soft and Hard Winter Wheats (Triticum aestivum L.) and Their Milling Fractions. J. Sci. Food Agric 86:477-485.
- Miliauskas G, Van Beek TA, Venskutonis PR, Linssen JP, De Waard P, Sudhölter EJ 2004. Antioxidant activity of Potentilla fruticosa. J. Sci. Food Agric 84:1997-2009.
- Oyaizu M 1986. Studies on Products of Browning Reaction. The Japanese Journal of Nutrition and Dietetics. 44:307-315.
- Ökdem B, Karatoprak GŞ, Koşar M 2018. Anti-inflammatory Activity and Phenolic Composition of a Traditional Medicinal Plant From Turkey: Potentilla recta L. Indian J. Tradit. Knowl 17(4):691-698
- Pitz HdS, Pereira A, Balasius MB, Voytena APL, Affonso RCL, Trevisan ACD, Ribeiro-do-Valle RM, Maraschin M 2016. In Vitro Evaluation of The Antioxidant Activity and Wound Healing Properties of Jaboticaba (Plinia Peruviana) Fruit Peel Hydroalcoholic Extract. Oxid Med Cell Longev 2016.
- Punjataewakupt A, Napavichayanun S, Aramwit P 2019. The Downside of Antimicrobial Agents for Wound Healing. Eur. J. Clin. Microbiol. Infect. Dis 38:39-54.
- Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C 1999. Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization assay. Free Radic. Biol. Med. 26:1231-1237.
- Saio V, Syiem D, Sharma R 2012. Effect of Potentilla fulgens on Lipid Peroxidation and Antioxidant Status in Alloxan-Induced Diabetic Mice. J Basic Clin Pharm 3:249.
- Sarikurkcu C, Zengin G, Aktümsek A, Ceylan O, Şanda MA 2015. Onopordum anatolicum Tohumlarinin Antioksidan Aktiviteleri. Selçuk Univ Fen Fak Fen Derg 41:89-96. Schwentker A, Vodovotz Y, Weller R, Billiar TR 2002. Nitric Oxide and Wound Repair: Role of Cytokines? Nitric Oxide 7:1-10.
- Sut S, Dall’Acqua S, Uysal S, Zengin G, Aktumsek A, Picot-Allain C, Mahomoodally F 2019. LC-MS, NMR Fingerprint of Potentilla argentea and Potentilla recta Extracts and Their In vitro Biopharmaceutical Assessment. Ind Crops Prod 131:125-133.
- Şöhretoğlu D, Kırmızıbekmez H. 2011. Polyphenols from Potentilla recta. Biochem. Syst. Ecol 39:132-134.
- Şöhretoğlu D, Zeynep Ü, Sabuncuoğlu S 2015. Assessment of Radical Scavenging Activities and Antiproliferative Properties of Two Cinquefoil (Potentilla) Species with Their Phytochemical Contents. Acta Medica 46:72-78.
- Talekar YP, Apte KG, Paygude SV, Tondare PR, Parab PB 2017. Studies on Wound Healing Potential of Polyherbal Formulation Using In Vitro and In Vivo Assays. J. Ayurveda Integr. Med 8:73-81.
- Tomczyk M, Latté KP 2009. Potentilla—A Review of Its Phytochemical and Pharmacological Profile. J Ethnopharmacol 122:184-204.
- Tomczyk M, Leszczyńska K, Jakoniuk P 2008. Antimicrobial Activity of Potentilla species. Fitoterapia 79:592-594.
- Tomczyk M, Paduch R, Wiater A, Pleszczyńska M, Kandefer-Szerszeń M, Szczodrak J 2013. The Influence of Aqueous Extracts of Selected Potentilla Species on Normal Human Colon Cells. Acta Pol Pharm 70:523-531.
- Tomczyk M, Wiater A, Pleszczyńska M 2011. In Vitro Anticariogenic Effects of Aerial Parts of Potentilla recta and Its Phytochemical Profile. Phytother Res 25:343-350.
- Tomic-Canic M, Burgess JL, O’Neill KE, Strbo N, Pastar I 2020. Skin Microbiota and its Interplay with Wound Healing. Am. J. Clin. Dermatol. 21:36-43.
- Tomovic MT, Cupara SM, Popovic-Milenkovic MT, Ljujic BT, Kostic MJ, Jankovic SM 2015. Antioxidant and Anti-inflammatory Activity of Potentilla reptans L. Acta Pol Pharm 72:137-145.
- Tosun A, Bahadir Ö, Altanlar N 2006. Antimicrobial Activity of Some Plants Used in Folk Medicine in Turkey. Turk J Pharm Sci 3:167-176.
- Uysal S, Aktumsek A 2015. A Phytochemical Study on Potentilla anatolica: An Endemic Turkish Plant Ind Crops Prod 76:1001-1007.
- Uysal S, Zengin G, Locatelli M, Bahadori MB, Mocan A, Bellagamba G, De Luca E, Mollica A, Aktumsek A 2017. Cytotoxic and Enzyme Inhibitory Potential of Two Potentilla Species (P. speciosa L. and P. reptans Willd.) and Their Chemical Composition. Front Pharmacol 8:290.
- Wang S-S, Wang D-M, Pu W-J, Li D-W 2013. Phytochemical Profiles, Antioxidant and Antimicrobial Activities of Three Potentilla Species. BMC Complement Altern Med 13:321.
- Wayne P 2009. Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Disk Diffusion Susceptibility Tests 19th ed. approved standard CLSI document M100-S19 29:M100-S121.
- Wolcott RD, Hanson JD, Rees EJ, Koenig LD, Phillips CD, Wolcott RA, Cox SB, White JS 2016. Analysis of The Chronic Wound Microbiota of 2,963 Patients by 16S rDNAPyrosequencing. Wound Repair Regen 24:163-174.
- Yarsan E 2014 Lipid Peroksidasyon Olayı ve Önlenmesine Yönelik Uygulamalar. Van Vet J 9:89-95.
- Yu D, Pu W, Li D, Wang D, Liu Q, Wang Y 2016. Phenolic Compounds and Antioxidant Activity of Different Organs of Potentilla fruticosa L. From Two Main Production Areas of China. Chem. Biodivers 13:1140-1148.
- Zhang LL, Lin YM 2009. AntioxidantTannins From Syzygium cumini Fruit. Afr. J. Biotechnol 8(10): 2301-2309.
Year 2022,
Volume: 25 Issue: 3, 439 - 448, 30.06.2022
Abstract
In this study, the antioxidant activity and effectiveness against pathogenic microorganisms in the skin were investigated to evaluate the wound healing mechanisms of Potentilla recta L. (Rosaceae), which is traditionally used in wound treatment. The antioxidant activity was determined using 1,1-Diphenyl-2-picrylhydrazyl (DPPH•) and2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+) radical scavenging activities, inhibition of β-carotene bleaching, protection of 2-deoxyribose and bovine brain-derived phospholipids against hydroxyl radical-mediated degradation assay, at different concentrations ranging from 0.001 to 2 mg mL-1. By the microdilution method, antimicrobial activity was evaluated on Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans. Besides, viability enhancing effects on murine fibroblast cells (L929) were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The key parameters for the extract included the following: DPPH• IC50: 0.19 mg mL-1, TEAC: 0.76 mmol L-1 Trolox, reduction: 0.87 mmol g-1 AsscE, and protection of lipid peroxidation IC50: 0.07 mg mL-1. A strong effect on S. epidermidis was observed with 79% inhibition at a concentration of 125 mg mL-1, but did not show toxicity to L929 cells below 250 mg mL-1 concentration. The results proved that the potent antioxidant activity combined with the antimicrobial activity supports traditional use.
Keywords
Project Number
TCD-2015-5609
References
- Amri B, Martino E, Vitulo F, Corana F, Bettaieb-Ben Kaâb L, Rui M, Rossi D, Mori M, Rossi S, Collina S 2017. Marrubium Vulgare L. Leave Extract: Phytochemical Composition, Antioxidant and Wound Healing Properties. Molecules 22:1851.
- Aruoma OI, Spencer JP, Warren D, Jenner P, Butler J, Halliwell B 1997. Characterization of Food Antioxidants, Illustrated Using Commercial Garlic and Ginger Preparations. Food Chem 60:149-156.
- Baytop T 1999. Turkiye’de Bitkiler ile Tedavi: Geçmipte ve Bugün-ilaveli ikinci baski Therapy with Medicinal Plants in Turkey: Past and Presnt extended 2nd Edn, Nobel Tip Kitaberleri Ltd, Turkey: Çapa-Istanbul 375sy.
- Bolla SR, Al-subaie AM, Al-Jindan RY, Balakrishna JP, Ravi PK, Veeraraghavan VP, Pillai AA, Gollapalli SSR, Joseph JP, Surapaneni KM 2019. In vitro wound healing potency of methanolic leaf extract of Aristolochia saccata is possibly mediated by its stimulatory effect on collagen-1 expression. Heliyon 5(5):e01648.
- Carter P 1971. Spectrophotometric Determination of Serum Iron at The Submicrogram Level With A New Reagent (Ferrozine). Anal.Biochem 40:450-458.
- Edwards JV, Howley P, Cohen IK 2004. In Vitro Inhibition of Human Neutrophil Elastase by Oleic Acid Albumin Formulations From Derivatized Cotton Wound Dressings. Int. J. Pharm 284:1-12.
- Folta KM, Gardiner SE 2009. Genetics and Genomics of Rosaceae. Springer-Verlag New York 6:315-335.
- Gangwar M, Gautam MK, Ghildiyal S, Nath G, Goel RK 2015. Mallotus philippinensis Muell. Arg Fruit Glandular Hairs Extract Promotes Wound Healing on Different Wound Model in Rats. BMC Complement Altern Med 15:123.
- Grochowski DM, Uysal S, Aktümsek A, Granica S, Zengin G, Ceylan R, Locatell M, Tomczyk M 2017. In Vitro Enzyme Inhibitory Properties, Antioxidant Activities, and Phytochemical Profile of Potentilla thuringiaca. Phytochem. Lett 20:365-372.
- Güner A, Aslan S 2012. Türkiye Bitkileri Listesi:(Damarlı Bitkiler). Nezahat Gökyiǧit Botanik Bahçesi Yayınları İstanbul 803 sy.
- Halliwell B, Gutteridge JM, Aruoma OI 1987. The Deoxyribose Method:A Simple “Test-tube” Assay For Determination of Rate Constants For Reactions of Hydroxyl Radicals. Anal.Biochem 165:215-219.
- Kang Y, Park Y, Lee G 1996. The Nitrite Scavenging and Electron Donating Activity of Phenol Compounds. Korean J Food Sci Technol 28:232-239.
- Karatoprak GŞ, Yücel Ç, Göger F, Sobarzo-Sánchez E, Akkol EK 2020. Potential Antioxidant and Enzyme Inhibitory Effects of Nanoliposomal Formulation Prepared from Salvia aramiensis Rech. f. Extract. Antioxidants 9:293.
- Kundu A, Ghosh A, Singh NK, Singh GK, Seth A, Maurya SK, Hemalatha S, Laloo D 2016. Wound Healing Activity of The Ethanol Root Extract and Polyphenolic Rich Fraction From Potentilla fulgens. Pharm.Biol 54:2383-2393.
- Leopoldini M, Marino T, Russo N, Toscano M 2004. Antioxidant Properties of Phenolic Compounds: H-Atom Versus Electron Transfer Mechanism. J. Phys. Chem. A 108:4916-4922. Liyana‐Pathirana CM, Shahidi F 2006. Antioxidant Properties of Commercial Soft and Hard Winter Wheats (Triticum aestivum L.) and Their Milling Fractions. J. Sci. Food Agric 86:477-485.
- Miliauskas G, Van Beek TA, Venskutonis PR, Linssen JP, De Waard P, Sudhölter EJ 2004. Antioxidant activity of Potentilla fruticosa. J. Sci. Food Agric 84:1997-2009.
- Oyaizu M 1986. Studies on Products of Browning Reaction. The Japanese Journal of Nutrition and Dietetics. 44:307-315.
- Ökdem B, Karatoprak GŞ, Koşar M 2018. Anti-inflammatory Activity and Phenolic Composition of a Traditional Medicinal Plant From Turkey: Potentilla recta L. Indian J. Tradit. Knowl 17(4):691-698
- Pitz HdS, Pereira A, Balasius MB, Voytena APL, Affonso RCL, Trevisan ACD, Ribeiro-do-Valle RM, Maraschin M 2016. In Vitro Evaluation of The Antioxidant Activity and Wound Healing Properties of Jaboticaba (Plinia Peruviana) Fruit Peel Hydroalcoholic Extract. Oxid Med Cell Longev 2016.
- Punjataewakupt A, Napavichayanun S, Aramwit P 2019. The Downside of Antimicrobial Agents for Wound Healing. Eur. J. Clin. Microbiol. Infect. Dis 38:39-54.
- Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C 1999. Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization assay. Free Radic. Biol. Med. 26:1231-1237.
- Saio V, Syiem D, Sharma R 2012. Effect of Potentilla fulgens on Lipid Peroxidation and Antioxidant Status in Alloxan-Induced Diabetic Mice. J Basic Clin Pharm 3:249.
- Sarikurkcu C, Zengin G, Aktümsek A, Ceylan O, Şanda MA 2015. Onopordum anatolicum Tohumlarinin Antioksidan Aktiviteleri. Selçuk Univ Fen Fak Fen Derg 41:89-96. Schwentker A, Vodovotz Y, Weller R, Billiar TR 2002. Nitric Oxide and Wound Repair: Role of Cytokines? Nitric Oxide 7:1-10.
- Sut S, Dall’Acqua S, Uysal S, Zengin G, Aktumsek A, Picot-Allain C, Mahomoodally F 2019. LC-MS, NMR Fingerprint of Potentilla argentea and Potentilla recta Extracts and Their In vitro Biopharmaceutical Assessment. Ind Crops Prod 131:125-133.
- Şöhretoğlu D, Kırmızıbekmez H. 2011. Polyphenols from Potentilla recta. Biochem. Syst. Ecol 39:132-134.
- Şöhretoğlu D, Zeynep Ü, Sabuncuoğlu S 2015. Assessment of Radical Scavenging Activities and Antiproliferative Properties of Two Cinquefoil (Potentilla) Species with Their Phytochemical Contents. Acta Medica 46:72-78.
- Talekar YP, Apte KG, Paygude SV, Tondare PR, Parab PB 2017. Studies on Wound Healing Potential of Polyherbal Formulation Using In Vitro and In Vivo Assays. J. Ayurveda Integr. Med 8:73-81.
- Tomczyk M, Latté KP 2009. Potentilla—A Review of Its Phytochemical and Pharmacological Profile. J Ethnopharmacol 122:184-204.
- Tomczyk M, Leszczyńska K, Jakoniuk P 2008. Antimicrobial Activity of Potentilla species. Fitoterapia 79:592-594.
- Tomczyk M, Paduch R, Wiater A, Pleszczyńska M, Kandefer-Szerszeń M, Szczodrak J 2013. The Influence of Aqueous Extracts of Selected Potentilla Species on Normal Human Colon Cells. Acta Pol Pharm 70:523-531.
- Tomczyk M, Wiater A, Pleszczyńska M 2011. In Vitro Anticariogenic Effects of Aerial Parts of Potentilla recta and Its Phytochemical Profile. Phytother Res 25:343-350.
- Tomic-Canic M, Burgess JL, O’Neill KE, Strbo N, Pastar I 2020. Skin Microbiota and its Interplay with Wound Healing. Am. J. Clin. Dermatol. 21:36-43.
- Tomovic MT, Cupara SM, Popovic-Milenkovic MT, Ljujic BT, Kostic MJ, Jankovic SM 2015. Antioxidant and Anti-inflammatory Activity of Potentilla reptans L. Acta Pol Pharm 72:137-145.
- Tosun A, Bahadir Ö, Altanlar N 2006. Antimicrobial Activity of Some Plants Used in Folk Medicine in Turkey. Turk J Pharm Sci 3:167-176.
- Uysal S, Aktumsek A 2015. A Phytochemical Study on Potentilla anatolica: An Endemic Turkish Plant Ind Crops Prod 76:1001-1007.
- Uysal S, Zengin G, Locatelli M, Bahadori MB, Mocan A, Bellagamba G, De Luca E, Mollica A, Aktumsek A 2017. Cytotoxic and Enzyme Inhibitory Potential of Two Potentilla Species (P. speciosa L. and P. reptans Willd.) and Their Chemical Composition. Front Pharmacol 8:290.
- Wang S-S, Wang D-M, Pu W-J, Li D-W 2013. Phytochemical Profiles, Antioxidant and Antimicrobial Activities of Three Potentilla Species. BMC Complement Altern Med 13:321.
- Wayne P 2009. Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Disk Diffusion Susceptibility Tests 19th ed. approved standard CLSI document M100-S19 29:M100-S121.
- Wolcott RD, Hanson JD, Rees EJ, Koenig LD, Phillips CD, Wolcott RA, Cox SB, White JS 2016. Analysis of The Chronic Wound Microbiota of 2,963 Patients by 16S rDNAPyrosequencing. Wound Repair Regen 24:163-174.
- Yarsan E 2014 Lipid Peroksidasyon Olayı ve Önlenmesine Yönelik Uygulamalar. Van Vet J 9:89-95.
- Yu D, Pu W, Li D, Wang D, Liu Q, Wang Y 2016. Phenolic Compounds and Antioxidant Activity of Different Organs of Potentilla fruticosa L. From Two Main Production Areas of China. Chem. Biodivers 13:1140-1148.
- Zhang LL, Lin YM 2009. AntioxidantTannins From Syzygium cumini Fruit. Afr. J. Biotechnol 8(10): 2301-2309.
There are 42 citations in total.
Details
| Primary Language | English |
|---|---|
| Subjects | Structural Biology |
| Journal Section | RESEARCH ARTICLE |
| Authors | |
| Project Number | TCD-2015-5609 |
| Publication Date | June 30, 2022 |
| Submission Date | March 10, 2021 |
| Acceptance Date | July 1, 2021 |
| Published in Issue | Year 2022Volume: 25 Issue: 3 |
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