Investigation of Antienzyme, DNA protection/breaking and Antimicrobial Activity of Firethorn (Pyracantha coccinea) Fruit and Leaf Extracts

Abstract

Firethorn (Pyracantha coccinea) plant has the potential to be used in the treatment of many diseases thanks to its secondary metabolites. However, there are very few studies in the literature on the use of P. coccinea in pharmacology. Therefore, in this study, we aimed to investigate the antienzyme activity of P. coccinea under in vitro conditions by preparing fruit and leaf methanol extracts. The effect of P. coccinea leaf and fruit extract on Glutathione-S transferase (GST) and Tyrosinase (TYR) enzyme activities, which are known in the literature to be related to cancer diagnosis and treatment, was investigated. In this study, for the first time, the DNA protection/breaking effect of fruit and leaf methanol extracts of P. coccinea was investigated. Additionally, the antimicrobial activity of both extracts against Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29242), Escherichia coli (ATCC 25922), Candida albicans (ATCC 10231) microorganisms was investigated. It was determined that P. coccinea leaf extract inhibited GST at micromolar level. It was determined that both extracts did not have a sufficient effect on TYR enzyme activity. It was determined that the extracts did not cause DNA breakage and had a protection effect of 1 mg/mL concentration. However, it did not show antimicrobial activity against all microorganisms studied. As a result, we think that P. coccinea plant can be a natural source for candidate drug studies, but the full potential of the plant should be revealed with additional preclinical and clinical studies.

Keywords

• Tyrosinase
• Glutathione-S transferase
• Enzyme Activity

Ateş Dikeni (Pyracantha coccinea) Meyve ve Yaprak Ekstrelerinin, Antienzim, DNA koruma/kırılma ve Antimikrobiyal Aktivitesinin Araştırılması

Öz

Ateş dikeni (Pyracantha coccinea) bitkisi sahip olduğu sekonder metabolitler sayesinde birçok hastalığın tedavisinde kullanılma potansiyeli taşımaktadır. Ancak literatürde P. coccinea’nın farmakolojide kullanımı ile ilgili çok az sayıda çalışma vardır. Bu nedenle bu çalışmada P. coccinea’nın meyve ve yaprak metanol ekstreleri hazırlanarak antienzim aktivitesi in vitro şartlarda araştırılmak istenmiştir. Bu sebeple kanser tanı ve tedavisi ile ilişkisi olduğu literatürlerce bilinen Glutatyon-S transferaz (GST) ve Tirozinaz (TYR) enzim aktiviteleri üzerine P. coccinea’nın yaprak ve meyve ekstresinin etkisi belirlenmiştir. İlk kez bu çalışmada P. coccinea’nin meyve ve yaprak metanol ekstreleri DNA koruma/kırılma etkisi ve her iki ekstrenin Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29242), Escherichia coli (ATCC 25922), Candida albicans (ATCC 10231) mikroorganizmalarına karşı antimikrobiyal etkinliği araştırılmıştır. P. coccinea’nın yaprak ekstresinin GST’yi mikromolar düzeyde inhibe ettiği tespit edilmiştir. Her iki ekstrenin TYR enzim aktivitesi üzerinde yeterli etki göstermediği belirlenmiştir. Ayrıca ekstrelerin DNA kırılmasına yol açmadığı ve 1 mg/mL konsantrasyonda koruma etkisine sahip olduğu gözlemlenmiştir. Ancak ekstreler çalışılan tüm mikroorganizmalara karşı antimikrobiyal etkinliğe sahip olmadığı görülmüştür. Sonuç olarak P. coccinea’nın bitkisinin aday ilaç çalışmaları için doğal kaynak olabileceğini buna karşın ilave preklinik ve klinik çalışmalarla bitkinin tam potansiyelinin ortaya çıkarılması gerektiği düşünülmektedir.

Anahtar Kelimeler

• Tirozinaz
• Glutatyon S Transferaz
• Enzim aktivitesi

Kaynakça

1. Akkemik E., Aybek A., Felek, İ. Effects of cefan melon cucumis melo l seedextracts on human erythrocyte carbonicanhydrase i-ii enzymes. Applied Ecology And Environmental Research, 2019; 17(6): 14699–14713.
2. Akkemik E., Fidan, M., Balaban, M., Inal B. ICP-OES And LC-ESI-MM/MM Analyses, enzyme inhibition and DNA protection potential of pelargonium quercetorum agnew. Studia Universitatis Babes-Bolyai, Chemia, 2022; 67(4).
3. Anonim. 2024 Https://Www.Pfizer.Com.Tr/Pfizerde-Bilim/Arge/Ar-Ge-Ana-Sayfa
4. Aslam S., Gupta V., Carbonic anhydrase inhibitors. [Updated 2023 Apr 17]. In: Statpearls [Internet]. Treasure Island (Fl): Statpearls Publishing; 2024 Jan-. Available From: Https://Www.Ncbi.Nlm.Nih.Gov/Books/Nbk557736/
5. Atalar MN., Aras A., Türkan F., Barlak N., Yildiko Ü., Karatas OF., Alma MH. The effects of daucus carota extract against pc3, pnt1a prostate cells, acetylcholinesterase, glutathione s‐transferase, and α‐glycosidase; an in vitro–in silico study. Journal of Food Biochemistry, 2021; 45(12): E13975.
6. Baber MA., Crist CM., Devolve NL., Patrone J.D. Tyrosinase inhibitors: a perspective. Molecules 2023; 28, 5762.
7. Balaban M., Akkemi̇k E. Investigation of natural inhibitors of liver glutathione s-transferase enzyme of shabut (barbus grypus) fish, International Siirt Conference On Scientific Research Siirt University, November 5-7 2021, 144s. Siirt, Türkiye
8. Bhattarai S., Sharma BK., Subedi N., Ranabhat S., Baral MP. Burden of serious bacterial infections and multidrug-resistant organisms in an adult population of nepal: a comparative analysis of minimally invasive tissue sampling informed mortality surveillance of community and hospital deaths. Clin Infect Dis. 2021; S415-S421.

9. Board PG., Menon D., Glutathione transferases, regulators of cellular metabolism and physiology, biochim. Biophys. Acta, 2013; 1830, 3267-3288.
10. Booth J., Boyland E., and Sims AP. An enzyme from rat liver catalysing conjugations with glutathione, Biochem. J., 1961; 79, 516
11. Brenner M., Hearing V.J. The protective role of melanin against uv damage in human skin. photochem. Photobiol. 2008, 84, 539–549.
12. Calay Ö., Tirozinaz Enziminin Bazı Tıbbi Bitkiler Tarafından Inhibisyonu, İstanbul Üniversitesi, Master's Thesis, Fen Bilimleri Enstitüsü; Biyokimya Abd, 73s İstanbul, Türkiye, 2010.
13. Chang TS., An Updated Review Of Tyrosinase Inhibitors. Int. J. Mol. Sci. 2009, 10, 2440–2475.
14. Seo SY., Sharma VK., Sharma N., Mushroom tyrosinase: recent prospects. J. Agric. Food Chem. 2003, 51, 2837–2853. Cooksey CJ., Garratt P J., Land EJ., Pavel S., Ramsden CA., Riley PA. Et Al., Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase, Journal Of Biological Chemistry, 1997; 272, 26226–26235.
15. Çöteli E., Karataş F., Ateş dikeninin (pyracantha coccinea roemer var. lalandi) kırmızı meyvelerindeki a, e, c vitamini, β-karoten, likopen, glutatyon ve malondialdehit miktarlarının araştırılması, Fırat Üniv. Fen Bilimleri Dergisi, 2017; 29(1): 41-46.
16. De Almeida SM., Lafayette EA., Da Silva LP., Amorim CA., De Oliveira TB., Ruiz AL., De Carvalho JE., De Moura RO., Beltrão EI., De Lima MC., De Carvalho JL., Synthesis, dna bnding, and antiproliferative activity of novel acridine-tiosemikarbazon derivatives, Int J Mol Sci. 2015; 16(6): 13023-13042.
17. Deering RW., Chen J., Sun J., Ma H., Dubert J., Barja JL., Seeram NP., Wang H., Rowley DC., N-acyl dehydrotyrosines, tyrosinase inhibitors from the marine bacterium thalassotalea sp. Pp2-459. J. Nat. Prod. 2016, 79, 447–450.
18. Duckworth HW., Coleman JE., J. Biol. Chem. 1970; 245, 1613-1625; sigma quality control test procedure, enzymatic assay of tyrosinase (Ec 1.14. 18. 1)
19. Gasparetti C., Nordlund E., Jänis J., Buchert J., Kruus K., Extracellular tyrosinase from the fungus trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from agaricus bisporus. Biochim. Biophys. Acta Bba Proteins Proteom. 2012, 1824, 598–607.
20. GBD 2019 Demographics collaborators. global age-sex-specific fertility, mor- tality, healthy life expectancy (hale), and population estimates in 204 countries and territories, 1950–2019: a comprehensive demographic analysis for the global burden of disease study 2019. Lancet 2020; 396:1160-203.
21. Hałdys K., Goldeman W., Jewgi ´Nski M., Woli ´Nska E., Anger-Góra N., Rossowska J., Latajka R., Halogenated aromatic thiosemicarbazones as potent inhibitors of tyrosinase and melanogenesis. bioorg. Chem. 2020, 94, 103419.
22. Hamada SI., Kamada M., Furumoto H., Hirao, T., and Aono T., Expression of glutathione s-transferase-π in human ovarian cancer as an indicator of resistance to chemotherapy, gynecol. Oncol., 1994; 52, 313-319.
23. Hayes, J. D., Flanagan, J. U., And Jowsey, I. R. (2005) Glutathione Transferases, Ann. Rev. Pharmacol. Tox Icol., 45, 51-88, Doi: 10.1146/Annurev.Pharmtox.45. 120403.095857.
24. Irtegün Kandemir, S., Aktepe, N., Baran, A. Et Al. Determination Of Chemical Composition And Antioxidant, Cytotoxic, Antimicrobial, And Enzyme Inhibition Activities Of Rumex Acetosella L. Plant Extract. Chem. Pap. (2024).
25. Karacaer NT., The medicinal effects of different solvent extracts of pyracantha coccinea roem. fruits: heavy metal content, antioxidant, and antimicrobial properties. Bezmialem Science, 2023; 11(1).
26. Khan AA., Akhtar S., Husain Q., Direct immobilization of polyphenol oxidases on celite 545 from ammonium sulphate fractionated proteins of potato (solanum tuberosum). Journal Of Molecular Catalysis B: Enzymatic 2006; 40(1-2): 58-63.
27. Luo W., Kinsey M., Schiffman JD., Lessnick SL., Glutathione s-transferases in pediatric cancer, front. Oncol., 2011; 1, 39.
28. Matoba Y., Kihara S., Bando, N., Yoshitsu, H., Sakaguchi, M., Kayama, K., Yanagisawa, S., Ogura, T., Sugiyama, M., Catalytic mechanism of the tyrosinase reaction toward the tyr 98 residue in the caddie protein. Plos Biol. 2018, 16, E3000077. Molecules 2023, 28, 57-62
29. Murray Cjl., Abbafati C., Abbas Km., Et al. Five insights from the global burden of disease study 2019. Lancet 2020; 396:1135-1159.
30. Özaslan MS., Investigation of potential effects of some indole compounds on the glutathione s-transferase enzyme. Biochemistry Moscow, 2024; 89, 553–561
31. Parvez S., Kang M., Chung H-S., Bae H., Naturally Occurring Tyrosinase Inhibitors: Mechanism And Applications In Skin Health, Cosmetic And Agriculture Industries, Phytotherapy Research, 2007; 21, 805-816.
32. Popoviciu Dr., Negreanu-Pirjol T., Motelica L., Negreanu-Pirjol Bs. Carotenoids, flavonoids, total phenolic compounds content and antioxidant activity of ındigenous pyracantha coccinea M. roem. Fruits. Rev Chim 2020; 71: 258-266.
33. Ramadan Mohamed A.G., Izatin türevlerinin dna koruma, etkileşim ve antimikrobiyal aktivitesinin analizleri ve seftriakson-fenilalanin kombinasyonunun sinerjistik etkilerinin değerlendirilmesi. Kastamonu Üniversitesi Fen Bilimleri Enstitüsü, Doktora Tezi Genetik Ve Biyomühendislik Ana Bilim Dali, 105s, Kastamonu, Türkiye, 2019.
34. Riley PA., Melanin Int J. Biochem. Cell Biol. 1997, 29, 1235–1239.
35. Robert LS., Marc FL., Michael VD., Chapter 26 - Carbonic anhydrase inhibitors, editor(s): robert l stamper, marc f lieberman, michael v drake, becker-shaffer's diagnosis and therapy of the glaucomas (eighth edition), Mosby, 2009, Pages 407-419.
36. Sadeghi Z., Valizadeh J., Azizian Shermeh O., Akaberi M., Antioxidant activity and total phenolic content of boerhavia elegans (choisy) grown in baluchistan, Iran. Avicenna Journal of Phytomedicine, 2015; 5(1): 1-9.
37. Şakiroğlu H., Kuşburnu meyvasından ızole edilen polifenol oksidaz enziminin kinetik ve elektroforetik özelliklerinin ıncelenmesi. Atatürk Üniversitesi, Fen Bilimleri Enstitüsü, Biyokimya ABD, 121s, Erzurum, Türkiye, 1994.
38. Sarikurkcu C., Tepe B., Biological activity and phytochemistry of firethorn (pyracantha coccinea m.j. roemer). J Funct Foods 2015; 19: 669-675.
39. Singh RR., Reindl KM., Glutathione s-transferases in cancer, Antioxidants, 2021; 10, 701.
40. Smit N., Vicanova J., Pavel S., The hunt for natural skin whitening agents. Int. J. Mol. Sci. 2009, 10, 5326–5349. Temiz A., Genel Mikrobiyoloji Uygulama Teknikleri (5. Baskı). Hatiboğlu Yayınevi, Ankara, Türkiye, 2010.
41. Tew KD., Glutathione-associated enzymes in anticancer drug resistance, Cancer Res., 1994; 54, 4313-4320.
42. Tew KD., Townsend D.M. Glutathione-S-transferases as determinants of cell survival and death. Antioxid. Redox Signal., 2012; 17, 1728-1737.
43. Tunç K., Semerci AB., Okur I., Antioxidant activity of the fruits of pyracantha coccinea using ethanolic extract method. Food And Health, 2020; 6(1): 35-40.
44. Turu D., Bozyel, M. E., Candan, K., Yakan, M. A., Benek, A., Canli, K. (2020). In vitro antimicrobial and antioxidant activities of pyracantha coccinea fruits ethanol extract. Vitro, 4, 89-93.
45. Ucar-Türker A., Birinci-Yildirim A., Pehlivan-Karakas F., Antibacterial and antitumor activities of some wild fruits grown in turkey. Biotechnology & Biotechnological Equipment, 2012; 26(1): 2765-2772.
46. Wong TC., Luh BS., Whitaker JR., Isolation and characterization of polyphenol oxidase isozymes of clingstone peach. Plant Physiology, 1971; 48(1): 19-23.
47. World Health Organization. The Top Ten Causes Of Death. 2020. Https://Www.Who. Int/News-Room/Fact-Sheets/Detail/The-Top-10-Causes-Of-Death. Accessed 25 April 2021.
48. Yang, P., Ebbert, J. O., Sun, Z., And Weinshilboum, R. W. (2006) Role of the glutathione metabolic pathway ın lung cancer treatment and prognosis: A review, J. Clin. Oncol., 24, 1761-1769,
49. Yeniçeri ŞA., Balaban M., Hallaç B., Akkemik E., Investigation of the antienzyme and antimicrobial properties of the fruit extracts of the oleaster (elaeagnus angustifolia l.) under in vitro conditions, proceedings Of Ikstc 2023 - 2nd International Karatekin Science and Technology Conference, 21-22 December 2023,281s, Çankırı, Türkiye
50. Yu F., Pan Z., Qu B., Yu X., Xu K., Deng Y., Liang F., Identification of a tyrosinase gene and its functional analysis in melanin synthesis of pteria penguin. Gene 2018, 656, 1–8.
51. Zaidi KU., Ali AS., Ali SA., Naaz I., Microbial Tyrosinases: Promising Enzymes For Pharmaceutical, Food Bioprocessing, And Environmental Industry. Biochem. Res. Int. 2014, 2014, 854687.
52. Zou Z., Xi W., Hu Y., Nie C., Zhou Z., Antioxidant activity of citrus fruits. Food Chemistry, 2016; 196, 885-896.