Effect of Sour Cherry Fruit on Urease, Chymotrypsin and Trypsin Enzymes

Abstract

Aim: The aim of this study was to determine urease, chymotrypsin, and trypsin inhibitory activity of sour cherry. Method: In this study, which was conducted between July 2024 and January 2025, 80% ethanol extract was prepared from sour cherry fruits collected from Çubuk district (Ankara), and fractionated with n-hexane, chloroform, ethyl acetate, and n-butanol. Then, urease, chymotrypsin, and trypsin inhibitory activities of extracts and fractions were determined. Additionally, phenolic content of ethanol extract and ethyl acetate fraction, the strongest activity was observed, were analyzed by HPLC. Results: Ethyl acetate, n-butanol, and remaining water fractions inhibited trypsin enzyme by more than 95%. Similarly, the strongest chymotrypsin and urease inhibitory activity was observed in ethyl acetate fraction (%84,07 and 42,36% inhibition, respectively). HPLC analysis revealed that ethanol extract contained 0,1082% and 0,0512% chlorogenic acid and rutin, respectively, while ethyl acetate fraction contained 0,165% and 0,079% chlorogenic acid and rutin, respectively. Conclusion: The inhibitory effects of ethanol extract and different fractions obtained from sour cherry fruit against urease, chymotrypsin, and trypsin were evaluated for the first time. The findings revealed that especially ethyl acetate fraction showed high inhibitory activity on these enzymes. HPLC analysis shows that chlorogenic acid and rutin are present in ethanol extract and ethyl acetate fraction, and these compounds may contribute to inhibitory activity. Accordingly, it was thought that sour cherry fruit could be evaluated among natural urease and serine protease inhibitor candidates that can be used in prevention or treatment of ulcers, obesity, and inflammatory diseases such as pancreatitis.

Keywords

• Chymotrypsin
• Prunus cerasus
• sour cherry
• trypsin
• urease

Vişne Meyvesinin Üreaz, Kimotripsin ve Tripsin Enzimleri Üzerine Etkisi

Abstract

Amaç: Bu araştırmada vişne meyvesinin (Prunus cerasus L.) üreaz, kimotripsin ve tripsin inhibe edici aktivitesinin tespiti amaçlanmıştır. Yöntem: 2024 yılı Temmuz ve 2025 Ocak ayları arasında gerçekleştirilen bu çalışmada Ankara’nın Çubuk ilçesinden toplanan vişne meyvelerinden %80 etanol ekstresi hazırlanmış ve hazırlanan ekstre sırasıyla n-hekzan, kloroform, etil asetat ve n-bütanol ile fraksiyonlanmıştır. Daha sonra elde edilen ekstre ve fraksiyonların in vitro üreaz, kimotripsin ve tripsin inhibe edici aktiviteleri belirlenmiştir. Ayrıca etanol ekstresi ve en kuvvetli aktivitenin gözlendiği etil asetat fraksiyonunun fenolik içeriği YPSK (Yüksek Performanslı Sıvı Kromatografisi) ile analiz edilmiştir. Bulgular: Etil asetat, n-bütanol ve kalan su fraksiyonları tripsin enzimini %95’ten yüksek oranlarda inhibe etmiştir. Yine benzer şekilde en kuvvetli kimotripsin ve üreaz inhibe edici aktivite etil asetat fraksiyonunda gözlemlenmiştir (sırasıyla %84,07 ve %42,36 inhibisyon). YPSK analizi ile etanol ekstresinin sırasıyla %0,1082 ve %0,0512 oranlarında; etilasetat fraksiyonunun ise sırasıyla %0,165 ve %0,079 oranında klorojenik asit ve rutin içerdiği tespit edilmiştir. Sonuç: Vişne meyvesinden elde edilen %80 etanol ekstresi ve farklı fraksiyonların üreaz, kimotripsin ve tripsin enzimlerine karşı inhibitör etkileri ilk kez değerlendirilmiştir. Elde edilen bulgular, özellikle etil asetat fraksiyonunun bu enzimler üzerinde yüksek inhibe edici aktivite gösterdiğini ortaya koymuştur. YPSK analizi hem etanol ekstresinde hem de etil asetat fraksiyonunda klorojenik asit ve rutin gibi fenolik bileşiklerin bulunduğunu ve bu bileşiklerin inhibisyon aktivitesine katkı sağlayabileceğini göstermiştir. Bu doğrultuda, vişne meyvesinin ülser, obezite ve pankreatit gibi inflamatuvar hastalıkların önlenmesi veya tedavisinde kullanılabilecek doğal üreaz ve serin proteaz inhibitörü adayları arasında değerlendirilebileceği düşünülmüştür.

Keywords

• Kimotripsin
• Prunus cerasus
• Rutin
• Tripsin
• Üreaz

References

1. 1. Yılmaz FM, et al. Sour cherry by-products: compositions, functional properties, and recovery potentials – a review. Crit Rev Food Sci Nutr 2019;59(22):3549-63.
2. 2. Rawlings ND. Peptidase inhibitors in the MEROPS database. Biochimie 2010;92:1463-83.
3. 3. Hedstrom L. Serine protease mechanism and specificity. Chem Rev 2002;102:4501-24.
4. 4. Patel SA. Critical review on serine protease: key immune manipulator and pathology mediator. Aller Immunopathol 2017;45(6):579-91.
5. 5. Safavi F, Rostami A. Role of serine proteases in inflammation: Bowman–Birk protease inhibitor (BBI) as a potential therapy for autoimmune diseases. Experiment Mol Pathol 2012;93(3):428-33.
6. 6. Sharony R, et al. Protein targets of inflammatory serine proteases and cardiovascular disease. J Inflam 2010;30(7):45.
7. 7. Kuo CS, et al. Inhibition of serine protease activity protects against high-fat diet-induced inflammation and insulin resistance. Sci Rep 2020;10:1725.
8. 8. Albarazanji K, et al. Intestinal serine protease inhibition increases FGF21 and improves metabolism in obese mice. Am J Physiol- Gastro Liver Physiol 2019;316(5):G653-G67.

9. 9. Owen CA. Serine proteinases. In: Laurent GJ, Shapiro SD, editors. Encyclopedia of Respiratory Medicine. Academic Press; 2006. pp:1-10.
10. 10. Amtul Z, et al. Cysteine based novel noncompetitive inhibitors of urease(s)-distinctive inhibition susceptibility of microbial and plant ureases. Bioorg Med Chem 2006;14:6737-44.
11. 11. Yılmaz Ö, Okcu N. Helicobacter pylori ve gastrointestinal sistemle ilişkili hastalıklar. The Eurasian J Med 2006;38:13-7.
12. 12. Rego YF, et al. A review on the development of urease inhibitors as antimicrobial agents against pathogenic bacteria. J Adv Res 2018;13:69-100.
13. 13. Modolo LV, et al. An overview on the potential of natural products as ureases inhibitors: a review. J Adv Res 2015;6:35-44.
14. 14. Gürbüz İ, et al. Folk medicine in Düzce province (Turkey). Turk J Bot 2019;43(6):769-84.
15. 15. Gunbatan T, et al. In vitro antioxidant, enzyme inhibitory and antimicrobial evaluation of Sideritis caesarea H. Duman, Aytac & Baser extracts. Nat Prod Commun 2023;18(4):1-11.
16. 16. Siddiqui SZ, et al. Synthetic N-[(substitutedsulfamoyl) phenyl] acetamides as moderate chymotrypsin inhibitors. Pakistan J Pharm Sci 2017;30(3):675-81.
17. 17. Günbatan T, et al. Chymotrypsin and trypsin inhibitory activity of some medicinal plants collected from Rize (Türkiye). Chem Biodivers 2024; 21(3):e202301879.
18. 18. Yoshii K, et al. Activating effects on trypsin, α-chymotrypsin, and lipase and inhibitory effects on α-amylase and α-glucosidase as provided by low-molecularweight compounds in the water extract of the earthworm Eisenia fetida. Enzyme Microb Tech 2018;118: 20-9.
19. 19. Gökbulut A, et al. Phenolic compounds characterization, carbohydrate digestive enzyme inhibitory and antioxidant activities of Hieracium pannosum Boiss. S Afr J Bot 2017;108:387-92.
20. 20. Nabil-Adam A, et al. Interaction of Jania rubens polyphenolic extract as an antidiabetic agent with a-amylase, lipase, and trypsin: In vitro evaluations and in silico studies. Catalysts 2023;13:443.
21. 21. Lekmine S, et al. Bioactive phenolics of Hyoscyamus muticus L. subsp. falezlez: A molecular and biochemical approach to antioxidant and urease inhibitory activities. Int J Mol Sci 2025;26:370.
22. 22. Shoukry SM, et al. Unraveling putative antiulcer phytoconstituents against Helicobacter pylori urease and human H + /K +-ATPase from Jacaranda mimosifolia using UPLC-MS/MS coupled to chemometrics and molecular docking. Microchem J 2023;189:10855.
23. 23. Raafat K, et al. Gastroprotective and anti-inflammatory effects of Prunus cerasus phytochemicals and their possible mechanisms of action. J Tradit Comp Med 2020;10(4):345-53.
24. 24. Tall JM, et al. Tart cherry anthocyanins suppress inflammation-induced pain behavior in rat. Behav Brain Res 2004;153(1):181-8.
25. 25. He Y, et al. Anti‐inflammatory and anti‐oxidative effects of cherries on Freund’s adjuvant‐induced arthritis in rats. Scandinavian J Rheumatol 2006;35(5):356-8.
26. 26. Howatson G, et al. Influence of tart cherry juice on indices of recovery following marathon running. Scandinavian J Med Sci Sports 2010;20(6):843-52.
27. 27. Carson CA. Tart cherry juice as a treatment for peripheral neuropathy. Integ Med 2015;14(1):48-9.
28. 28. Biro A, et al. Anthocyanin-rich sour cherry extract attenuates the lipopolysaccharide-induced endothelial inflammatory response. Molecules 2019;24:3427.
29. 29. Kang SY, et al. Tart cherry anthocyanins inhibit tumor development in Apc(Min) mice and reduce proliferation of human colon cancer cells. Cancer Lett 2003;194(1):13-9.
30. 30. Martin KR, Wooden A. Tart cherry juice induces differential dose-dependent effects on apoptosis, but not cellular proliferation, in MCF-7 human breast cancer cells. J Med Food 2012;15:945-54.
31. 31. Oğur R, et al. Report: investigation of anti-cancer effects of cherry in vitro. Pakistan J Pharm Sci 2014;27:587-92.
32. 32. Martin KR, et al. The effect of 100% tart cherry juice on serum uric acid levels, biomarkers of inflammation and cardiovascular disease risk factors. FASEB J 2011;25:339.2
33. 33. Ataie-Jafari A, et al. Effects of sour cherry juice on blood glucose and some cardiovascular risk factors improvements in diabetic women. Nutr Food Sci 2008;38:355-60.
34. 34. Keane KM, et al. Effects of Montmorency tart cherry (Prunus Cerasus L.) consumption on vascular function in men with early hypertension. The Am J Clin Nutr 2016;103(6):1531-9.
35. 35. Karaçelik AA. Phytochemical profiling, antioxidant activities and in vitro/in silico enzyme inhibitory potentials of apricot cultivars grown in Igdır/Turkey. S Afr J Bot 2023;156:257-67.
36. 36. Dong H, et al. Potentialities of Dandelion (Taraxacum Mongolicum Hand.-Mazz.) Flower extracts on gastric protection against helicobacter pylori and characterization of its bioactive constituents. Chem Biodivers 2024;21:e202400140.
37. 37. Raza MA, Shahwar D. Trypsin inhibitory potential and microbial transformation of rutin isolated from Citrus sinensis. Med Chem Res 2013;22:3698-702.