EFFECT OF WHEATGRASS ON HUMAN LYMPHOCYTE CELLS

Abstract

Objective While chemotherapeutic drugs are effective at eliminating cancer cells, they can also damage healthy cells. The aim of this study was to analyze the phenolic component content of wheatgrass extract (Triticum aestivum L.) and to determine the effect of this extract against DNA damage caused by cisplatin and etoposide used in chemotherapeutic treatment in healthy cells will. Material and Method Sprouted wheat methanol extract was prepared and analysis of phenolic components was performed by HPLC (high performance liquid chromatography). IC50 (half maximal inhibitory concentration) and LD50 (median lethal dose) values were calculated by applying a cell viability assay based on wheat extract concentration. Cells were incubated at these determined concentration levels and the presence of DNA damage was assessed by the Comet method. Results As a result of the phenol component analysis, p-hydroxybenzoic acid was determined in the highest amount and o-coumaric acid in the lowest amount. The values for wheatgrass extract, etoposide and cisplatin at different concentrations applied to lymphocyte cells were found to be IC50=204.6 μg/mL, LD50=15.84 μg/mL and LD50=24.51 μg/mL, respectively. As a result of comet analysis, it was found that etoposide LD50 and etoposide LD50+wheatgrass extract IC50 group were statistically significant (p<0.05), compared to the control group, there was no statistical significance between etoposide LD50 and etoposide LD50+wheatgrass extract IC50 group (p>0.05). Similar to this result, cisplatin LD50 and cisplatin LD50+wheatgrass extract IC50 group were found to be statistically significant compared to the control group (p<0.05), while there was no statistical significance between etoposide LD50 and etoposide LD50+wheatgrass extract IC50 group (p> 0.05). Conclusion Although wheatgrass was observed in our study to cause a reduction in DNA damage caused by etoposide and cisplatin, no statistical significance was found.

Keywords

• Wheatgrass
• Comet method
• HPLC
• MTT

BUĞDAY ÇİMİNİN İNSAN LENFOSİT HÜCRELERİ ÜZERİNE ETKİSİ

Abstract

Amaç Kemoterapötik ilaçlar kanser hücrelerinin ortadan kaldırılmasında etkili iken aynı zamanda sağlıklı hücrelerde de hasar oluşturabilmektedir. Bu çalışmada, buğday çimi (Triticum aestivum L.) ekstraktının fenolik bileşen içeriğinin analizi ve bu ekstraktın kemoterapötik tedavide kullanılan sisplatin ve etoposid’in sağlıklı hücrelerde oluşturduğu DNA hasarına karşı etkisinin belirlenmesi amaçlanmıştır. Gereç ve Yöntem Çimlendirilmiş buğdayların metanol ekstraktı hazırlanarak HPLC (yüksek performanslı sıvı kromatografisi) ile fenolik bileşen analizi yapıldı. Buğday ekstraktı konsantrasyonuna bağlı hücre canlılık testi uygulanarak IC50 (Yarı maksimum inhibitör konsantrasyonu) ve LD50 (ortalama öldürücü doz) değerleri hesaplandı. Belirlenen bu konsantrasyon değerleri ile hücreler inkübe edilerek DNA hasarı varlığı Comet metodu ile değerlendirildi. Bulgular Fenolik bileşen analizi sonucunda p-hidroksibenzoik asit en yüksek miktarda, o-kumarik asit ise en düşük düzeyde tespit edildi. Lenfosit hücrelerine uygulanan farklı konsantrasyonlardaki buğday çimi ekstraktı, etoposid ve sisplatin için değerler sırasıyla IC50=204,6 μg/mL, LD50=15,84 μg/mL ve LD50=24,51 μg/mL olarak bulundu. Comet analizi sonucunda kontrol grubuna kıyasla, etoposid LD50 ve etoposid LD50+buğday çimi ekstraktı IC50 grubu istatistiksel olarak anlamlı bulunurken (p<0,05), etoposid LD50 ve etoposid LD50+buğday çimi ekstraktı IC50 grubu arasında istatistiksel olarak anlamlılık bulunamadı (p>0,05). Bu sonuca benzer olarak kontrol grubuna kıyasla, sisplatin LD50 ve sisplatin LD50+buğday çimi ekstraktı IC50 grubu istatistiksel olarak anlamlı bulunurken (p<0,05), etoposid LD50 ve etoposid LD50+buğday çimi ekstraktı IC50 grubu arasında istatistiksel olarak anlamlılık saptanmadı (p>0,05). Sonuç Çalışmamızda buğday çiminin etoposid ve sisplatin nedeni ile oluşan DNA hasarında azalmaya neden olduğu görülmüş olmasına rağmen istatistiksel olarak anlamlılık saptanmamıştır.

Keywords

• Buğday çimi
• Comet metodu
• HPLC
• MTT

Thanks

Çalışmamıza ait bazı deneysel aşamaların gerçekleştirilmesinde cihaz kullanılması ile destek olan Süleyman Demirel Üniversitesi Yenilikçi Teknolojiler Uygulama ve Araştırma Merkezi (YETEM)'e teşekkür ederiz.

References

1. 1. Rommasi F, Esfandiari N. Liposomal nanomedicine: applications for drug delivery in cancer therapy. Nanoscale Research Letters. 2021;16(1):1-20.
2. 2. Sun C-Y, Zhang Q-Y, Zheng G-J, Feng B. Phytochemicals: Current strategy to sensitize cancer cells to cisplatin. Biomedicine & Pharmacotherapy. 2019;110:518-27.
3. 3. Fang C-y, Lou D-y, Zhou L-q, Wang J-c, Yang B, He Q-j, et al. Natural products: Potential treatments for cisplatin-induced nephrotoxicity. Acta Pharmacologica Sinica. 2021;42(12):1951- 69.
4. 4. Tchounwou PB, Dasari S, Noubissi FK, Ray P, Kumar S. Advances in our understanding of the molecular mechanisms of action of cisplatin in cancer therapy. Journal of experimental pharmacology. 2021;13:303.
5. 5. Kim SS, Wengier DL, Ragland CJ, Sattely ES. Transcriptional Reactivation of Lignin Biosynthesis for the Heterologous Production of Etoposide Aglycone in Nicotiana benthamiana. ACS synthetic biology. 2022;11(10):3379-87.
6. 6. Zhang W, Gou P, Dupret J-M, Chomienne C, Rodrigues-Lima F. Etoposide, an anticancer drug involved in therapy-related secondary leukemia: Enzymes at play. Translational oncology. 2021;14(10):101169.
7. 7. Gore RD, Palaskar SJ, Bartake AR. Wheatgrass: Green blood can help to fight cancer. Journal of Clinical and Diagnostic Research: JCDR. 2017;11(6):ZC40.
8. 8. Adams M, Jewell A. The use of complementary and alternative medicine by cancer patients. International Seminars in Surgical Oncology. 2007;4:10.

9. 9. Hassan N, Siddique MS. Wheat Grass (Triticum aestivum L.) Benefits Health in a Pandemic Scenario. Journal for Research in Applied Sciences and Biotechnology. 2022;1(1):24-9.
10. 10. Patel JB. Anticancer & cytotoxic potential of aqueous extract of Triticum aestivum on hela cell line. Journal of Drug Delivery and Therapeutics. 2016;6(3):84-9.
11. 11. Kaur N, Singh B, Kaur A, Yadav MP, Singh N, Ahlawat AK, et al. Effect of growing conditions on proximate, mineral, amino acid, phenolic composition and antioxidant properties of wheatgrass from different wheat (Triticum aestivum L.) varieties. Food Chemistry. 2021;341:128201.
12. 12. Garg M, Sharma A, Vats S, Tiwari V, Kumari A, Mishra V, et al. Vitamins in cereals: a critical review of content, health effects, processing losses, bioaccessibility, fortification, and biofortification strategies for their improvement. Frontiers in nutrition. 2021;8:254.
13. 13. Caponio F, Alloggio V, Gomes T. Phenolic compounds of virgin olive oil: influence of paste preparation techniques. Food Chemistry. 1999;64(2):203-9.
14. 14. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental cell research. 1988;175(1):184-91.
15. 15. Molaae N, Mosayebi G, Pishdadian A, Ejtehadifar M, Ganji A. Evaluating the proliferation of human PeripheralBlood mononuclear cells using MTT assay. International Journal of Basic Science in Medicine. 2017;2(1):25-8.
16. 16. Alitheen NB, Oon CL, Keong YS, Chuan TK, Li HK, Yong HW. Cytotoxic effects of commercial wheatgrass and fiber towards human acute promyelocytic leukemia cells (HL60). Pakistan Journal of Pharmaceutical Sciences. 2011;24(3): 243-250
17. 17. Ghumman A, Singh N, Kaur A. Chemical, nutritional and phenolic composition of wheatgrass and pulse shoots. International journal of food science & technology. 2017;52(10):2191-200.
18. 18. Rodríguez FC, Gallagher E, Rai DK, Burgess CM. Nutritional and physiochemical properties of wheatgrass juice and preservation strategies. Food Chemistry Advances. 2022:100136.
19. 19. Choi M-H, Lee MY, Yang S-H, Shin H-J, Jeon YJ. Hydrophobic fractions of Triticum aestivum L. extracts contain polyphenols and alleviate inflammation by regulating nuclear factor-kappa B. Biotechnology and Bioprocess Engineering. 2021;26(1):93-106.
20. 20. Rosa L, Moreno-Escamilla J, Rodrigo-García J, Alvarez-Parrilla E. Phenolic compounds. Postharvest physiology and biochemistry of fruits and vegetables. 2019:253-71.
21. 21. Eissa HA, Mohamed SS, Hussein A. Nutritional value and impact of wheatgrass juice (Green Blood Therapy) on increasing fertility in male albino rats. Bulletin of the National Research Centre. 2020;44(1):1-11.
22. 22. Hebbani AV, Bulle S, Kanu VR, Balachandrababu Malini A, Reddy VD, Chakravarthula VN. Nephro-protective activity of wheatgrass juice against alcohol-induced oxidative damage in rats. Toxicology Mechanisms and Methods. 2020;30(9):679-86.
23. 23. Skoczylas Ł, Korus A, Tabaszewska M, Gędoś K, Szczepańska E. Evaluation of the quality of fresh and frozen wheatgrass juices depending on the time of grass harvest. Journal of Food Processing and Preservation. 2018;42(1):e13401.
24. 24. Thakur N, Dhaliwal HS, Sharma V. Qualitative and Quantitative RP-HPLC-PDA Method of Analysis of Polyphenols in Lyophilized Wheat Seedling Juice Powder. International Journal on Emerging Technologies 11(2): 36-43
25. 25. Shakya G, Balasubramanian S, Rajagopalan R. Methanol extract of wheatgrass induces G1 cell cycle arrest in a p53-dependent manner and down regulates the expression of cyclin D1 in human laryngeal cancer cells-an in vitro and in silico approach. Pharmacognosy Magazine. 2015;11(42):139.
26. 26. Fjällskog MLH, Granberg DP, Welin SL, Eriksson C, Öberg KE, Janson ET, et al. Treatment with cisplatin and etoposide in patients with neuroendocrine tumors. Cancer: Interdisciplinary International Journal of the American Cancer Society. 2001;92(5):1101-7.
27. 27. Maranchie JK. Silencing of Nox4 enhances cisplatin chemosensitivity of renal cell carcinoma. The Journal of Urology. 2008;179(4S):37-37.
28. 28. Mirmalek SA, Azizi MA, Jangholi E, Yadollah-Damavandi S, Javidi MA, Parsa Y, et al. Cytotoxic and apoptogenic effect of hypericin, the bioactive component of Hypericum perforatum on the MCF-7 human breast cancer cell line. Cancer cell international. 2015;16(1):1-9.
29. 29. Gibb RK, Taylor DD, Wan T, O'Connor DM, Doering DL, Gerçel- Taylor Ç. Apoptosis as a measure of chemosensitivity to cisplatin and taxol therapy in ovarian cancer cell lines. Gynecologic oncology. 1997;65(1):13-22.
30. 30. Hosoyamada M, Obinata M, Suzuki M, Endou H. Cisplatin-induced toxicity in immortalized renal cell lines established from transgenic mice harboring temperature sensitive SV40 large T-antigen gene. Archives of toxicology. 1996;70(5):284-92.
31. 31. Hazman Ö, Evin H, Bozkurt MF, Ciğerci İH. Hazman, Ömer, et al. "Two faces of arbutin in hepatocellular carcinoma (HepG2) cells: Anticarcinogenic effect in high concentration and protective effect against cisplatin toxicity through its antioxidant and anti-inflammatory activity in low concentration. Biologia. 2022; 77 :225–239
32. 32. Shu X, Fan C, Long B, Zhou X, Wang Y. The anti-cancer effects of cisplatin on hepatic cancer are associated with modulation of miRNA-21 and miRNA-122 expression. Eur Rev Med Pharmacol Sci. 2016;20(21):4459-65.
33. 33. Parihar A, Parihar MS, Ghafourifar P. Significance of mitochondrial calcium and nitric oxide for apoptosis of human breast cancer cells induced by tamoxifen and etoposide. International journal of molecular medicine. 2008;21(3):317-24.
34. 34. Ardeshiry LA, Rezaie TM, Mortazavi SA, Barzegar M, Moghadamnia SH, Rezaee MB. Study of anti cancer property of Scrophularia striata extract on the human astrocytoma cell line (1321). 2010; 9 (4): 403-410.
35. 35. Mozaffari F, Lindemalm C, Choudhury A, Granstam-Björneklett H, Helander I, Lekander M, et al. NK-cell and T-cell functions in patients with breast cancer: effects of surgery and adjuvant chemo-and radiotherapy. British journal of cancer. 2007;97(1):105-11.
36. 36. Wijayahadi N, Haron M, Stanslas J, Yusuf Z. Changes in cellular immunity during chemotherapy for primary breast cancer with anthracycline regimens. Journal of chemotherapy. 2007;19(6):716-23.
37. 37. Murta EFC, de Andrade JM, Falcio RP, Bighetti S. Lymphocyte subpopulations in patients with advanced breast cancer submitted to neoadjuvant chemotherapy. Tumori Journal. 2000;86(5):403-7.
38. 38. Sabbioni ME, Bernhard J, Siegrist H-P, Schmitz S-FH, Gertsch MC, Thürlimann B, et al. Does subjective burden of early breast cancer and its treatment affect immune measures during adjuvant therapy? Breast cancer research and treatment. 2004;87(1):75-86.
39. 39. Onyema OO, Decoster L, Njemini R, Forti LN, Bautmans I, De Waele M, et al. Chemotherapy-induced changes and immunosenescence of CD8+ T-cells in patients with breast cancer. Anticancer research. 2015;35(3):1481-9.
40. 40. Kumar A, Fillmore HL, Kadian R, Broaddus WC, Tye GW, Van Meter TE. The Alkylphospholipid Perifosine Induces Apoptosis and p21-Mediated Cell Cycle Arrest in MedulloblastomaPerifosine Induces Apoptosis and Mitotic Arrest. Molecular Cancer Research. 2009;7(11):1813-21.
41. 41. Suman G, Jamil K. Application of human lymphocytes for evaluating toxicity of anti-cancer drugs. Int J Pharmacol. 2006;2(4):374-81.
42. 42. Atha DH, Coskun E, Erdem O, Tona A, Reipa V, Nelson BC. Genotoxic effects of etoposide, bleomycin, and ethyl methanesulfonate on cultured CHO cells: Analysis by GC-MS/MS and comet assay. Journal of nucleic acids. 2020;2020:1-10