Mineral Pitch Attenuates Oxidative Stress-Induced Eryptosis in Human Erythrocytes via Antioxidants and Calcium-Modulatory Mechanism

Authors

  • Muhammad Najamul Hassan Department of Biochemistry, University of Agriculture, Faisalabad, Punjab, Pakistan.
  • Muhammad Rasheed Department of Biochemistry, University of Agriculture, Faisalabad, Punjab, Pakistan.
  • Namra Butt Department of Biochemistry, University of Agriculture, Faisalabad, Punjab, Pakistan.
  • Nida Anwar Department of Botany, University of Agriculture, Faisalabad, Punjab, Pakistan.
  • Ahmad Shahab Department of Biochemistry, University of Agriculture, Faisalabad, Punjab, Pakistan.
  • Maryam Ashiq Department of Biochemistry, University of Agriculture, Faisalabad, Punjab, Pakistan.
  • Wazir Hamid Ali Department of Biochemistry, Government College University Faisalabad, Punjab, Pakistan.
  • Kashif Jilani Department of Biochemistry, University of Agriculture, Faisalabad, Punjab, Pakistan.

DOI:

https://doi.org/10.70749/ijbr.v3i6.1690

Keywords:

Mineral Pitch, Eryptosis, Antioxidant, Oxidative Stress, Apoptosis

Abstract

Background: Erythrocytes are highly susceptible to oxidative stress, which can induce eryptosis programmed cell death mechanism characterized by hemolysis, membrane blebbing, and cell shrinkage. Mineral Pitch (Asphaltum punjabium), a traditional medicinal substance, has demonstrated antioxidant potential, but its effect on erythrocyte integrity under oxidative stress has not been thoroughly investigated. Objective: This study aimed to evaluate the antioxidant and cytoprotective effects of Mineral Pitch on human erythrocytes exposed to oxidative stress induced by hyperosmotic sucrose. Methods: Human erythrocytes were treated with sucrose (350–550 mM) to induce oxidative stress and then exposed to Mineral Pitch at concentrations of 0.5, 1.0, and 1.5 g/10 mL. Hemolysis percentage, antioxidant enzyme activities (SOD, GPx, and CAT), mean corpuscular volume (MCV), and calcium channel involvement were analyzed using standard biochemical assays and ELISA. Results: Sucrose elevated hemolysis from 0.16% to 0.24%, while Mineral Pitch significantly reduced it to 0.16% at 1.5 g/10 mL. GPx activity, which decreased to 213.8 U/gHb under stress, was restored to 335 U/gHb. SOD activity improved from 877 U/gHb to 794 U/gHb, and CAT activity increased from 34.6 U/gHb to 44 U/gHb following treatment. MCV dropped to 68 fL with sucrose but was preserved at 85 fL with Mineral Pitch. Furthermore, the use of amlodipine confirmed a calcium-dependent mechanism in eryptosis, modulated by Mineral Pitch. Conclusion: Mineral Pitch demonstrates potent antioxidant and cytoprotective effects on erythrocytes under oxidative stress. It stabilizes cell membranes, restores antioxidant defenses, and regulates calcium-mediated eryptosis. These findings suggest its potential as a natural therapeutic agent against oxidative hematological damage.

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References

Lang, F., Lang, E., & Föller, M. (2012). Physiology and pathophysiology of Eryptosis. Transfusion Medicine and Hemotherapy, 39(5), 308-314.

https://doi.org/10.1159/000342534

Klinken, S. P. (2002). Red blood cells. The International Journal of Biochemistry & Cell Biology, 34(12), 1513-1518.

https://doi.org/10.1016/s1357-2725(02)00087-0

Hamasaki, N., & Yamamoto, M. (2000). Red blood cell function and blood storage. Vox Sanguinis, 79(4), 191-197.

https://doi.org/10.1159/000056729

Doulatov, S., Notta, F., Laurenti, E., & Dick, J. (2012). Hematopoiesis: A human perspective. Cell Stem Cell, 10(2), 120-136.

https://doi.org/10.1016/j.stem.2012.01.006

Palis, J. (2014). Primitive and definitive erythropoiesis in mammals. Frontiers in Physiology, 5.

https://doi.org/10.3389/fphys.2014.00003

Bhavsar, S. K., Hosseinzadeh, Z., Brenner, D., Honisch, S., Jilani, K., Liu, G., Szteyn, K., Sopjani, M., Mak, T. W., Shumilina, E., & Lang, F. (2014). Energy-sensitive regulation of Na+/K+-ATPase by Janus kinase 2. American Journal of Physiology-Cell Physiology, 306(4), C374-C384.

https://doi.org/10.1152/ajpcell.00320.2013

Hamidi, M., & Tajerzadeh, H. (2003). Carrier erythrocytes: An overview. Drug Delivery, 10(1), 9-20.

https://doi.org/10.1080/713840329

Andolfo, I., Russo, R., Gambale, A., & Iolascon, A. (2016). New insights on hereditary erythrocyte membrane defects. Haematologica, 101(11), 1284-1294.

https://doi.org/10.3324/haematol.2016.142463

Helms, C. C., Gladwin, M. T., & Kim-Shapiro, D. B. (2018). Erythrocytes and vascular function: Oxygen and nitric oxide. Frontiers in Physiology, 9.

https://doi.org/10.3389/fphys.2018.00125

Thiagarajan, P., Parker, C. J., & Prchal, J. T. (2021). How do red blood cells die? Frontiers in Physiology, 12.

https://doi.org/10.3389/fphys.2021.655393

Wu, H., Liu, X., Jaenisch, R., & Lodish, H. F. (1995). Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell, 83(1), 59-67.

https://doi.org/10.1016/0092-8674(95)90234-1

Valent, P., Büsche, G., Theurl, I., Uras, I. Z., Germing, U., Stauder, R., Sotlar, K., Füreder, W., Bettelheim, P., Pfeilstöcker, M., Oberbauer, R., Sperr, W. R., Geissler, K., Schwaller, J., Moriggl, R., Béné, M. C., Jäger, U., Horny, H., & Hermine, O. (2018). Normal and pathological erythropoiesis in adults: From gene regulation to targeted treatment concepts. Haematologica, 103(10), 1593-1603.

https://doi.org/10.3324/haematol.2018.192518

Wang, J., Wagner-Britz, L., Bogdanova, A., Ruppenthal, S., Wiesen, K., Kaiser, E., Tian, Q., Krause, E., Bernhardt, I., Lipp, P., Philipp, S. E., & Kaestner, L. (2013). Morphologically homogeneous red blood cells present a heterogeneous response to hormonal stimulation. PLoS ONE, 8(6), e67697.

https://doi.org/10.1371/journal.pone.0067697

Fowler, V. M. (2013). The human erythrocyte plasma membrane. Current Topics in Membranes, 39-88.

https://doi.org/10.1016/b978-0-12-417027-8.00002-7

Li, H., & Lykotrafitis, G. (2014). Erythrocyte membrane model with explicit description of the lipid Bilayer and the Spectrin network. Biophysical Journal, 107(3), 642-653.

https://doi.org/10.1016/j.bpj.2014.06.031

Mischitelli, M., Jemaàa, M., Fezai, M., Almasry, M., Lang, F., & Faggio, C. (2017). Stimulation of erythrocyte cell membrane scrambling by Adarotene. Cellular Physiology and Biochemistry, 41(2), 519-529.

https://doi.org/10.1159/000456942

Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., Squadrito, F., Altavilla, D., & Bitto, A. (2017). Oxidative stress: Harms and benefits for human health. Oxidative Medicine and Cellular Longevity, 2017(1).

https://doi.org/10.1155/2017/8416763

Aslam, H. M., Sohail, A., Shahid, A., Khan, M. A., Muhammad Umar Sharif, Kausar, R., Nawab, S., Farooq, W., Jilani, K., & Rasheed, M. (2024). Levofloxacin induces erythrocyte contraction leading to red cell death. Drug Target Insights, 18(1), 78-83.

https://doi.org/10.33393/dti.2024.3060

Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., Squadrito, F., Altavilla, D., & Bitto, A. (2017). Oxidative stress: Harms and benefits for human health. Oxidative Medicine and Cellular Longevity, 2017(1).

https://doi.org/10.1155/2017/8416763

Seifried, H. E., Anderson, D. E., Fisher, E. I., & Milner, J. A. (2007). A review of the interaction among dietary antioxidants and reactive oxygen species. The Journal of Nutritional Biochemistry, 18(9), 567-579.

https://doi.org/10.1016/j.jnutbio.2006.10.007

Cadenas, E., & Davies, K. J. (2000). Mitochondrial free radical generation, oxidative stress, and aging11This article is dedicated to the memory of our dear friend, colleague, and mentor Lars Ernster (1920–1998), in gratitude for all he gave to us. Free Radical Biology and Medicine, 29(3-4), 222-230.

https://doi.org/10.1016/s0891-5849(00)00317-8

Tu, B. P., & Weissman, J. S. (2004). Oxidative protein folding in eukaryotes. The Journal of Cell Biology, 164(3), 341-346.

https://doi.org/10.1083/jcb.200311055

Sikandar, M., Bari Khan, M. A., Jilani, K., & Xing, L. (2025). Effects of fluconazole on oxidative stress and cellular well-being in erythrocytes. Biochemical and Biophysical Research Communications, 770, 152006.

https://doi.org/10.1016/j.bbrc.2025.152006

Halliwell, B. (2007). Biochemistry of oxidative stress. Biochemical society transactions, 35(5), 1147-1150.

Pham-Huy, L. A., He, H., & Pham-Huy, C. (2008). Free radicals, antioxidants in disease and health. International journal of biomedical science: IJBS, 4(2), 89.

https://pmc.ncbi.nlm.nih.gov/articles/PMC3614697/

Wilcox, C. S., & Gutterman, D. (2005). Focus on oxidative stress in the cardiovascular and renal systems. American Journal of Physiology-Heart and Circulatory Physiology, 288(1), H3-H6.

https://doi.org/10.1152/ajpheart.00854.2004

Naveed, A., Jilani, K., Siddique, A. B., Akbar, M., Riaz, M., Mushtaq, Z., Sikandar, M., Ilyas, S., Bibi, I., Asghar, A., Rasool, G., & Irfan, M. (2020). Induction of erythrocyte shrinkage by omeprazole. Dose-Response, 18(3).

https://doi.org/10.1177/1559325820946941

Betteridge, D. J. (2000). What is oxidative stress? Metabolism, 49(2), 3-8.

https://doi.org/10.1016/s0026-0495(00)80077-3

Tsutsui, H., Kinugawa, S., & Matsushima, S. (2011). Oxidative stress and heart failure. American Journal of Physiology-Heart and Circulatory Physiology, 301(6), H2181-H2190.

https://doi.org/10.1152/ajpheart.00554.2011

Gulcin, İ. (2020). Antioxidants and antioxidant methods: An updated overview. Archives of Toxicology, 94(3), 651-715.

https://doi.org/10.1007/s00204-020-02689-3

Zbidah, M., Lupescu, A., Jilani, K., Fajol, A., Michael, D., Qadri, S. M., & Lang, F. (2011). Apigenin-induced suicidal erythrocyte death. Journal of Agricultural and Food Chemistry, 60(1), 533-538.

https://doi.org/10.1021/jf204107f

Roy, A. E., & Sreeni, T. V. (2022). Conceptual review on Shilajathu - An ayurvedic perspective. International Journal of Ayurveda and Pharma Research, 19-25.

https://doi.org/10.47070/ijapr.v10i7.2416

Cagno, V., Donalisio, M., Civra, A., Cagliero, C., Rubiolo, P., & Lembo, D. (2015). In vitro evaluation of the antiviral properties of Shilajit and investigation of its mechanisms of action. Journal of Ethnopharmacology, 166, 129-134.

https://doi.org/10.1016/j.jep.2015.03.019

Carrasco-Gallardo, C., Guzmán, L., & Maccioni, R. B. (2012). Shilajit: A natural Phytocomplex with potential Procognitive activity. International Journal of Alzheimer's Disease, 2012, 1-4.

https://doi.org/10.1155/2012/674142

Stohs, S. J. (2013). Safety and efficacy of Shilajit (Mumie, Moomiyo). Phytotherapy Research, 28(4), 475-479.

https://doi.org/10.1002/ptr.5018

Jafari, M., Forootanfar, H., Ameri, A., Foroutanfar, A., Adeli-Sardou, M., Rahimi, H. R., ... & Shakibaie, M. (2019). Antioxidant, cytotoxic and hyperalgesia-suppressing activity of a native Shilajit obtained from Bahr Aseman mountains. Pakistan Journal of Pharmaceutical Sciences, 32(5).

Jambi, E. J., & Abdulaziz Alshubaily, F. (2022). Shilajit potentiates the effect of chemotherapeutic drugs and mitigates metastasis induced liver and kidney damages in osteosarcoma rats. Saudi Journal of Biological Sciences, 29(9), 103393.

https://doi.org/10.1016/j.sjbs.2022.103393

Hadi, S., Ahmed, S. H., Talib, N., Hussein, H. A., & Al-Karkhi, I. H. T. (2020). Alcoholic Extract of Shilajit as Anti Protein Denaturation, Anti Blood Hemolysis, and Anti Microbial. Indian Journal of Forensic Medicine & Toxicology, 14(1).

https://doi.org/10.37506/v14/i1/2020/ijfmt/192929

Curtin, J. F., Donovan, M., & Cotter, T. G. (2002). Regulation and measurement of oxidative stress in apoptosis. Journal of Immunological Methods, 265(1-2), 49-72.

https://doi.org/10.1016/s0022-1759(02)00070-4

Isler, M., Delibas, N., Guclu, M., Gultekin, F., Sutcu, R., Bahceci, M., & Kosar, A. (2002). Superoxide dismutase and glutathione peroxidase in erythrocytes of patients with iron deficiency anemia: effects of different treatment modalities. Croatian medical journal, 43(1), 16-19.

Peskin, A. V., & Winterbourn, C. C. (2017). Assay of superoxide dismutase activity in a plate assay using WST-1. Free Radical Biology and Medicine, 103, 188-191.

https://doi.org/10.1016/j.freeradbiomed.2016.12.033

Rana, S., Dixit, S., & Mittal, A. (2019). In Silico target identification and validation for antioxidant and anti-inflammatory activity of selective phytochemicals. Brazilian Archives of Biology and Technology, 62.

https://doi.org/10.1590/1678-4324-2019190048

Shabir, K., Jilani, K., Zbidah, M., Riaz, M., Bibi, I., & Asghar, A. (2019). Triggering of erythrocyte membrane blebbing by ciprofloxacin. Acta Poloniae Pharmaceutica - Drug Research, 76(5), 901-906.

https://doi.org/10.32383/appdr/110772

Ilyas, S., Srivastava, R. R., & Kim, H. (2020). Disinfection technology and strategies for COVID-19 hospital and bio-medical waste management. Science of The Total Environment, 749, 141652.

https://doi.org/10.1016/j.scitotenv.2020.141652

Chen, L. H., & Thacker, R. R. (1984). An increase in glutathione peroxidase activity induced by high supplementation of vitamin C in rats. Nutrition Research, 4(4), 657-664.

https://doi.org/10.1016/s0271-5317(84)80039-1

Phaechamud, T., Charoenteeraboon, J., Wetwitayaklung, P., Limmatvapirat, C., & Srichan, T. (2008). Some biological activities and safety of mineral pitch. Science, Engineering and Health Studies, 7-17.

https://doi.org/10.14456/sustj.2008.6

Pant, K., Gupta, P., Damania, P., Yadav, A. K., Gupta, A., Ashraf, A., & Venugopal, S. K. (2016). Mineral pitch induces apoptosis and inhibits proliferation via modulating reactive oxygen species in hepatic cancer cells. BMC Complementary and Alternative Medicine, 16(1).

https://doi.org/10.1186/s12906-016-1131-z

Zhang, X., & Hintze, T. H. (1998). Amlodipine releases nitric oxide from canine coronary Microvessels. Circulation, 97(6), 576-580.

https://doi.org/10.1161/01.cir.97.6.576

Jilani, K., Enkel, S., Bissinger, R., Almilaji, A., Abed, M., & Lang, F. (2013). Fluoxetine induced suicidal erythrocyte death. Toxins, 5(7), 1230-1243.

https://doi.org/10.3390/toxins5071230

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Published

2025-06-30

How to Cite

Hassan, M. N., Rasheed, M., Butt, N., Anwar, N., Shahab, A., Ashiq, M., Ali, W. H., & Jilani, K. (2025). Mineral Pitch Attenuates Oxidative Stress-Induced Eryptosis in Human Erythrocytes via Antioxidants and Calcium-Modulatory Mechanism. Indus Journal of Bioscience Research, 3(6), 520-529. https://doi.org/10.70749/ijbr.v3i6.1690