DISRUPTING FIBROGENIC PATHWAYS: INSILICO AND INVITRO EVALUATION OF KAEMPFEROL AND COUMESTROL AS POTENTIAL ANTI-FIBROTIC AGENTS
Main Article Content
Keywords
Coumestrol, Euphorbia hirta, Fibrogenic Pathways, Kaempferol , Lepidium sativum, Liver Fibrosis
Abstract
Background: Liver fibrosis is caused by chronic liver injury which is a reversible process of excessive extracellular matrix deposition. The continuous activation of hepatic stellate cells progresses to fibrosis, resulting in cirrhosis and hepatocellular carcinoma. Conventional treatment focuses only on symptoms, but natural phytochemicals as antifibrotic drugs offer a prospective option for novel therapeutic techniques that target fibrogenic pathways.
Objective: To explore the hepatoprotective potential of selected phytochemicals using computational and experimental approaches involving fibrogenic pathways.
Methodology: Bioactive compounds of medicinal plants Euphorbia hirta (whole plant) and Lepidium sativum (seeds) were studied. The 69 phytochemicals were molecular docked against IL6, AKT1, EGFR, and CASP3 proteins. Safety was assessed by ADMET profiling, while cytotoxicity, migration, invasion, and gene expression effects of Kaempferol and Coumestrol were assessed by in vitro assays. Statistical analysis was conducted using SPSS software.
Results: Kaempferol and Coumestrol were chosen from 69 phytochemicals based on high binding energies and atomic interactions with proteins. Coumestrol showed superior anti-invasive properties, and higher binding affinities with AKT1 (-6.4 kcal/mol), EGFR (-7.1 kcal/mol), and CASP3 (-7.1 kcal/mol) when compared to Kaempferol, plus no AMES toxicity and exceeded total clearance (8.085) of Kaempferol (6.868). Kaempferol exhibited greater cytotoxicity, colony-building inhibition, and stronger inhibition of cancer cell viability (38.8%) compared to Coumestrol (53.6%). Both bioactive compounds downregulated EGFR, AKT1, and IL6 while upregulating Caspase-3 expression, to efficiently target fibrogenic pathways.
Conclusion: The study concluded that Euphorbia hirta (whole plant) and Lepidium sativum (seeds) have the potential for hepatoprotection for liver fibrosis.
References
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31. Hussain S, Mustafa G, Ahmed S, Albeshr MF. Underlying mechanisms of Bergenia spp. to treat hepatocellular carcinoma using an integrated network pharmacology and molecular docking approach. Pharmaceuticals. 2023;16(9):1239.
32. Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports. 2017;7(1):42717.
33. Pires DE, Blundell TL, Ascher DB. pkCSM: predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. Journal Of Medicinal Chemistry. 2015;58(9):4066-72.
34. Azeem M, Mustafa G, Mahrosh HS. Virtual screening of phytochemicals by targeting multiple proteins of severe acute respiratory syndrome coronavirus 2: Molecular docking and molecular dynamics simulation studies. International Journal Of Immunopathology And Pharmacology. 2022;36:03946320221142793.
35. Haritha M, Sreerag M, Suresh CH. Quantifying the hydrogen-bond propensity of drugs and its relationship with Lipinski's rule of five. New Journal of Chemistry. 2024;48(11):4896-908.
36. Zhang Q, Chen L, Gao M, Wang S, Meng L, Guo L. Molecular docking and in vitro experiments verified that kaempferol induced apoptosis and inhibited human HepG2 cell proliferation by targeting BAX, CDK1, and JUN. Molecular and Cellular Biochemistry. 2023;478(4):767-80.
37. Yang G, Xing J, Aikemu B, Sun J, Zheng M. Kaempferol exhibits a synergistic effect with doxorubicin to inhibit proliferation, migration, and invasion of liver cancer. Oncology Reports. 2021;45(4):1-10.
38. Ju PC, Ho YC, Chen PN, Lee HL, Lai SY, Yang SF, et al. Kaempferol inhibits the cell migration of human hepatocellular carcinoma cells by suppressing MMP‐9 and Akt signaling. Environmental Toxicology. 2021;36(10):1981-9.
39. Alyami NM, Alyami HM, Almeer R. Using green biosynthesized kaempferol-coated sliver nanoparticles to inhibit cancer cells growth: An in vitro study using hepatocellular carcinoma (HepG2). Cancer Nanotechnology. 2022;13(1):26.
40. Pacheco I, Abril N, Zafra R, Morales-Prieto N, Hernández VM, Ruiz M, et al. Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica. Scientific Reports. 2019;9(1):1485.
41. Wang C-Z, Calway T, Yuan C-S. Herbal medicines as adjuvants for cancer therapeutics. The American journal of Chinese medicine. 2012;40(04):657-69.
42. Abid F, Saleem M, Leghari T, Rafi I, Maqbool T, Fatima F, et al. Evaluation of in vitro anticancer potential of pharmacological ethanolic plant extracts Acacia modesta and Opuntia monocantha against liver cancer cells. Brazilian Journal of Biology. 2022;84:e252526.
43. Wang J, Wu Z, Chen X, Sun Y, Ma S, Weng J, et al. Network Pharmacology, Molecular Docking Analysis and Molecular Dynamics Simulation of Scutellaria baicalensis in the Treatment of Liver Fibrosis. Current Pharmaceutical Design. 2024;30(17):1326-40.
44. Liu S, Liu X, Jiang S, Fu M, Hu J, Liu J, et al. Protective mechanism of Paeoniae Radix Alba against chemical liver injury based on network pharmacology, molecular docking, and in vitro experiments. Journal of Traditional Chinese Medical Sciences. 2024;11(1):55-66.
45. Raju C, Sankaranarayanan K, editors. In-Silico Investigation of Phyllanthus niruri Phytochemicals as Hepatic Fibrosis Modulators. Biology and Life Sciences Forum; 2025: MDPI.
46. Sharma V, Joseph C, Ghosh S, Agarwal A, Mishra MK, Sen E. Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Molecular Cancer Therapeutics. 2007;6(9):2544-53.
47. Hu B, Sun M, Liu J, Hong G, Lin Q. The preventative effect of Akt knockout on liver cancer through modulating NF-κB-regulated inflammation and Bad-related apoptosis signaling pathway. International Journal Of Oncology. 2016;48(4):1467-76.
48. Yang H, Wang J, Fan J-h, Zhang Y-q, Zhao J-x, Dai X-j, et al. Ilexgenin A exerts anti-inflammation and anti-angiogenesis effects through inhibition of STAT3 and PI3K pathways and exhibits synergistic effects with Sorafenib on hepatoma growth. Toxicology and Applied Pharmacology. 2017;315:90-101.
49. Zhang J, Chen Y, Lin J, Jia R, An T, Dong T, et al. Cyclovirobuxine D exerts anticancer effects by suppressing the EGFR-FAK-AKT/ERK1/2-slug signaling pathway in human hepatocellular carcinoma. DNA and Cell Biology. 2020;39(3):355-67.
2. Subramoniam A, Pushpangadan P. Development of phytomedicines for liver diseases. Indian Journal of Pharmacology. 1999;31(3):166-75.
3. Mustafa ME, Mansoor MM, Mohammed A, Babker AAA. Evaluation of platelets count and coagulation parameters among patients with liver disease. World Journal of Pharmaceutical Research. 2015;4(10):360-8.
4. Bataller R, Brenner DA. Liver fibrosis. The Journal of Clinical Investigation. 2005;115(2):209-18.
5. Sun C, Hu J-J, Pan Q, Cao Y, Fan J-G, Li G-M. Hepatic differentiation of rat induced pluripotent stem cells in vitro. World Journal of Gastroenterology: WJG. 2015;21(39):11118.
6. Cavalli M, Pan G, Nord H, Wallén Arzt E, Wallerman O, Wadelius C. Genetic prevention of hepatitis C virus‐induced liver fibrosis by allele‐specific downregulation of MERTK. Hepatology Research. 2017;47(8):826-30.
7. Li X, Jin Q, Xu H, Zhang Z, Zhou H, Yan D, et al. Chronic hepatitis B patients with high liver fibrosis levels should receive antiviral treatment. Experimental and Therapeutic Medicine. 2017;13(6):3624-30.
8. Yuan X, Duan S-Z, Cao J, Gao N, Xu J, Zhang L. Noninvasive inflammatory markers for assessing liver fibrosis stage in autoimmune hepatitis patients. European Journal Of Gastroenterology & Hepatology. 2019;31(11):1467-74.
9. Zhang A, Sun H, Wang X. Recent advances in natural products from plants for treatment of liver diseases. European Journal Of Medicinal Chemistry. 2013;63:570-7.
10. Lim Y-S, Kim WR. The global impact of hepatic fibrosis and end-stage liver disease. Clinics In Liver Disease. 2008;12(4):733-46.
11. Seki E, Schwabe RF. Hepatic inflammation and fibrosis: functional links and key pathways. Hepatology. 2015;61(3):1066-79.
12. Mann J, Mann DA. Transcriptional regulation of hepatic stellate cells. Advanced Drug Delivery Reviews. 2009;61(7-8):497-512.
13. Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annual Review Of Pathology: Mechanisms Of Disease. 2011;6(1):425-56.
14. Ellis EL, Mann DA. Clinical evidence for the regression of liver fibrosis. Journal Of Hepatology. 2012;56(5):1171-80.
15. Czaja AJ. Hepatic inflammation and progressive liver fibrosis in chronic liver disease. World Journal Of Gastroenterology: WJG. 2014;20(10):2515.
16. Calvaruso V, Craxì A. Regression of fibrosis after HBV antiviral therapy. Is cirrhosis reversible? Liver International. 2014;34:85-90.
17. Fallowfield JA. Therapeutic targets in liver fibrosis. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2011;300(5):G709-G15.
18. Glass LM, Dickson RC, Anderson JC, Suriawinata AA, Putra J, Berk BS, et al. Total body weight loss of≥ 10% is associated with improved hepatic fibrosis in patients with nonalcoholic steatohepatitis. Digestive Diseases And Sciences. 2015;60:1024-30.
19. Yamada K, Mizukoshi E, Seike T, Horii R, Kitahara M, Sunagozaka H, et al. Light alcohol consumption has the potential to suppress hepatocellular injury and liver fibrosis in non-alcoholic fatty liver disease. PLoS One. 2018;13(1):e0191026.
20. Damiris K, Tafesh ZH, Pyrsopoulos N. Efficacy and safety of anti-hepatic fibrosis drugs. World Journal of Gastroenterology. 2020;26(41):6304.
21. Dutt R, Garg V, Khatri N, Madan AK. Phytochemicals in anticancer drug development. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 2019;19(2):172-83.
22. Pal SK, Shukla Y. Herbal medicine: current status and the future. Asian Pacific Journal Of Cancer Prevention. 2003;4(4):281-8.
23. Koehn FE, Carter GT. The evolving role of natural products in drug discovery. Nature Reviews Drug Discovery. 2005;4(3):206-20.
24. Saklani A, Kutty SK. Plant-derived compounds in clinical trials. Drug Discovery Today. 2008;13(3-4):161-71.
25. Ghorani-Azam A, Sepahi S, Riahi-Zanjani B, Ghamsari AA, Mohajeri SA, Balali-Mood M. Plant toxins and acute medicinal plant poisoning in children: A systematic literature review. Journal Of Research In Medical Sciences. 2018;23(1):26.
26. Ru J, Li P, Wang J, Zhou W, Li B, Huang C, et al. TCMSP: a database of systems pharmacology for drug discovery from herbal medicines. Journal Of Cheminformatics. 2014;6:1-6.
27. Mohanraj K, Karthikeyan BS, Vivek-Ananth R, Chand RB, Aparna S, Mangalapandi P, et al. IMPPAT: A curated database of Indian Medicinal Plants, Phytochemistry and Therapeutics. Scientific Reports. 2018;8(1):4329.
28. Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, et al. PubChem 2019 update: improved access to chemical data. Nucleic Acids Research. 2019;47(D1):D1102-D9.
29. Althagafi I, El-Metwaly N, Farghaly TA. New series of thiazole derivatives: synthesis, structural elucidation, antimicrobial activity, molecular modeling and MOE docking. Molecules. 2019;24(9):1741.
30. Dhorajiwala TM, Halder ST, Samant L. Comparative in silico molecular docking analysis of l-threonine-3-dehydrogenase, a protein target against African trypanosomiasis using selected phytochemicals. Journal of Applied Biotechnology Reports. 2019;6(3):101-8.
31. Hussain S, Mustafa G, Ahmed S, Albeshr MF. Underlying mechanisms of Bergenia spp. to treat hepatocellular carcinoma using an integrated network pharmacology and molecular docking approach. Pharmaceuticals. 2023;16(9):1239.
32. Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports. 2017;7(1):42717.
33. Pires DE, Blundell TL, Ascher DB. pkCSM: predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. Journal Of Medicinal Chemistry. 2015;58(9):4066-72.
34. Azeem M, Mustafa G, Mahrosh HS. Virtual screening of phytochemicals by targeting multiple proteins of severe acute respiratory syndrome coronavirus 2: Molecular docking and molecular dynamics simulation studies. International Journal Of Immunopathology And Pharmacology. 2022;36:03946320221142793.
35. Haritha M, Sreerag M, Suresh CH. Quantifying the hydrogen-bond propensity of drugs and its relationship with Lipinski's rule of five. New Journal of Chemistry. 2024;48(11):4896-908.
36. Zhang Q, Chen L, Gao M, Wang S, Meng L, Guo L. Molecular docking and in vitro experiments verified that kaempferol induced apoptosis and inhibited human HepG2 cell proliferation by targeting BAX, CDK1, and JUN. Molecular and Cellular Biochemistry. 2023;478(4):767-80.
37. Yang G, Xing J, Aikemu B, Sun J, Zheng M. Kaempferol exhibits a synergistic effect with doxorubicin to inhibit proliferation, migration, and invasion of liver cancer. Oncology Reports. 2021;45(4):1-10.
38. Ju PC, Ho YC, Chen PN, Lee HL, Lai SY, Yang SF, et al. Kaempferol inhibits the cell migration of human hepatocellular carcinoma cells by suppressing MMP‐9 and Akt signaling. Environmental Toxicology. 2021;36(10):1981-9.
39. Alyami NM, Alyami HM, Almeer R. Using green biosynthesized kaempferol-coated sliver nanoparticles to inhibit cancer cells growth: An in vitro study using hepatocellular carcinoma (HepG2). Cancer Nanotechnology. 2022;13(1):26.
40. Pacheco I, Abril N, Zafra R, Morales-Prieto N, Hernández VM, Ruiz M, et al. Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica. Scientific Reports. 2019;9(1):1485.
41. Wang C-Z, Calway T, Yuan C-S. Herbal medicines as adjuvants for cancer therapeutics. The American journal of Chinese medicine. 2012;40(04):657-69.
42. Abid F, Saleem M, Leghari T, Rafi I, Maqbool T, Fatima F, et al. Evaluation of in vitro anticancer potential of pharmacological ethanolic plant extracts Acacia modesta and Opuntia monocantha against liver cancer cells. Brazilian Journal of Biology. 2022;84:e252526.
43. Wang J, Wu Z, Chen X, Sun Y, Ma S, Weng J, et al. Network Pharmacology, Molecular Docking Analysis and Molecular Dynamics Simulation of Scutellaria baicalensis in the Treatment of Liver Fibrosis. Current Pharmaceutical Design. 2024;30(17):1326-40.
44. Liu S, Liu X, Jiang S, Fu M, Hu J, Liu J, et al. Protective mechanism of Paeoniae Radix Alba against chemical liver injury based on network pharmacology, molecular docking, and in vitro experiments. Journal of Traditional Chinese Medical Sciences. 2024;11(1):55-66.
45. Raju C, Sankaranarayanan K, editors. In-Silico Investigation of Phyllanthus niruri Phytochemicals as Hepatic Fibrosis Modulators. Biology and Life Sciences Forum; 2025: MDPI.
46. Sharma V, Joseph C, Ghosh S, Agarwal A, Mishra MK, Sen E. Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Molecular Cancer Therapeutics. 2007;6(9):2544-53.
47. Hu B, Sun M, Liu J, Hong G, Lin Q. The preventative effect of Akt knockout on liver cancer through modulating NF-κB-regulated inflammation and Bad-related apoptosis signaling pathway. International Journal Of Oncology. 2016;48(4):1467-76.
48. Yang H, Wang J, Fan J-h, Zhang Y-q, Zhao J-x, Dai X-j, et al. Ilexgenin A exerts anti-inflammation and anti-angiogenesis effects through inhibition of STAT3 and PI3K pathways and exhibits synergistic effects with Sorafenib on hepatoma growth. Toxicology and Applied Pharmacology. 2017;315:90-101.
49. Zhang J, Chen Y, Lin J, Jia R, An T, Dong T, et al. Cyclovirobuxine D exerts anticancer effects by suppressing the EGFR-FAK-AKT/ERK1/2-slug signaling pathway in human hepatocellular carcinoma. DNA and Cell Biology. 2020;39(3):355-67.
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Jun 14, 2024
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Areeba Imtiaz, Syed Muhammad Ali Shah, & Dr Sultan Ayaz. (2024). DISRUPTING FIBROGENIC PATHWAYS: INSILICO AND INVITRO EVALUATION OF KAEMPFEROL AND COUMESTROL AS POTENTIAL ANTI-FIBROTIC AGENTS. Journal of Population Therapeutics and Clinical Pharmacology, 31(6), 3714-3729. https://doi.org/10.53555/5da3gn95
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Author Biographies
Areeba Imtiaz
PhD Scholar, Department of Eastern Medicine Government College University Faisalabad
Syed Muhammad Ali Shah
Assistant Professor, Department of Eastern Medicine Government College University Faisalabad
Dr Sultan Ayaz
Associate Professor & Chairperson Department of Eastern Medicine Government College University Faisalabad
How to Cite
Areeba Imtiaz, Syed Muhammad Ali Shah, & Dr Sultan Ayaz. (2024). DISRUPTING FIBROGENIC PATHWAYS: INSILICO AND INVITRO EVALUATION OF KAEMPFEROL AND COUMESTROL AS POTENTIAL ANTI-FIBROTIC AGENTS. Journal of Population Therapeutics and Clinical Pharmacology, 31(6), 3714-3729. https://doi.org/10.53555/5da3gn95