Silybum marianum and Trigonella foenum-graecum Exhibit Moderate In Vitro Antimalarial Properties against Plasmodium knowlesi
-
Norsyafiqah binti Ahmad Safri
,
Fei Wen Cheong
,
Nur Zulaikha Zulkefli
,
Abolghasem Siyadatpanah
,
Roghayeh Norouzi
,
Yee Ling Lau
Abstract
Background: Malaria remains a major global health burden, with increasing challenges posed by drug-resistant Plasmodium species. Another emerging threat is Plasmodium knowlesi, a zoonotic parasite responsible for rising human infections in Southeast Asia, particularly Malaysia. This study investigates the in vitro antimalarial activity of Silybum marianum (milk thistle) and Trigonella foenum-graecum (fenugreek) seed extracts against P. knowlesi, and their cytotoxicity against mammalian cells.
Methods: Using schizont maturation inhibition assay, P. knowlesi cultures were exposed to serial dilutions of both plant hydroethanolic extracts (0.20–100.00 μg/mL). Chloroquine was used as positive control. Cytotoxicity was evaluated on Vero cells using the MTT assay.
Results: Both extracts exhibited moderate antimalarial activity, with IC₅₀ values of 16.12 ± 1.02 μg/mL (S. marianum) and 17.08 ± 1.26 μg/mL (T. foenum-graecum). Cytotoxicity testing showed that the extracts were non-toxic to mammalian cells, with CC₅₀ values of 84.76 ± 15.12 μg/mL (S. marianum) and 72.75 ± 15.81 μg/mL (T. foenum-graecum). The selectivity indices were 5.26 and 4.26, respectively, indicating moderate selectivity towards the parasite over mammalian cells.
Conclusion: Both S. marianum and T. foenum-graecum demonstrate promising antimalarial potential against P. knowlesi in vitro, with acceptable cytotoxicity profiles. These findings support further investigation into their bioactive constituents and mechanism of action, offering preliminary evidence for their potential development as alternative antimalarial agents targeting zoonotic malaria.
1. World Health Organization. 2026. World malaria report 2025. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2025. Accessed 4 June 2026.
2. White NJ. Plasmodium knowlesi: The Fifth Human Malaria Parasite. Clin Infect Dis. 2008; 46(2):172–3.
3. Jajosky RP, Wu SC, Jajosky PG, et al. Plasmodium knowlesi (Pk) Malaria: A Review & Proposal of Therapeutically Rational Exchange (T-REX) of Pk-Resistant Red Blood Cells. Trop Med Infect Dis. 2023; 8(10):478.
4. Trape JF. The public health impact of chloroquine resistance in Africa. Am J Trop Med Hyg. 2001; 64(1-2 Suppl):12-7.
5. Alam MT, Bora H, Bharti PK, et al. Similar trends of pyrimethamine resistance-associated mutations in Plasmodium vivax and P. falciparum. Antimicrob Agents Chemother. 2007; 51(3): 857-63.
6. Rogers WO, Sem R, Tero T, et al. Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia. Malar J. 2009; 8:10.
7. Balikagala B, Fukuda N, Ikeda M, et al. Evidence of Artemisinin-Resistant Malaria in Africa. N Engl J Med. 2021; 385(13):1163-71.
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9. Hsu E. Reflections on the ‘discovery’ of the antimalarial qinghao. Br J Clin Pharmacol. 2006; 61(6): 666–70.
10. de Avelar CR, Nunes BVC, da Silva Sassaki B, et al. Efficacy of silymarin in patients with non-alcoholic fatty liver disease - the Siliver trial: a study protocol for a randomized controlled clinical trial. Trials. 2023; 24(1):177.
11. Zhang X, Liu M, Wang Z, et al. A review of the botany, phytochemistry, pharmacology, synthetic biology and comprehensive utilization of Silybum marianum. Front Pharmacol. 2024; 15:1417655.
12. Visuvanathan T, Than LTL, Stanslas J, et al. Revisiting Trigonella foenum-graecum L.: Pharmacology and Therapeutic Potentialities. Plants (Basel). 2022; 11(11):1450.
13. Raghuram TC, Sharma RD, Sivakumar B, et al. Effect of fenugreek seeds on intravenous glucose disposition in non‐insulin dependent diabetic patients. Phytother Res. 1994; 8(2):83–6.
14. Palaniswamy M, Pradeep BV, Sathya R, et al. In Vitro Anti‐Plasmodial Activity of Trigonella foenum – graecum L. Evid Based Complement Alternat Med. 2010; 7(4):441–5.
15. Mina PR., Kumar Y, Verma AK, et al. Silymarin, a polyphenolic flavonoid impede Plasmodium falciparum growth through interaction with heme. Nat Prod Res. 2020; 34(18):2647–51.
16. van Schalkwyk DA, Moon RW, Duffey M, et al. Ex vivo susceptibility to new antimalarial agents differs among human infecting Plasmodium species. Int J Parasitol Drugs Drug Resist. 2021; 17:5–11.
17. Fatih FA, Staines HM, Siner A, et al. Susceptibility of human Plasmodium knowlesi infections to anti-malarials. Malar J. 2013; 12:425.
18. Moon RW, Hall J, Rangkuti F, et al. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci U S A. 2013; 110(2):531-6.
19. World Health Organization. Malaria Unit. 2001. In vitro micro-test (Mark III) for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine and artemisinin instructions for use of the in vitro micro-test kit (Mark III), 2nd rev.
20. Rosoanaivo P, Deharo E, Ratsimamanga‐Urverg S, et al. Guidelines for the nonclinical evaluation of the efficacy of traditional antimalarials. In: Merlin W, Gerald B, Philippe R, editors. Traditional Medicinal Plants and Malaria. USA: CRC Press; 2004. p. 256‐68.
21. Kane NF, Kyama MC, Nganga JK, et al. Comparison of phytochemical profiles and antimalarial activities of Artemisia afra plant collected from five countries in Africa. S Afr J Bot. 2019; 125:126–133.
22. García-Huertas P, Pabón A, Arias C, et al. Evaluation of cytotoxic effect and genetic damage of standardized extracts of Solanum nudum with antiplasmodial activity. Biomedica. 2013; 33(1):78–87.
23. de Souza GE., Bueno RV, de Souza JO, et al. Antiplasmodial profile of selected compounds from Malaria Box: in vitro evaluation, speed of action and drug combination studies. Malar J. 2019; 18:447.
24. Kumar S, Guha M, Choubey V, et al. Antimalarial drugs inhibiting hemozoin (beta-hematin) formation: a mechanistic update. Life Sci. 2007; 80(9):813–28.
25. Llurba Montesino N, Kaiser M, Brun R, et al. An approved preparation of Silybum marianum contains constituents with activity against Plasmodium falciparum. Planta Med. 2016; 81(S 01):S1–381.
26. Francis GC, Eze NC, Imafidor HO. Evaluation of antiplasmodial effects of black seeds, fenugreek and coriander seeds in albino mice infected with Plasmodium berghei. Sci Afr. 2024; 23(1):205–18.
27. Priya V, Jananie RK, Vijayalakshmi K. GC/MS determination of bioactive components of Trigonella foenum grecum. J Chem Pharm Res. 2011; 3(5):35–40.
28. Nawaz A, Riaz T, Ahmad A. In-vitro evaluation of antiproliferative potential of various fractions of Silybum marianum using HeLa and HepG2 cell lines. Pak J Pharm Sci. 2021; 34(2(Supplementary)):755–60.
29. Al-Oqail MM, Farshori NN, Al-Sheddi ES, et al. In vitro cytotoxic activity of seed oil of fenugreek against various cancer cell lines. Asian Pac J Cancer Prev. 2013; 14(3):1829–32.
30. Al-Timimi LAN. Antibacterial and Anticancer Activities of Fenugreek Seed Extract. Asian Pac J Cancer Prev. 2019; 20(12):3771–6.
31. Babaei AH, Motamedifar M, Khalifat S, et al. In vitro study of antibacterial property and cytotoxic effects of aqueous, ethanolic, methanolic, and hydroalcoholic extracts of fenugreek seed. Pak J Med Health Sci. 2018; 12:906–10.
32. Wright CW, Allen D, Cai Y, et al. Alkaloids from Aspidosperma pachypterum as potential antimalarial agents. Phytother Res. 1992; 6(3):121-6.
2. White NJ. Plasmodium knowlesi: The Fifth Human Malaria Parasite. Clin Infect Dis. 2008; 46(2):172–3.
3. Jajosky RP, Wu SC, Jajosky PG, et al. Plasmodium knowlesi (Pk) Malaria: A Review & Proposal of Therapeutically Rational Exchange (T-REX) of Pk-Resistant Red Blood Cells. Trop Med Infect Dis. 2023; 8(10):478.
4. Trape JF. The public health impact of chloroquine resistance in Africa. Am J Trop Med Hyg. 2001; 64(1-2 Suppl):12-7.
5. Alam MT, Bora H, Bharti PK, et al. Similar trends of pyrimethamine resistance-associated mutations in Plasmodium vivax and P. falciparum. Antimicrob Agents Chemother. 2007; 51(3): 857-63.
6. Rogers WO, Sem R, Tero T, et al. Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia. Malar J. 2009; 8:10.
7. Balikagala B, Fukuda N, Ikeda M, et al. Evidence of Artemisinin-Resistant Malaria in Africa. N Engl J Med. 2021; 385(13):1163-71.
8. World Health Organization. 2025. Malaria: Artemisinin partial resistance. https://www.who.int/news-room/questions-and-answers/item/artemisinin-resistance. Accessed 4 June 2026.
9. Hsu E. Reflections on the ‘discovery’ of the antimalarial qinghao. Br J Clin Pharmacol. 2006; 61(6): 666–70.
10. de Avelar CR, Nunes BVC, da Silva Sassaki B, et al. Efficacy of silymarin in patients with non-alcoholic fatty liver disease - the Siliver trial: a study protocol for a randomized controlled clinical trial. Trials. 2023; 24(1):177.
11. Zhang X, Liu M, Wang Z, et al. A review of the botany, phytochemistry, pharmacology, synthetic biology and comprehensive utilization of Silybum marianum. Front Pharmacol. 2024; 15:1417655.
12. Visuvanathan T, Than LTL, Stanslas J, et al. Revisiting Trigonella foenum-graecum L.: Pharmacology and Therapeutic Potentialities. Plants (Basel). 2022; 11(11):1450.
13. Raghuram TC, Sharma RD, Sivakumar B, et al. Effect of fenugreek seeds on intravenous glucose disposition in non‐insulin dependent diabetic patients. Phytother Res. 1994; 8(2):83–6.
14. Palaniswamy M, Pradeep BV, Sathya R, et al. In Vitro Anti‐Plasmodial Activity of Trigonella foenum – graecum L. Evid Based Complement Alternat Med. 2010; 7(4):441–5.
15. Mina PR., Kumar Y, Verma AK, et al. Silymarin, a polyphenolic flavonoid impede Plasmodium falciparum growth through interaction with heme. Nat Prod Res. 2020; 34(18):2647–51.
16. van Schalkwyk DA, Moon RW, Duffey M, et al. Ex vivo susceptibility to new antimalarial agents differs among human infecting Plasmodium species. Int J Parasitol Drugs Drug Resist. 2021; 17:5–11.
17. Fatih FA, Staines HM, Siner A, et al. Susceptibility of human Plasmodium knowlesi infections to anti-malarials. Malar J. 2013; 12:425.
18. Moon RW, Hall J, Rangkuti F, et al. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci U S A. 2013; 110(2):531-6.
19. World Health Organization. Malaria Unit. 2001. In vitro micro-test (Mark III) for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine and artemisinin instructions for use of the in vitro micro-test kit (Mark III), 2nd rev.
20. Rosoanaivo P, Deharo E, Ratsimamanga‐Urverg S, et al. Guidelines for the nonclinical evaluation of the efficacy of traditional antimalarials. In: Merlin W, Gerald B, Philippe R, editors. Traditional Medicinal Plants and Malaria. USA: CRC Press; 2004. p. 256‐68.
21. Kane NF, Kyama MC, Nganga JK, et al. Comparison of phytochemical profiles and antimalarial activities of Artemisia afra plant collected from five countries in Africa. S Afr J Bot. 2019; 125:126–133.
22. García-Huertas P, Pabón A, Arias C, et al. Evaluation of cytotoxic effect and genetic damage of standardized extracts of Solanum nudum with antiplasmodial activity. Biomedica. 2013; 33(1):78–87.
23. de Souza GE., Bueno RV, de Souza JO, et al. Antiplasmodial profile of selected compounds from Malaria Box: in vitro evaluation, speed of action and drug combination studies. Malar J. 2019; 18:447.
24. Kumar S, Guha M, Choubey V, et al. Antimalarial drugs inhibiting hemozoin (beta-hematin) formation: a mechanistic update. Life Sci. 2007; 80(9):813–28.
25. Llurba Montesino N, Kaiser M, Brun R, et al. An approved preparation of Silybum marianum contains constituents with activity against Plasmodium falciparum. Planta Med. 2016; 81(S 01):S1–381.
26. Francis GC, Eze NC, Imafidor HO. Evaluation of antiplasmodial effects of black seeds, fenugreek and coriander seeds in albino mice infected with Plasmodium berghei. Sci Afr. 2024; 23(1):205–18.
27. Priya V, Jananie RK, Vijayalakshmi K. GC/MS determination of bioactive components of Trigonella foenum grecum. J Chem Pharm Res. 2011; 3(5):35–40.
28. Nawaz A, Riaz T, Ahmad A. In-vitro evaluation of antiproliferative potential of various fractions of Silybum marianum using HeLa and HepG2 cell lines. Pak J Pharm Sci. 2021; 34(2(Supplementary)):755–60.
29. Al-Oqail MM, Farshori NN, Al-Sheddi ES, et al. In vitro cytotoxic activity of seed oil of fenugreek against various cancer cell lines. Asian Pac J Cancer Prev. 2013; 14(3):1829–32.
30. Al-Timimi LAN. Antibacterial and Anticancer Activities of Fenugreek Seed Extract. Asian Pac J Cancer Prev. 2019; 20(12):3771–6.
31. Babaei AH, Motamedifar M, Khalifat S, et al. In vitro study of antibacterial property and cytotoxic effects of aqueous, ethanolic, methanolic, and hydroalcoholic extracts of fenugreek seed. Pak J Med Health Sci. 2018; 12:906–10.
32. Wright CW, Allen D, Cai Y, et al. Alkaloids from Aspidosperma pachypterum as potential antimalarial agents. Phytother Res. 1992; 6(3):121-6.
| Files | ||
| Issue | Vol 21 No 2 (2026) | |
| Section | Original Article(s) | |
| Keywords | ||
| Plasmodium knowlesi Silybum marianum Trigonella foenum-graecum Anti-plasmodial Cytotoxicity | ||
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How to Cite
1.
Ahmad Safri N, Cheong FW, Zulkefli NZ, Siyadatpanah A, Norouzi R, Lau YL. Silybum marianum and Trigonella foenum-graecum Exhibit Moderate In Vitro Antimalarial Properties against Plasmodium knowlesi. Iran J Parasitol. 2026;21(2):195-204.
