Document Type : Original Article


Department of Agricultural Biotechnology, Faculty of Agricultural and Natural Sciences, Imam Khomeini International University (IKIU), Qazvin, Iran



Background: Despite significant efforts, the artemisinin-based drugs are still very expensive due to the limited production of this metabolite within wild Artemisia spp . plants. Therefore, the current work set out to evaluate the effect of chitosan nanoparticles, as a novel elicitor to characterize the expression of genes functioning in artemisinin synthesis pathway using a comparative experimental investigation.
Methods: The suspensioncultures of A. anuua were exposed to 5, 10, 15 mg/L of chitosan nanoparticles (during 8, 24, 48 and 72 h upon treatment). The expression of DBR2, SQS, CYp , ADS, CPR and ALDH genes were quantified by qRT-PCR technique.
Results: Chitosan nanoparticles were effective in inducing artemisinin production at 15 mg/L after 8 h, and 5 and 10 mg/L after 72 h of elicitation, in which all the ADS, CYp , CPR, DBR2 and ALDH genes were upregulated except SQS.
Conclusion: The treatment of 5 mg/L after 72 h, when cells entered the stationary and then death phases, is recommended because it seems chitosan nanoparticles require more time to up-regulate the ADS, CYP and ALDH genes and thereby probably enhance the artemisinin content. The results suggest that chitosan nanoparticles can be used as a novel effective elicitor for artemisinin production.

Graphical Abstract

The effects of chitosan nanoparticles on genes expression of artemisinin synthase in suspension culture of Artemisia annua L: A comparative study


Main Subjects

1. Rasouli H, Hosseini-Ghazvini S M-B, Adibi H, Khodarahmi R. (2017). Differential α-amylase/α-glucosidase inhibitory activities of plant-derived phenolic compounds: a virtual screening perspective for the treatment of obesity and diabetes. Food & Function, 8(5): 1942-1954.
2. Rasouli H, Hosseini-Ghazvini S M-B, Khodarahmi R. (2019). Therapeutic potentials of the most studied flavonoids: highlighting antibacterial and antidiabetic functionalities Studies in natural products chemistry (Vol. 60, pp. 85-122): Elsevier.
3. Keikhaie K R, Fazeli-Nasab B, Jahantigh H R, Hassanshahian M. (2018). Antibacterial Activity of Ethyl Acetate and Methanol Extracts of Securigera securidaca, Withania sominefra, Rosmarinus officinalis and Aloe vera Plants against Important Human Pathogens. Journal of Medical Bacteriology, 7(1-2): 13-21.
4. Rasouli H, Hosseini Ghazvini S M B, Yarani R, Altıntaş A, Jooneghani S G N, Ramalho T C. (2020). Deciphering inhibitory activity of flavonoids against tau protein kinases: a coupled molecular docking and quantum chemical study. Journal of Biomolecular Structure and Dynamics: 1-14.
5. Rasouli H, Parvaneh S, Mahnam A, Rastegari-Pouyani M, Hoseinkhani Z, Mansouri K. (2017). Anti-angiogenic potential of trypsin inhibitor purified from Cucumis melo seeds: Homology modeling and molecular docking perspective. International journal of biological macromolecules, 96: 118-128.
6. Sobhanizade A, Solouki M, Fazeli-Nasab B. (2017). Optimization of callus induction and effects of biological and non-biological elicitors on content of phenol/flavonoid compounds in Nigella sativa under in-vitro conditions. Cell and Tissue Journal, 8(2): 165-184.
7. Judd R, Bagley M C, Li M, Zhu Y, Lei C, Yuzuak S, Ekelöf M, Pu G, Zhao X, Muddiman D C. (2019). Artemisinin biosynthesis in non-glandular trichome cells of Artemisia annua. Molecular plant, 12(5): 704-714.
8. Sebola T E, Uche-Okereafor N C, Mekuto L, Makatini M M, Green E, Mavumengwana V. (2020). Antibacterial and Anticancer Activity and Untargeted Secondary Metabolite Profiling of Crude Bacterial Endophyte Extracts from Crinum macowanii Baker Leaves. International Journal of Microbiology, 2020.
9. Shi P, Fu X, Liu M, Shen Q, Jiang W, Li L, Sun X, Tang K. (2017). Promotion of artemisinin content in Artemisia annua by overexpression of multiple artemisinin biosynthetic pathway genes. Plant Cell, Tissue and Organ Culture (PCTOC), 129(2): 251-259.
10. Wang J, Li J-l, Li J, Li J-x, Liu S-j, Huang L-q, Gao W-y. (2017). Production of active compounds in medicinal plants: from plant tissue culture to biosynthesis. Chinese Herbal Medicines, 9(2): 115-125.
11. Nielsen E, Temporiti M E E, Cella R. (2019). Improvement of phytochemical production by plant cells and organ culture and by genetic engineering. Plant cell reports, 38(10): 1199-1215.
12. Mahendran D, Geetha N, Venkatachalam P. (2019). Role of Silver Nitrate and Silver Nanoparticles on Tissue Culture Medium and Enhanced the Plant Growth and Development In vitro Plant Breeding towards Novel Agronomic Traits (pp. 59-74): Springer.
13. Chung I-M, Rajakumar G, Thiruvengadam M. (2018). Effect of silver nanoparticles on phenolic compounds production and biological activities in hairy root cultures of Cucumis anguria. Acta Biologica Hungarica, 69(1): 97-109.
14. Alam P, Albalawi T H. (2020). In vitro alteration of artemisinin biosynthesis in Artemisia annua L during treatment with methyl jasmonate. Tropical Journal of Pharmaceutical Research, 19(1): 33-37.
15. Zarayneh S, Sepahi A A, Jonoobi M, Rasouli H. (2018). Comparative antibacterial effects of cellulose nanofiber, chitosan nanofiber, chitosan/cellulose combination and chitosan alone against bacterial contamination of Iranian banknotes. International Journal of Biological Macromolecules, 118: 1045-1054.
16. Babaei Z, Solouki M, Fazeli-Nasab B. (2018). Investigating The Effect of Biological and non-Biological Elicitor on Expression of Hyp-1 Gene in Hypericum perforatum. Modern Genetics, 13(4): 543-549. Article Code: MGJ-17-B-00487
17. Murashige T, Skoog F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant, 15(3): 473-497.
18. Ghasemi B, Hosseini R, Dehghan Nayeri F. (2015). Effects of cobalt nanoparticles on artemisinin production and gene expression in Artemisia annua. Turkish Journal of Botany, 39(5): 769-777.
19. Ghassemi B, Hosseini R, Dehghan nayeri F. (2015). The Effect of Nano Cobalt and Nano Chitosan on Artemisinin production and expression of SQS and DBR2 genes in Artemisia annua. [Research]. Genetic Engineering and Biosafety Journal, 4(1): 25-39.
20. Pfaffl M W. (2001). A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research, 29(9): e45-e45.
21. Mallona I, Díez-Villanueva A, Martín B, Peinado M A. (2017). Chainy: an universal tool for standardized relative quantification in real-time PCR. Bioinformatics, 33(9): 1411-1413.
22. Ali M, Abbasi B H, Ahmad N, Khan H, Ali G S. (2017). Strategies to enhance biologically active-secondary metabolites in cell cultures of Artemisia–current trends. Critical reviews in biotechnology, 37(7): 833-851.
23. Wang Y, Weathers P. (2007). Sugars proportionately affect artemisinin production. Plant Cell Rep, 26(7): 1073-1081.
24. Shen Q, Zhang L, Liao Z, Wang S, Yan T, Shi P, Liu M, Fu X, Pan Q, Wang Y. (2018). The genome of Artemisia annua provides insight into the evolution of Asteraceae family and artemisinin biosynthesis. Molecular plant, 11(6): 776-788.
25. Firsov A, Mitiouchkina T, Shaloiko L, Pushin A, Vainstein A, Dolgov S. (2020). Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes. Plants, 9(4): 537.
26. Tang K, Shen Q, Yan T, Fu X. (2014). Transgenic approach to increase artemisinin content in Artemisia annua L. Plant Cell Rep, 33(4): 605-615.
27. Saifi M, Khan S, Kiran U, Fatima S, Abdin M Z. (2020). Transgenic technology to improve therapeutic efficacy of medicinal plants. Transgenic Technology Based Value Addition in Plant Biotechnology: 207.
28. Lei C, Ma D, Pu G, Qiu X, Du Z, Wang H, Li G, Ye H, Liu B. (2011). Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L. Ind Crops Prod, 33(1): 176-182.
29. Ma Y N, Xu D B, Yan X, Wu Z K, Kayani S I, Shen Q, Fu X Q, Xie L H, Hao X L, Hassani D. (2021). JA and ABA activated AaGSW1‐AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua. Plant Biotechnology Journal.
30. Perassolo M, Cardillo A B, Busto V D, Giulietti A M, Talou J R. (2018). Biosynthesis of sesquiterpene lactones in plants and metabolic engineering for their biotechnological production Sesquiterpene Lactones (pp. 47-91): Springer.
31. Xie L, Yan T, Li L, Chen M, Ma Y, Hao X, Fu X, Shen Q, Huang Y, Qin W. (2020). The WRKY transcription factor AaGSW2 promotes glandular trichome initiation in Artemisia annua. Journal of Experimental Botany.
32. Du Y, Yang B, Yi Z, Hu L, Li M. (2020). Engineering Saccharomyces cerevisiae coculture platform for the production of flavonoids. Journal of agricultural and food chemistry, 68(7): 2146-2154.
33. Salehi M, Karimzadeh G, Naghavi M R, Badi H N, Monfared S R. (2018). Expression of key genes affecting artemisinin content in five Artemisia species. Scientific reports, 8(1): 1-11.
34. Ma D, Li G, Zhu Y, Xie D-Y. (2017). Overexpression and suppression of Artemisia annua 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1 gene (AaHDR1) differentially regulate artemisinin and terpenoid biosynthesis. Front Plant Sci, 8: 77.
35. Yin H, Kjaer A, Fretté X C, Du Y, Christensen L P, Jensen M, Grevsen K. (2012). Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L. Process Biochem, 47(11): 1559-1562.
36. Putalun W, Luealon W, De-Eknamkul W, Tanaka H, Shoyama Y. (2007). Improvement of artemisinin production by chitosan in hairy root cultures of Artemisia annua L. Biotechnology letters, 29(7): 1143-1146.
37. Jiao J, Gai Q-Y, Wang X, Qin Q-P, Wang Z-Y, Liu J, Fu Y-J. (2018). Chitosan elicitation of Isatis tinctoria L. hairy root cultures for enhancing flavonoid productivity and gene expression and related antioxidant activity. Industrial crops and products, 124: 28-35.
38. Ahmed K B M, Khan M M A, Siddiqui H, Jahan A. (2020). Chitosan and its oligosaccharides, a promising option for sustainable crop production-a review. Carbohydrate polymers, 227: 115331.
39. Baldi A, Dixit V. (2008). Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua. Bioresour Technol, 99(11): 4609-4614.
40. Darki B S, Shabani L, Pourvaez R, Mostafa S. (2019). Effects of CuSO4 and AgNO3 on artemisinin and phenolic compound in shoot cultures of Artemisia annua L. Journal of Plant Process and Function, 8(31): 2.
41. Rasouli H, Norooznezhad A H, Rashidi T, Hoseinkhani Z, Mahnam A, Tarlan M, Moasefi N, Mostafaei A, Mansouri K. (2018). Comparative in vitro/theoretical studies on the anti-angiogenic activity of date pollen hydro-alcoholic extract: Highlighting the important roles of its hot polyphenols. BioImpacts: Bi, 8(4): 281.
42. Jahani M, Azadbakht M, Rasouli H, Yarani R, Rezazadeh D, Salari N, Mansouri K. (2019). L-arginine/5-fluorouracil combination treatment approaches cells selectively: rescuing endothelial cells while killing MDA-MB-468 breast cancer cells. Food and Chemical Toxicology, 123: 399-411.
43. Rasouli H, Popović-Djordjević J, Sayyed R, Zarayneh S, Jafari M, Fazeli-Nasab B. (2020). Nanoparticles: a new threat to crop plants and soil Rhizobia? Sustainable Agriculture Reviews 41 (pp. 201-214): Springer.
44. Rasouli H. (2019). Devil's hand conceals behind the obscure side of AgNPs: A letter to the editor. International Journal of Biological Macromolecules, 125: 510-513.