Document Type : Full Paper

Authors

• Elham Amjadi ¹
• Ali Ganjeali ²
• Abolfazl Shakeri ³
• Mehrdad Lahouti ⁴
• Saeed Tavakoli ⁵

¹ Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

² Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad. Iran.

³ Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

⁴ Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.

⁵ Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran.

Abstract

Keywords

• free radical
• phenolic compounds
• phenology
• silymarin

Main Subjects

• Medicinal plant science

Extended Abstract

Introduction

     The milk thistle (Silybum marianum (L.) Gaertn.) contains silymarin, a flavonolignan found in its seeds, which is used for the treatment of liver, digestive, biliary, and poisoning diseases. Elicitors are substances that can boost the synthesis of secondary metabolites in plants by activating defense mechanisms. Aspergillus niger is known as the main source of citric acid production, Pseudomonas putida can produce significant quantities of organic acids through the decomposing organic substances in the soil and subsequently providing more phosphorus in the soil for absorption by plants. The yeast Yarrowia lipolytica is recognized as the host of naringenin biosynthesis, which serve as a precursor in the synthesis of flavonoids. Silver nitrate enhances the expression of certain genes and various intracellular enzymes by inhibiting ethylene activity, thereby affecting the synthesis of secondary metabolites in plants. Considering the importance of milk thistle as a medicinal plant, this research was undertaken to explore the potential for enhancing the production of medicinal compounds, particularly silymarin and antioxidant compounds, through the application of various biotic and abiotic elicitors at different phenological stages.

Materials and methods

Twenty-day-old seedlings of milk thistle cultivated in sterile soil in a greenhouse were subjected to four different treatments. These included Pseudomonas putida, Yarrowia lipolytica, each at a concentration of 0.5 McFarland standards, approximately 1.5×10⁸ bacteria or yeast, and Aspergillus niger at a concentration of 1.2 × 10⁷ spores per ml. These treatments were added to the rhizosphere in a volume of 10 ml. Additionally, silver nitrate treatment (at a concentration of 100 ppm) was applied through leaf spraying during both vegetative and reproductive stages. The plants aerial parts were collected at two stages namely vegetative and reproductive stages as well as seeds to examine their biochemical characteristics. A methanolic extract prepared by dissolving 1 g of plant powder in 10 ml of 80% methanol to assess the total phenol, flavonoid, phenolic acid content, IC_50, as well as silymarin content.

Results and Discussion

The results revealed that the highest content of total phenol in the areal parts at vegetative stage was attributed to the application of silver nitrate (5.33 mg GAE g^-1 DW), whereas in the reproductive stage, the highest content was associated with the control treatment (5.11 mg GAE g^-1 DW). Furthermore, in seeds, the highest level was obtained through the use of yeast (12.94 mg GAE g^-1 DW). The maximum content of flavonoids during the vegetative stage was linked to silver nitrate (5.5 mg QE g^-1 DW), while in the reproductive stage it was related to fungi application (3.4 mg QE g^-1 DW), and in the seeds, was belonged to yeast application (4.5 mg QE g^-1 DW). The highest content of phenolic acid in the vegetative stage (2.73 mg CEA g^-1 DW) was related to the application of bacteria, and in the reproductive stage, it was related to fungi (1.62 mg CEA g^-1 DW), and in the seeds, it was attributed to yeast (3.5 mg CEA g^-1 DW). The studied bio stimulants are sources for citric acid production and are able to produce and accumulate phenolic and flavonoids compounds within the vacuole, thereby enhancing antioxidant activity. Certain heavy metals, such as silver nitrate, protect plants against oxidative damage by the promoting of phenolic compound synthesis, mitigating free radical damage and improving the compatibility of plants with their environment.

The highest antioxidant activity and value of DPPH (IC₅₀) were obtained in both the vegetative (85.99 µg ml^-1) and reproductive stages (65.45 µg ml^-1) using silver nitrate trearment, while in seed production stage (37.54 µg ml^-1) was assigned to yeast treatment. By changing the plasma membrane, yeast can synthesize secondary metabolites and activate a defensive response against reactive oxygen species. ‌.

The application of silver nitrate resulted in the highest synthesis of silymarin in the aerial parts of the plant during both the vegetative (9.96%) and reproductive (6.64%) stages. Furthermore, the application of bacteria significantly enhanced the silymarin content in the seeds. Heavy metals bind to receptors on the surface of the plasma membrane, initiate a network of signal transmissions and cause the overexpression of all genes involved in the synthesis of secondary metabolites, including silymarin. Bacteria, as external producers of auxin and gibberellin hormones, induce flavonoid synthesis pathway, which in turn promotes the synthesis of taxifolin and silymarin. Yeast increases the synthesis of phenylalanine, as a precursor in the silymarin synthesis pathway.

Conclusion

The results indicated that the total phenol content of aerial parts of plant increased from the vegetative to the reproductive stage and subsequently in the seed; however, it was different for total flavonoid content. It seems that elicitors have diverse effects on various stages of plant growth, attributable to their specific roles in the biosynthetic pathways of different compounds.

Author Contributions

1. Amjadi: Conceptualization, Investigation, Formal analysis & Writing - original draft. A. Ganjeali: Conceptualization, Investigation, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Final approval of the article. A. Shakeri: Methodology, Writing - original draft & editing. M. Lahoti: Investigation, Methodology. S. Tavakoli: Investigation, Methodology.

Data Availability Statement

All data supporting the findings of this study are available within the paper and are available from the corresponding author on reasonable request.

Acknowledgements

We sincerely thank all individuals who provided technical assistance during the execution of this project.

We are special thankful from Ferdowsi University of Mashhad, Ministry of Science, Research, and Technology, Iran to financial support (grant no. 3/55205).

We are grateful to I. R of the Iran Meteorogicat Organization for providing the meteorological data and the Academic Center of Education, Culture and Research (ACECR) for the analysis and processing of the medicinal plants.

Ethical considerations

This article does not contain any studies involving human and animal subjects. The authors avoided data fabrication, falsification, plagiarism and misconduct.

Conflict of interest

The corresponding author declare that there is no Conflict of interest.