Changes in Growth and Secondary Metabolite Production in Cannabis sativa L. in Response to Simultaneous Treatment with Plasma-Activated Water, Melatonin, and Nano-Zinc

Engineering Nanoscience Environmental Engineering Chemistry Environmental Chemistry Biochemistry Botany Life Sciences
Cannabis sativa plasma-activated water melatonin nano-zinc secondary metabolites

Academic Background

Industrial hemp (Cannabis sativa L.) holds significant value in the pharmaceutical and industrial sectors due to its rich secondary metabolites, such as cannabinoids, terpenoids, and flavonoids. Cannabinoids, including tetrahydrocannabinol (THC) and cannabidiol (CBD), exhibit notable pharmacological activities, with THC being psychoactive and CBD possessing anti-inflammatory, anti-anxiety, and neuroprotective properties. However, the production of secondary metabolites in industrial hemp is influenced by various environmental factors. While traditional chemical fertilizers can enhance yield, they may negatively impact the environment. Therefore, finding a green and efficient alternative to optimize the growth and secondary metabolite production of industrial hemp has become a research focus.

In recent years, plasma-activated water (PAW), melatonin (MT), and zinc oxide nanoparticles (ZnO-NPs) have shown potential in regulating plant growth and enhancing secondary metabolite accumulation. PAW promotes plant growth and secondary metabolite synthesis by generating reactive oxygen and nitrogen species (RONS). MT, as a plant hormone, regulates physiological and biochemical processes in plants. ZnO-NPs enhance plant growth and stress resistance by improving nutrient uptake and antioxidant enzyme activity. However, the combined effects of these three substances on industrial hemp have not been systematically studied. Therefore, this research aims to explore the impact of combined PAW, MT, and ZnO-NPs treatments on the growth and secondary metabolite production of industrial hemp.

Source of the Paper

This paper was co-authored by Hakimeh Oloumi, Fatemeh Nasibi, Zakie Poorsheikhali, and Leila Malekpourzadeh, affiliated with the Department of Ecology, Graduate University of Advanced Technology, Kerman, Iran, and the Biology Department, Shahid Bahonar University of Kerman, Kerman, Iran. The paper was accepted on March 26, 2025, and published in Bionanoscience with the DOI 10.1007/s12668-025-01917-3.

Research Process

1. Experimental Design and Material Preparation

This study employed a completely randomized block design and was conducted in 2023 in the greenhouse of the Graduate University of Advanced Technology, Kerman, Iran. Seeds of the industrial hemp cultivar ‘Mashhad’ were obtained from Pakan Bazr Company in Isfahan, Iran. The seeds were sterilized with sodium hypochlorite, soaked in distilled water for 24 hours, and then sown in acid-washed soil at a depth of 2 cm. Greenhouse conditions were set to 16 hours of light and 8 hours of darkness, and plants were irrigated with a complete nutrient solution.

2. Preparation and Analysis of Plasma-Activated Water (PAW)

PAW was generated using a homemade plasma reactor with two coaxial cylindrical electrodes spaced 5 mm apart, with the outer electrode serving as a dielectric mounted on the surface of a Pyrex tube. The reactor was operated using a high-voltage DC pulse (200 Hz), and the gas flow rate was regulated by a digital flow controller, supplying 99% pure oxygen. PAW samples were prepared by exposing distilled water to the plasma reactor for 10 and 20 minutes. The concentration of reactive oxygen species in PAW was measured using a spectrometer (Avantes AvaSpec) and a UV light source (Bloorazma deuterium lamp).

3. MT, ZnO-NPs, and PAW Treatments

When the plants developed 3–4 leaves, they were divided into two groups: one group was sprayed with 12 ppm MT, while the other group received distilled water as a control. MT treatment was applied three times at 5-day intervals. Subsequently, each group was treated with 0, 5, and 10 ppm ZnO-NPs, as well as 10-minute and 20-minute PAW treatments. PAW and ZnO-NPs treatments were also applied every 5 days for two weeks.

4. Measurement of Growth and Physiological Parameters

After harvest, root length, shoot length, fresh weight, and dry weight of the plants were measured. Leaf relative water content (RWC) was determined using the immersion method, and the membrane stability index (MSI) was measured using the conductivity method. Total chlorophyll content was measured using a portable chlorophyll meter (SPAD-502+).

5. Analysis of Secondary Metabolites

Phenolic compounds and flavonoid content were determined using the Folin-Ciocâlteu reagent method and aluminum chloride colorimetric method, respectively. Alkaloid content was measured using the ethanol/acetic acid extraction method. CBD and THC content were analyzed using high-performance liquid chromatography (HPLC).

Main Results

1. Significant Improvement in Growth Parameters

Combined treatments significantly enhanced root length, shoot length, fresh weight, and dry weight of industrial hemp. The combination of MT, 10-minute PAW, and 5 ppm ZnO-NPs increased root length by 184%, while MT, 20-minute PAW, and 10 ppm ZnO-NPs increased shoot length and shoot dry weight by 79% and 81%, respectively.

2. Improvement in Physiological Parameters

The 20-minute PAW treatment increased leaf relative water content (RWC) by 32% and the membrane stability index (MSI) by 25%. The combination of MT and 5 ppm ZnO-NPs increased total chlorophyll content by 44%.

3. Increase in Secondary Metabolites

The combination of MT and 20-minute PAW increased total phenolic compounds by 65%, while 10-minute PAW and 10 ppm ZnO-NPs increased alkaloid content by 86%. CBD and THC content also significantly increased, with the combination of MT, 10-minute PAW, and 5 ppm ZnO-NPs resulting in the highest THC levels.

Conclusion

This study demonstrates that the combined treatment of PAW, MT, and ZnO-NPs synergistically promotes the growth and secondary metabolite production of industrial hemp. This combined treatment not only significantly improves physiological parameters but also substantially increases the content of secondary metabolites, particularly CBD and THC. The findings provide a new approach for the green and efficient cultivation of industrial hemp, with significant agricultural and pharmaceutical applications.

Research Highlights

  1. Synergistic Effects: This is the first systematic study on the combined effects of PAW, MT, and ZnO-NPs on industrial hemp, revealing their synergistic interactions.
  2. Green and Efficient: Offers an environmentally friendly alternative to traditional chemical fertilizers.
  3. Optimization of Secondary Metabolites: Significantly enhances the content of CBD and THC in industrial hemp, providing high-quality raw materials for pharmaceutical applications.

Other Valuable Information

The limitations of this study include its confinement to greenhouse conditions, necessitating further validation through field trials. Additionally, the research focused solely on leaves; future studies could extend to other tissues such as stems, flowers, and seeds to comprehensively evaluate the effects of combined treatments.