Introduction

Cannabis sativa L., an annual plant species native to Asian regions, primarily exhibits dioecious characteristics and boasts an extensive history of cultivation owing to its diverse applications ranging from fiber production and nutritional seeds to medicinal and psychoactive purposes1. Contemporary scientific and commercial attention has grown substantially as novel utilizations continue to emerge across various sectors including dietary supplements, functional food ingredients, and health-promoting products2. The plant’s therapeutic potential has particularly driven its global agricultural expansion, especially for pharmaceutical-grade cannabis production serving the burgeoning medical marijuana sector3. The flowering tops of medicinal varieties contain numerous bioactive compounds including cannabinoids, terpenes, and flavonoid derivatives that account for their pharmacological effects4. C. sativa produces more than 113 different cannabinoids, with the most abundant being cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), Δ9-tetrahydrocannabinolic acid (THCA), and cannabichromenic acid (CBCA). These acidic compounds can undergo decarboxylation to form their neutral counterparts: cannabigerol (CBG), cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC), and cannabichromene (CBC), respectively5. Research demonstrates that these valuable secondary metabolites show significant variation depending on growth environment and farming practices6,7,8,9,10,11, creating challenges for the pharmaceutical sector which requires consistent, standardized plant material - an objective currently hindered by insufficient understanding of how external factors influence plant biochemistry and physiology.

Deep-water culture (DWC), a subset of hydroponic cultivation, offers a sustainable and efficient approach for plant growth in challenging environments12. Compared to traditional soil-based agriculture, hydroponic systems demonstrate superior productivity, enhanced synthesis of bioactive compounds, and reduced susceptibility to pests and diseases13,14,15. One of the major advantages of hydroponics is the precise regulation of pH and nutrient levels, allowing for optimized plant nutrition. Hydroponic methods are particularly useful for studying the influence of different nitrogen forms on plant morphology, physiology, and secondary metabolite production, as they eliminate soil-based nitrogen variability16. Researchers have employed these systems to compare nitrogen utilization efficiency across plant species, focusing on nitrate (NO3−) and ammonium (NH4+) as primary nitrogen sources17.

Nitrogen plays a pivotal role in agriculture, being the most critical nutrient for crop growth and soil fertility18. As a fundamental component of chlorophyll, nitrogen is indispensable for photosynthesis, directly influencing plant development, yield, and quality19. Cannabis is one of the plants in which nitrogen plays an important role in the content of its main secondary metabolites, and as nitrogen increases in the growth medium, the amount of Δ9-tetrahydrocannabinolic acid (THC) also increases20. Additionally, nitrogen availability affects key enzymatic processes in biosynthetic pathways, such as the phenylpropanoid pathway, which governs the production of medicinal phytochemicals21. Plants primarily absorb nitrogen in the form of nitrate (NO3−) or ammonium (NH4+)22. Studies on medicinal species, including Echinacea purpurea23, Cannabis sativa24, Dracocephalum moldavica25, and Erythropalum scandens26, have demonstrated varying responses to these nitrogen forms in terms of growth and phytochemical profiles. While NO3− is the preferred nitrogen source for most plants, NH4+ uptake occurs when nitrate availability is limited. However, exclusive reliance on NH4+ can lead to stunted growth, root system alterations, and even toxicity symptoms such as leaf chlorosis27. The optimal NH4+:NO3− ratio for plant growth depends on multiple factors, including environmental conditions, developmental stage, genetic traits, and nitrogen accessibility28,29. Notably, NH4+ absorption acidifies the rhizosphere, as each NH4+ ion taken up by roots is exchanged for an H+ ion, lowering the surrounding pH30,31. Research has shown that secondary metabolites, including rosmarinic acid, flavonoids, ursolic acid, and oleanolic acid, peak at a 75:25 NH4+:NO3− ratio, with declines observed at higher or lower ammonium concentrations32. Similarly, Prunella vulgaris exhibited maximal biomass production under a 75:25 NH4+:NO3− treatment33. Plant species exhibit distinct nitrogen preferences: crops like corn, beans, peas, and tomatoes thrive on nitrate-dominant fertilizers, whereas tea plants favor ammonium-rich nutrition34,35.

Cannabis sativa has emerged as a significant horticultural crop due to its legalization for medicinal and recreational use in various countries, including Canada and USA35. However, scientific knowledge on optimal cultivation practices—particularly regarding nitrogen nutrition—remains limited. Nitrogen is a vital macronutrient, but improper levels (especially excessive NH4+ or NO3−) can harm plant metabolism and yield. Currently, there is scarce research on how NH4+/NO3− ratios affect C. sativa’s growth, secondary metabolism, and phytochemical production (such as cannabinoids and phenolics). Proper nutrient management in hydroponic systems (such as DWC) is critical to maximize biomass and bioactive compound synthesis. This study investigates Impact of NH4+:NO3− ratios and solution strength on phytochemicals and antioxidants in Cannabis sativa L. flowers organ grown in DWC systems to optimize cultivation practices.

Materials and methods

Plant materials

In 2023, a study was carried out in the greenhouse facilities of the Horticultural Sciences Department at Urmia University, Iran. This project investigated how different ratios of ammonium to nitrate in the nutrient solution would influence the growth and quality of medicinal cannabis plants. The study employed a completely randomized design (CRD). Each treatment was replicated three times (n = 3 biological replicates). Within each biological replicate, two technical observations were recorded, resulting in a total of six data points per treatment. The specific ratios tested were: 20 NH4+:80 NO3−, 40 NH4+:60 NO3−, 60 NH4+:40 NO3−, and three additional variations (1/2 20NH4+:80NO3−, 1/2 40 NH4+:60 NO3−, and 1/2 60 NH4+:40 NO3−). The seeds, sourced from a reliable supplier, were first planted in small cell trays. The growing medium for germination was a blend of fine peat moss and perlite. Once the seedlings had developed three to four leaves, they were moved to their main growing setup, a floating culture system (DWC). The different nutrient treatments began immediately after this transfer. Throughout the experiment, the greenhouse environment was carefully maintained. Daytime temperatures were kept at around 29 °C, dropping to 21 °C at night, with a relative humidity of 55%. The plants received a light intensity ranging from 560 to 640 µmol m−2 s−1. The plants were grown in large, three-layered polyethylene containers, each holding 200 L of nutrient solution. A blower and air diffusers provided constant aeration to the solution. Each container held 8 plants, which floated on the solution surface on a polystyrene sheet (Fig. 1). When the plants reached the flowering stage (Fig. 1), samples were collected to measure morphological various traits and phytochemical analysis. Table 1 shows the composition of the nutrient solutions for the two N-form treatments.

Fig. 1
figure 1

Experimental setup and the flowering stage of cannabis plants. Each container held 8 plants, which floated on the surface of the nutrient solution on a polystyrene sheet.

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Table 1 Composition of the nutrient solutions for the two N-form treatments.
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Growth and morphological traits

Plant height (cm) was measured using a meter tape, while the dry weights of the aerial parts and roots (g) were determined using a precision balance.

Preparation of methanolic extract

The extraction procedure involved homogenizing 1 g of dried aerial tissue in 10 mL of 80% aqueous methanol. Sonication was performed for 45 min at 25 °C (40 Hz, Elmasonic, Germany). Following sonication, the mixtures were centrifuged (4000 rpm, 5 min), and the clarified supernatants were collected. The final extracts were stored at 4 °C in amber vials to protect from light, pending further analysis36.

Total phenolic content (TPC)

The Folin–Ciocalteu method, with slight modifications, was employed to determine the TPC (based on Slinkard and Singleton’s protocol)37. Briefly, 5 µl of the extract solution was mixed with 1 ml of a 1:10 diluted Folin-Ciocalteu reagent, followed by the addition of 480 µl of 7.5% sodium carbonate. The mixture was allowed to stand at room temperature for 30 min, after which the absorbance was measured at 760 nm using a UV–vis spectrophotometer (UNICO, China). The TPC was calculated using a gallic acid standard curve and expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW).

Total flavonoid content (TFC)

The TFC of the methanolic extract from cannabis was determined following the method described by Shin et al.38. Initially, 5 µl of the methanolic extract was mixed with 200 µl of 5% NaNO2, followed by the addition of 300 µl of 10% AlCl3 after 5 min. After another 5 min, 0.2 ml of 1 mM NaOH was added, and the solution was diluted to 1 ml with distilled water. The absorbance was measured at 380 nm. The TFC was quantified using a quercetin standard curve and expressed as milligrams of quercetin equivalents per gram of dry weight (mg QUE/g DW).

Antioxidant activity DPPH assay

The antioxidant activity of the methanolic extract of Cannabis sativa L. was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. In this method39, a specific quantity of the extract was combined with 2000 µl of a DPPH solution, prepared by dissolving 0.006 g of DPPH in 150 ml of 80% methanol. After incubating the mixture at room temperature for 30 min, the absorbance of the solution was measured at a wavelength of 517 nm. The percentage of DPPH radical inhibition was calculated using the following formula:

$${\text{DPPH Sc }}\left( \% \right){\text{ }}=\left[ {\left( {{\text{Ab}}{{\text{s}}_{{\text{control}}}}-{\text{Ab}}{{\text{s}}_{{\text{sample}}}}} \right)/{\text{Ab}}{{\text{s}}_{{\text{control}}}}} \right]{\text{ }} \times {\text{1}}00$$

Analysis of cannabinoid compounds using GC-MS

The cannabinoid compounds in Cannabis sativa L. were analyzed using gas chromatography coupled with mass spectrometry (GC-MS) (Agilent Technologies, USA, 7890B GC System/5977A MSD). The analysis focused on quantifying cannabinoid content, specifically delta-9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabichromene (CBC), cannabinol (CBN), and delta-8-tetrahydrocannabinol (Δ8-THC). Dried flower samples were ground into a fine powder and thoroughly mixed. A 200 mg portion of each powdered sample was placed in a flask and extracted with 3 ml of hexane using an ultrasonic bath for 10 min. After extraction, the mixture was filtered, and 1 ml of the filtrate was transferred to a vial for individual analysis by gas chromatography-mass spectrometry (GC-MS). A 1 µl sample was injected into the system, utilizing an HP-5 MS column (30 m × 0.25 mm, 0.25 μm). The injector and auxiliary temperatures were both set to 280 °C. The column temperature program began at 50 °C, held for 1 min, then increased at a rate of 10 °C per minute until reaching 260 °C, where it was maintained for 8 min. The mass spectrometer operated in electron ionization (EI) mode with an ionization energy of 70 electron volts. The mass range analyzed was 50 to 550 atomic mass units (amu). The ionization source and quadrupole temperatures were set to 230 °C and 150 °C, respectively. High-purity helium (99.9995%) was used as the carrier gas at a pressure of 34 psi and a flow rate of 1 ml/min. Compound identification was achieved by comparing the mass spectra with reference data from the NIST and Wiley libraries, as well as by evaluating retention indices and fragmentation patterns reported in the literature.

Statistical analysis

The acquired data were analyzed using one-way ANOVA (with three replications) integrated in SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). The means were evaluated with Tukey’s honestly significant difference (Tukey’s HSD) test at a significance level of p < 0.05.

Results and discussion

NH4 +:NO3 − ratios’ effects on morphological characteristics

Cannabis sativa L. demonstrated optimal biomass production and morphological characteristics when exposed to NH4+:NO3− ratios of 40:60 at full solution strength and 60:40 at half solution strength, while the lowest values were observed at a 60:40 ratio with full solution strength (Fig. 2). During mid-growth stages, plants supplied with NH4+:NO3− ratios of 40:60 at half solution strength and 20:80 at both half and full solution strength exhibited favorable growth. Research has consistently shown that aerobic plants generally thrive better with a combination of NH4+ and NO3− as nitrogen sources40,41. Unlike NO3−-N, NH4+-N does not require a reduction process within the plant before being incorporated into amino forms (-NH2), thereby conserving plant energy. This may explain the enhanced biomass observed at a 60:40 ratio with half solution strength. Conversely, increased NH4+ levels can lead to reduced plant height, fewer lateral branches, lower fresh weight of aerial organs and roots, and decreased dry matter content. Studies have highlighted that higher nitrate ratios often improve growth traits, as excessive ammonium can be toxic and inhibit growth in many species. For instance, in Citrullus lanatus, higher nitrate-to-ammonium ratios correlated with increased biomass, while ammonium dominance reduced biomass accumulation in tomato seedlings. Additionally, NH4+ can impair root system development, negatively impacting overall plant growth. Nitrogen fertilization is a critical factor in Ocimum basilicum production, where ammonium availability, even in the presence of nitrate, significantly restricts growth42,43. Ammonium toxicity may arise from various mechanisms, including disrupted acid-base balance, reduced protein glycosylation, environmental acidification, and energy costs associated with expelling excess ammonium. Certain plant species exhibit root sensitivity to high NH4+ levels, potentially due to disrupted auxin transport or ethylene signaling, which can inhibit root growth. Photosynthesis, a vital process for plant growth, is also adversely affected by ammonium as the primary nitrogen source, leading to reduced net photosynthesis rates. High NH4+ concentrations in leaf tissues can disrupt chloroplast function, uncoupling electron transport from phosphorylation, further impairing plant growth.

Fig. 2
figure 2

Plant height (cm) and dry weights of above-ground parts and roots (g) of Cannabis sativa L. under different NH4+:NO3− ratios and nutrient solution strengths. Data are means ± standard error (SE) of three biological replicates (n = 3). Error bars represent ± SE. Different letters above the bars indicate statistically significant differences among treatments (p < 0.05, Tukey’s HSD test).

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Effect of the NH4 +:NO3 − ratios on TPC and TFC

It is well known that phenolic compounds and flavonoids are crucial bioactive compounds with wide interest over the years due to their human health benefits. The results showed that the various ratios of NH4+:NO3− influenced TPC (Fig. 3) and TFC (Fig. 4) in Cannabis sativa L. (p < 0.01). The maximum TFC and TPC values in the flower organ were found at an NH4+:NO3−− concentration of 60:40 with half solution strength.

Fig. 3
figure 3

Total phenolic content in the flower organ of Cannabis sativa under different ammonium-to-nitrate ratios and full or half ionic strength treatments. Data are means ± standard error (SE) of three biological replicates (n = 3). Error bars represent ± SE. Different letters above the bars indicate statistically significant differences among treatments (p < 0.05, Tukey’s HSD test).

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Fig. 4
figure 4

Total flavonoid content in the flower organ of Cannabis sativa L. under different ammonium-to-nitrate ratios and full or half ionic strength treatments. Data are means ± standard error (SE) of three biological replicates (n = 3). Error bars represent ± SE. Different letters above the bars indicate statistically significant differences among treatments (p < 0.05, Tukey’s HSD test).

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The relationship between nitrogen source and phenolic compound accumulation in our study revealed a nuanced interaction with overall solution strength. Under full-strength nutrient conditions, phenolic biosynthesis was predominantly enhanced at lower NH4+:NO3− ratios. This can be explained by the fact that in a nutrient-replete environment, the primary driver for phenolic induction is likely a mild nitrogen form-related stress. While nitrate assimilation is energetically costly, it maintains a stable cellular pH. In contrast, even moderate levels of ammonium under optimal nutrition can impose a subtle metabolic stress, potentially through slight disruptions in pH homeostasis or by generating a low level of reactive oxygen species (ROS), thereby triggering the antioxidant system and the synthesis of phenolic compounds as a protective measure44. However, a pivotal shift in this response was observed under half-strength solution strength. In this scenario of generalized nutrient limitation, the highest phenolic levels were associated with higher NH4+:NO3− ratios. We propose that under combined stress—where energy and carbon are already limited due to overall nutrient scarcity—the additional metabolic burden imposed by high ammonium becomes a critical secondary stressor. Ammonium assimilation requires substantial carbon skeletons for detoxification and incorporation, further diverting resources away from growth45. This creates a pronounced carbon sink that, coupled with the existing nutrient stress, strongly upregulates the phenylpropanoid pathway as a primary outlet for the redirected carbon flux. Furthermore, the intense energy demand and potential for proton toxicity associated with high ammonium uptake in a nutrient-starved root system likely exacerbate oxidative stress, providing a potent synergistic signal for the massive induction of phenolic antioxidants46. Therefore, the switch in the optimal ratio for phenolic accumulation under different solution strengths underscores a fundamental shift in plant strategy: from a targeted response to nitrogen form under ample nutrition to an amplified, synergistic defense activation under combined nutrient and ammonium stress.

The observed increase in phenolic compounds in response to reduced macronutrient and micronutrient strength can be attributed to a confluence of physiological and biochemical mechanisms. Primarily, nutrient limitation, particularly of nitrogen, shifts the plant’s carbon allocation strategy. According to the Carbon-Nutrient Balance hypothesis, constrained nitrogen availability limits protein synthesis, leading to an accumulation of carbon skeletons that are subsequently channeled into the shikimic acid and phenylpropanoid pathways, thereby enhancing the production of carbon-based secondary metabolites like phenolics47. Concurrently, the mild oxidative stress induced by nutrient deficiency results in the generation of reactive oxygen species (ROS), prompting a counteractive upregulation of the plant’s antioxidant defense system, in which phenolic compounds play a crucial role48. This biosynthetic response is further potentiated by hormonal signaling shifts, where stress hormones such as jasmonic acid and ethylene upregulate key biosynthetic genes, including phenylalanine ammonia-lyase (PAL)49. Furthermore, alterations in the availability of specific micronutrients, which act as cofactors for enzymes involved in both the synthesis and degradation of phenolics, can alter the metabolic equilibrium, favoring accumulation50. Therefore, the elevation of phenolic content is best interpreted as a multifaceted adaptive strategy, where the plant reallocates resources from primary growth to chemical defense, ensuring survival under suboptimal nutritional conditions.

The significant increase in total phenolic and flavonoid content (TPC and TFC) observed under elevated NH4+:NO3− ratios under half strength (60:40 treatment) can be mechanistically explained through its impact on nitrogen assimilation and carbon metabolism. High ammonium concentration disrupts the ionic balance, leading to cytosolic acidosis and an increased energy demand for its detoxification via the GS/GOGAT cycle (glutamine synthetase/glutamate synthase)51. This metabolic burden can inhibit the activity of phosphoenolpyruvate carboxylase (PEPC), a key enzyme anaplerotic enzyme replenishing the TCA cycle, while simultaneously upregulating phenylalanine ammonia-lyase (PAL), the gateway enzyme of the phenylpropanoid pathway52. Consequently, the carbon flux is diverted away from the TCA cycle and toward the synthesis of phenolic precursors like phenylalanine.

Furthermore, ammonium stress is well-documented to induce a state of ‘carbon surplus’ or ‘C/N imbalance’. As primary nitrogen assimilation becomes constrained or more energy-intensive, excess carbon skeletons (such as phosphoenolpyruvate and erythrose-4-phosphate) are shunted into the shikimate and phenylpropanoid pathways, directly fueling the production of phenolics and flavonoids53. This transcriptional reprogramming is often evidenced by the upregulation of genes encoding PAL, chalcone synthase (CHS), and flavonoid glycosyltransferases under ammonium nutrition, as shown in transcriptomic studies54. Therefore, the elevated TPC and TFC in our study are not merely a general stress response but a direct physiological consequence of altered nitrogen form, which reconfigures central metabolism to favor the biosynthesis of secondary metabolites as a likely adaptive mechanism.

Antioxidant activity by DPPH assays

The study revealed that different NH4+:NO3− ratios significantly influenced the antioxidant activity of Cannabis sativa L., as determined by DPPH assays (p < 0.01). Specifically, the flower organ exhibited the highest antioxidant activity at NH4+:NO3− ratios of 60:40 and 40:60, both at half solution strength, as illustrated in Fig. 5.

Fig. 5
figure 5

Antioxidant activity in the flower organ of Cannabis sativa L. under different ammonium-to-nitrate ratios and full or half ionic strength treatments. Data are means ± standard error (SE) of three biological replicates (n = 3). Error bars represent ± SE. Different letters above the bars indicate statistically significant differences among treatments (p < 0.05, Tukey’s HSD test).

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The interplay between nitrogen form and solution strength in modulating antioxidant activity revealed a context-dependent response in our study. Under full-strength nutrient conditions, the highest antioxidant activity was associated with lower NH4+:NO3− ratios. In this nutrient-sufficient environment, the primary stimulus for antioxidant induction appears to be the metabolic cost and mild nitrosative stress linked to nitrate reduction. While preferable to ammonium toxicity, nitrate assimilation still generates reactive nitrogen species (RNS) alongside ROS, necessitating a robust antioxidant system to maintain cellular redox homeostasis55. The superior growth and biomass often observed with nitrate nutrition likely support a greater metabolic capacity to synthesize these protective compounds without compromising growth. Conversely, a pivotal shift occurred under half-strength solution strength, where the highest antioxidant activity was induced by higher NH4+:NO3− ratios56. We posit that under generalized nutrient stress, the plant’s physiological priority shifts from growth to survival. The combination of nutrient scarcity and high ammonium imposes a severe, multi-faceted stress. High ammonium uptake under these conditions likely leads to a rapid acidification of the rhizosphere and cytoplasm, disrupts the proton gradient, and uncouples photophosphorylation, resulting in a burst of oxidative stress. This synergistic effect—where ammonium toxicity amplifies the stress from nutrient deficiency—creates a powerful, non-linear trigger for the massive upregulation of the entire antioxidant system as a crucial survival mechanism. This shift highlights a strategic reallocation: under ample nutrition, antioxidants manage routine metabolic byproducts, whereas under combined stress, they are deployed as an essential defense against severe cellular damage51,52,53,54,55,56,57,58.

GC-MS analysis

Chromatographic analysis of Cannabis flowers reveals a significant influence of ammonium-to-nitrate ratios and solution strength on cannabinoid biosynthesis (Fig. 6). The data demonstrate that the NH4+:NO3− ratio of 40:60 under full solution strength promoted optimal production of major cannabinoids, including delta-9-THC (28.46%) and CBD (10.09%). This optimal ratio likely reflects balanced nitrogen assimilation that supports the terpenophenolic pathway without imposing ammonium toxicity. The complete suppression of cannabinoids at the 60:40 ratio under full ionic strength clearly indicates ammonium toxicity. This can be attributed to several physiological disruptions: the high energy cost of ammonium detoxification and assimilation competes directly with the carbon-intensive cannabinoid biosynthesis pathway. Additionally, ammonium-induced cytoplasmic acidosis may inhibit key enzymes such as olivetol synthase and geranyl pyrophosphate: olivetol ate geranyl transferase, which are crucial for cannabinoid formation.

Fig. 6
figure 6

Amount of cannabinoid compounds in flower organ.

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Interestingly, under half solution strength conditions, the 40:60 ratio maintained relatively high cannabinoid production (23.79% delta-9-THC), while the 60:40 ratio showed severe reduction. This demonstrates that proper nitrogen form proportioning can partially mitigate the effects of overall nutrient stress on secondary metabolism. However, the combination of high ammonium and reduced solution strength creates synergistic stress that severely compromises the plant’s ability to produce cannabinoids. These findings align with ecological theories of resource allocation, where plants under moderate stress often increase secondary metabolite production, but under severe or combined stresses, they prioritize survival over chemical defense. The differential response of cannabinoid profiles to nitrogen treatments suggests distinct regulatory points in the cannabinoid biosynthetic pathway that respond differently to nitrogen form and availability47.

Conclusion

This study demonstrates that the phytochemical composition of Cannabis sativa L. flowers is governed by the interaction between the NH4+:NO3− ratio and the overall nutrient solution strength, rather than by nitrogen form alone. Two distinct metabolic responses were identified. Under full-strength nutrition, a balanced 40:60 NH4+:NO3− ratio optimizes cannabinoid biosynthesis, supporting efficient nitrogen assimilation and creating favorable physiological conditions for producing delta-9-THC and CBD. In contrast, the combination of high ammonium and low solution strength acts as a potent trigger for phenolic and antioxidant accumulation, reflecting a coordinated stress-induced activation of the phenylpropanoid pathway. Overall, these findings provide a practical framework for precision nutrient management in controlled cannabis cultivation. By adjusting both nitrogen form and solution strength, producers can purposefully steer metabolic pathways—either enhancing cannabinoid yield for pharmaceutical applications or elevating phenolic antioxidants for nutraceutical and functional-ingredient markets. This dual-pathway model offers a clear foundation for targeted, reliable, and value-oriented production strategies in modern cannabis industries.