Effect of organic and chemical fertilizers on growth and active constituents of Dodonaea viscosa - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice BMC Plant Biol . 2026 Apr 15;26:688. doi: 10.1186/s12870-026-08645-0 Search in PMC Search in PubMed View in NLM Catalog Add to search Effect of organic and chemical fertilizers on growth and active constituents of Dodonaea viscosa Fanar Hashum Al-hashemi Fanar Hashum Al-hashemi 1 Department of Horticulture and Landscape Design, Collage of Agriculture and Forestry, Mosul university, Mosul, Iraq Find articles by Fanar Hashum Al-hashemi 1 , Heidar Meftahizade Heidar Meftahizade 2 Department of Horticultural Sciences, Faculty of Agriculture & Natural Resources, Ardakan University, P.O. Box 184, Ardakan, Iran Find articles by Heidar Meftahizade 2, ✉ Author information Article notes Copyright and License information 1 Department of Horticulture and Landscape Design, Collage of Agriculture and Forestry, Mosul university, Mosul, Iraq 2 Department of Horticultural Sciences, Faculty of Agriculture & Natural Resources, Ardakan University, P.O. Box 184, Ardakan, Iran ✉ Corresponding author. Received 2025 Aug 17; Accepted 2026 Mar 24; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13085368 PMID: 41987065 Abstract The aim of this study was to investigate the effects of two fertilizers: organic fertilizer (humic acid, HA) at four levels (0, 0.75, 1.5, and 2 mL·L⁻¹) and chemical (NPK 20:20:20) at three levels (0, 0.5, and 1 g per pot). The vegetative growth parameters (plant height, leaf number, branch number, stem diameter, fresh and dry weight) and biochemical characteristics total chlorophyll, nitrogen, phosphorus, and potassium content, protein, and total phenolic acids were analyzed. Foliar application of HA at 2 mL·L⁻¹ + NPK 0.5 g per pot significantly improved the vegetative growth traits (plant height, leaf number, branch number, and stem diameter). The nutrient content (NPK) and protein increased at NPK (1 g per pot). The study also quantified total phenolic compounds and assessed antioxidant activity via DPPH, showing that D. viscosa leaves have high phenolic content and act as a strong natural antioxidant resource. The highest antioxidant activity (85.5 µmol·L⁻¹) was found with the ethyl acetate extract at 200 µmol·L⁻¹. The combination of HA and NPK significantly increased the phenolic acids, especially quercetin, gallic acid, and cinnamic acid. GC-MS identified eleven compounds within the organic layer of the extract, with myrcene (43.431%), eugenol. In conclusion, the optimal HA and NPK combination significantly enhanced vegetative growth, biomass, and secondary metabolite production in Dodonaea viscosa . These outcomes support the hypothesis that combined foliar application of HA and NPK produces synergistic effects on growth and phytochemical biosynthesis. Keywords: Growth parameters, Phenolic compounds, Antioxidant activity, DPPH assay, GC-MS analysis, Secondary metabolites Introduction Dodonaea viscosa L. is an evergreen, fast-growing shrub belonging to the Sapindaceae family, native to Australia and widely distributed across warm regions of South Africa, North America, and South Asia [ 1 ]. Due to its ecological adaptability, rapid growth, and ease of propagation by seeds and cuttings, it is widely cultivated as a hedge and landscape plant [ 2 ]. D. viscosa is also an important medicinal species, traditionally used for treating inflammation, microbial infections, muscle spasms, pain, and skin injuries, and its parts have been formulated into ointments and topical preparations [ 3 , 4 ]. Phytochemical analyses have revealed diverse bioactive constituents such as phenolics, flavonoids, alkaloids, terpenoids, tannins, steroids, fixed oils, and carbohydrates [ 1 , 5 ]. Among them, phenolic compounds are particularly valuable due to their strong antioxidant, anti-inflammatory, anticancer, antidiabetic, cardioprotective, and neuroprotective properties [ 6 ]. Due to increasing demand for medicinal plants and constraints associated with limited natural resources, there is growing interest in sustainable cultivation strategies that enhance plant growth and phytochemical productivity. Foliar fertilization has emerged as an efficient approach for supplying nutrients and plant growth-enhancing compounds because absorption occurs directly through leaf tissues, bypassing soil limitations such as salinity, nutrient fixation, and poor microbial activity [ 7 ]. Among foliar bio stimulants, humic acid (HA), a major component of humic substances, has gained substantial attention due to its rich content of reactive carboxyl and phenolic groups, which improve cation exchange capacity, nutrient chelation, root growth, physiological performance, and stress tolerance [ 8 , 9 ]. Furthermore, HA can enhance nutrient retention and reduce leaching or volatilization, thereby improving fertilizer use efficiency and stimulating plant metabolic pathways related to chlorophyll formation and secondary metabolite biosynthesis [ 10 ]. Mineral NPK fertilizers supply key macronutrients that are required for both physiological and biochemical functions. Nitrogen is critical for the production of amino acids, proteins, and chlorophyll; phosphorus is important in ATP and nucleic acid physiological processes; potassium aids in osmotic balance, activating enzymes, and translocating carbohydrates [ 11 ]. Studies have observed associations between optimized NPK fertilization for enhanced chlorophyll biosynthesis, photosynthetic functioning, and phenolic and volatile compound accumulation in others’ medicinal species [ 12 – 14 ].The adequate availability of NPK that supports chlorophyll biosynthesis indirectly facilitates carbon acquisitions necessary for shikimic acid pathway activity and is therefore considered an important factor in supporting phenolic metabolism [ 15 ]. There is growing evidence that the co-application of HA and NPK fertilizers results in physiological and metabolic performances that exceed applying HA and NPK fertilizers alone. The co-application of HA and NPK fertilizers improved chlorophyll content [ 16 ], nutrient uptake [ 17 ] and increased the levels of various biomass or secondary metabolites [ 18 ] in horticultural crops. To date, however, despite the medicinal properties and high amount of phytochemicals found in D. viscosa , there are no studies that have investigated the integrated foliar application of HA and NPK fertilizers on vegetative growth and secondary metabolite profile. Therefore, this study is the first to investigate the synergistic effects of foliar-applied humic acid and NPK fertilizer on both growth traits and secondary metabolite profiles of D. viscosa which providing new insights and potential guidelines for sustainable production of medicinal and aromatic plants. Despite the recognized roles of humic acid and NPK fertilizers in improving plant growth, the mechanistic linkage between vegetative performance, nutrient accumulation, and secondary metabolite biosynthesis in medicinal shrubs remains insufficiently explored. In particular, it is unclear whether integrated fertilization strategies can induce coordinated physiological and biochemical responses rather than isolated treatment effects. if found that the combined application of humic acid and NPK fertilizers would exert synergistic effects on Dodonaea viscosa by simultaneously enhancing vegetative growth, nutrient uptake efficiency, and phenylpropanoid-based secondary metabolite biosynthesis, thereby improving antioxidant capacity. Therefore, the objective of this study was to test a hypothesis-driven framework linking integrated humic acid–NPK fertilization to coordinated growth, nutritional, and secondary metabolic responses in Dodonaea viscosa . Materials and methods Experimental design The experiment was conducted under greenhouse conditions at the Department of Horticulture and Landscape Design, University of Mosul, Iraq, between 15 September 2022 and 15 October 2023 using a randomized complete block design (RCBD). Individual pots were considered as experimental units, and each treatment was replicated ten times. (Table 1 ) Treatments were randomly assigned within each block to minimize environmental variability The environmental conditions were continuously monitored, with day temperatures ranging from 14 to 41 °C and relative humidity from 26 to 68%. Light intensity was approximately 60–70% of full sunlight. Soil samples were analyzed before planting and were classified as loamy sand with pH 7.52, EC 0.53 mmhos cm⁻¹, CaCO₃ 19.81%, and available N 15.56 mg kg⁻¹, P 63.6 mg kg⁻¹, and K 1.8 mg kg⁻¹.To ensure the accuracy of irrigation water analysis, the ionic charge balance between total cations and total anions was calculated. The charge balance error was maintained within acceptable analytical limits (± 5%), in accordance with standard water quality assessment procedures. Any minor discrepancies were attributed to analytical uncertainty and unmeasured trace ions. Table 1. Average of Temperature and Humidity inside the lath house Month Temperature (°C) Humidity (%) Great Small September 34.40 21.60 34 October 32.30 15.00 43 November 22.50 8.50 52 December 17.00 2.60 63 January 14.30 1.30 68 February 15.60 2.60 63 March 19.60 8.40 60 April 25.20 11.20 58 May 34.10 18.80 57 June 40.20 23.30 26 July 41.70 22.40 27.1 August 34.20 24.23 29 September 35.40 25.30 35 October 32.10 22.10 45 Open in a new tab The spraying of HA continued till wetness. Both spraying of HA and addition of chemical fertilizer were applied every month starting on November. Chemical Fertilizer (Nano NPK 20:20:20) at three levels: 0, 0.5, 1 g pot − 1 , was added with water irrigation (about 200 ml NPK.pot − 1 ) (Table 1 ). Plant material and experimental conditions Dodonaea viscosa L. seedlings were used in this study. The plant material was collected from healthy and uniform shrubs growing on the campus of the University of Mosul, Iraq. The species belongs to the family Sapindaceae and is characterized as a perennial evergreen shrub with simple, alternate, lanceolate leaves and small greenish flowers. The local cultivar used in this study was identified and authenticated by the Department of Horticulture and Landscape Design, College of Agriculture and Forestry, University of Mosul. Seeds were collected, cleaned, and germinated in the greenhouse under controlled conditions (25 ± 2 °C, 65–70% relative humidity, 14 h photoperiod). After the appearance of the fifth leaf, uniform seedlings (approximately 10 cm height) were transplanted into 25 cm plastic pots containing a soil: peat moss mixture (1:1 v/v). Vegetative growth measurements Vegetative growth traits were recorded at monthly intervals during the experimental period (from November 2022 to October 2023). The parameters measured included plant height (cm), number of leaves per plant, number of branches per plant, and stem diameter (mm). Plant height and stem diameter were measured using a graduated ruler and digital caliper, respectively, while the number of leaves and branches were counted manually. These parameters were selected following the methods described by [ 19 , 20 ], with minor modifications. Chlorophyll measurement Total chlorophyll content was determined in fresh leaves using the acetone extraction method described by [ 21 ] with slight modifications. Leaf samples (0.5 g) were homogenized in 80% acetone, centrifuged at 4000 rpm for 10 min, and the absorbance of the supernatant was measured at 645 and 663 nm using a UV–Vis spectrophotometer (Shimadzu, Japan). Total chlorophyll (mg g⁻¹ FW) was calculated using the following Eq. (1): 1 where A is the absorbance at the respective wavelength, V is the extract volume (mL), and W is the sample weight (g). Measurements were taken at the same time as other vegetative parameters. Irrigation water analysis The irrigation water used throughout the experiment was analyzed prior to and during the growing period to ensure consistent quality. Samples were collected monthly and analyzed according to the methods of the American Public Health Association (APHA, 2017). The parameters measured included electrical conductivity (EC = 0.58 dsm -1 ), pH = 7.34, total dissolved solids (TDS), and concentrations of major cations (Ca²⁺=47 mg l -1 , Mg²⁺=24 mg l -1 , Na⁺=12 mg l -1 , K⁺=4 mg l -1 ) and anions (Cl⁻=11 mg l -1 , SO₄²⁻=22 mg l - , HCO₃⁻=38 mg l -1 ). All analyses were conducted in the Soil and Water Laboratory, College of Agriculture and Forestry, University of Mosul. The irrigation water was classified as low salinity, suitable for horticultural use (EC < 0.8 dS m⁻¹). The seedlings were treated with the following fertilizers: Humic acid (HA) at four levels: (0, 0.75, 1.5, 2) ml L − 1 . It’s sprayed until wetness occurred every month, the first addition implemented during the month of November and Chemical Fertilizer (Nano NPK 20:20:20) at three levels: 0, 0.5, 1 g pot − 1 , this fertilizer was added with water irrigation (about 200 ml NPK.pot − 1 ) once a month and the first addition was done in November. Determination of N, P, K contents The solution was analyzed after the chemically digestion process to determine the percentage of N, P and K in fresh leaves according to [ 22 ]. Crude protein The percentage of total protein in leaves was estimated According to the source [ 22 ] and elucidated by the Eq. (2): 2 the total protein of fresh leaves was respected. Preparation of plant extracts Ten grams of dry leaves was grinded and mixed with 200 ml of many solvents with different polarities (Hot water, Ethanol, Ethyl acetate, Chloroform, Petroleum ether) by using dark flasks with magnetic stirrer for 72 h and the extraction process was based the degree of boiling solvent, then the extracts after 72 h, the extracts has been filtered through filter paper (Whatman No.1), and the vestige was re-extracted with the same volume of solvents for 24 h and evaporated under vacuum at 60 °C to estimate antioxidant activity [ 23 ]. Preparation of the extract for oil The leaves were collected from the field, then dried in the shade and crushed in to powder with a weight about 50 g, then the powder was mixed with distilled water (500) ml in a circul flask. The distillation process was carried out throw the Clevenger tool, two hrs after boiling, we obtained the distillation water which contained the oil, then the diethyl ether was added at a rate, then the diethyl ether was added at a rate (2 × 25) ml for the purpose of separating the oil. The oil was saved at 4 °C until the analysis was begun. Essential oil Essential oil constituents were analyzed using gas chromatography–mass spectrometry (GC–MS; Shimadzu QP2010). One microliter of the essential oil solution was injected into a capillary column (30 m × 0.30 mm × 0.20 μm). The carrier gas was helium at a flow rate of 1 mL min⁻¹, and the oven temperature program was 70–210 °C. Identification of compounds was confirmed by comparing the mass spectra with the NIST library and by matching retention indices (RI) with published data (see Industrial Crops and Products, 2014, 62:403–409). The major constituents identified were myrcene (43.43%), eugenol (20.12%), and α-terpineol (15.50%), confirming the essential oil composition of D. viscosa . Phenolic compounds The extraction was evaluated by soxhelt for 3 h at 65º C used (25 g) dry leaves, then this extract was evaporated at 60º C until 15 ml. After evaporation, the remains of sample were re-extracted with 250 ml of absolute ethanol by using soxhlet apparatus for 72 h at 78 °C. The resulting extract was filtered and evaporated, then the concentrated crude was carried out for acid hydrolysis (5 ml of crade was mixed with 20 ml of 1 N HCl for 1 h in bath water at 100 °C (Harborne, 1973). The mixture was separated with (2 × 25 ml) of ethyl acetate (C 4 H 8 O 2 ) by using separation funnel, then the upper layer was taken and identified by HPLC-Technique (Japan 2010, Sahimadzu, Lc), the column was C 18 (4.6 × 220) mm at a flow rate 1.5 ml min − 1 , the mobile phase consisting of water: acetionitrate (20:80) v/v, UV absorbance at 320 nm. The concentration of the separated compounds was calculated according to the Eq. (3): 3 Determination of total phenolic content Total phenolic content (TPC) was estimated according to the method reported by [ 24 ] by taking 0.5 g of dry leaves, and then 20 ml of ethanol was added to it and left for 24 h and placed in the electric vibrator for three times and for five minutes each time, and the sample was filtered and 1 ml of filtrate was taken and 0.2 ml of Folin-Ciocalteu reagent, 2 ml distilled water and 1 ml of sodium carbonate solution concentration of 15% until the color changes, then the absorption was measured by the spectrophotometer at a wavelength of 765 nm and according to the following Eq. (4): Phenols (mg g − 1 dry weight) = 4 Antioxidant activity Total free radical scavenging capacity of the extract was evaluated by using DPPH, which is called (2,2-diphenyl-1-picryl-hydrazyl). Its chemical formula is (C 18 H 12 N 5 O 6 ). The color is blackish-greenish powder, insoluble in water [ 25 ]. 15.8 mg of DPPH was weighed and dissolved in 200 ml of methanol; different concentration of the phenolic extracts was prepared in three replicates; 50,100,150,200. µ ml − 1 , ascorbic acid was used as a control sample. Then 1 ml of DPPH solution was added to each concentration as well as sample control then the samples were incubated at room temperature measured at a wavelength of 517 nm with a spectrophotometer device of British origin with a single cell and the following Eq. (5) was applied [ 26 ]. 5 Ample.bB: Absorbance of control sa Abs: Absorbance of sample (average absorption of the three absorption values of each dilution). Statistical analysis Data were analyzed using SPSS software version 25.0 (IBM Corp., Armonk, NY, USA). The experiment was conducted in a two-factor randomized complete block design (RCBD), with humic acid (four levels) and NPK fertilizer (three levels) as factors. Analysis of variance (ANOVA) was performed to determine the effects of treatments and their interactions on all measured parameters. Means were compared using Duncan’s multiple range test (DMRT) at a 5% probability level ( P ≤ 0.05), following the procedures described by Gomez and Gomez (1984). Relationships among traits were examined through Pearson correlation coefficients, and the results were graphically presented using ggplot2 in R Software. All graphs were produced using GraphPad Prism version 9.5. Although statistical significance was used to identify treatment effects, the interpretation of results was not based solely on P-values. Observed differences were evaluated in the context of biological relevance and consistency across measured parameters. Variability among replicates was considered when interpreting treatment responses. Results Plant height The results evaluate the effects of treatments of humic acid (HA) at concentrations of 0, 0.75, 1.5, and 2 ml L⁻¹, and NPK fertilizer at three concentrations of 0, 0.5, and 1 g pot⁻¹ on plant height. The results showed that all treatments significantly increased plant height compared with the control. The HA at 1.5 with NPK at 0.5 apply treatment exhibited the highest length, with a 25.79% increase relative to the control, The HA at 0.75 with NPK at 0 and HA at 2 with NPK at 0 treatments recorded moderate performances, with increases of 14.22% and 10.97%, respectively. The HA at 0 with NPK at 0.5, HA at 2 with NPK at 0.5, and HA at 2 with NPK at1 treatments exhibited relatively lower increases of 11.31%, 7.64%, and 8.48%, respectively, compared with the control, but still confirmed the positive effects of the treatments (Fig. 1 A). Fig. 1. Open in a new tab Effect of Humic acid on four levels (0, 0.75, 1.5, 2) ml L − 1 and chemical (NPK 20:20:20) at three levels (0, 0.5, 1) g pot − 1 in ( A ) plant height (PHe), ( B ) leaf number (LN), ( C ) branch number (BN) and ( D ) stem diameter (SD) of Dodonaea Viscosa Leaf number The HA at 1.5 with NPK at 0.5 treatment recorded the highest leaf number, with a 19.79% increase over the control. In contrast, the HA0.75NPK0.5 treatment exhibited the lowest effect on leaf number, with only a 3.24% increase. Other treatments also showed considerable changes, with HA at 1.5 with NPK at 0 (18.49%), HA at 0.75 with NPK at 0 (15.80%), and HA at 2 with NPK at 0.5 (12.87%) ranked next. The HA at 0 with NPK at 1 and HA at 1.5 with NPK at 1 treatments both resulted in an 11.23% increase, while HA at 2 with NPK at1 showed a 10.96% increase, indicating moderate performance. Furthermore, the HA at 0.75 with NPK at 1 and HA at 2 with NPK at 0 treatments, with increases of 8.90% and 8.16%, respectively, along with HA at 0 with NPK at 0.5 (5.38%), also confirmed the positive effect of the treatments on leaf number (Fig. 1 B). Branches/plant Number of plant branches were significant compared with the control. The HA1.5NPK0.5 treatment recorded the highest number of branches, with a 69.37% increase relative to the control. In contrast, the HA0NPK0.5 treatment showed the lowest effect, with a 17.03% increase. Moreover, branch number increased by 62.52% in HA0.75NPK0 (Fig. 1 C). Stem diameter Based on the obtained data, the combined treatments of humic acid (HA) and NPK fertilizer also significantly increased stem length across all treatments compared with the control. The HA0.75NPK0 treatment exhibited the greatest effect, with a 70.43% increase, while the HA0.75NPK0.5 treatment showed the lowest effect, with only a 10.53% increase. Furthermore, (Fig. 1 D). Chlorophyll content According to the results of this experiment, the combined treatments of humic acid (HA) and NPK fertilizer had a significant effect on the chlorophyll content of Dodonaea viscosa . The highest chlorophyll content was recorded under the HA1.5NPK0.5 treatment, with a 26.24% increase compared with the control, while the HA2NPK1 treatment showed the lowest response, with a 3.38% decrease. (Fig. 2 A). Fig. 2. Open in a new tab Effect of Humic acid at four levels (0, 0.75, 1.5, 2) ml L − 1 and chemical (NPK 20:20:20) at three levels (0, 0.5, 1) g pot − 1 in ( A ) total chlorophyll (TChl), ( B ) fresh weight (FW), ( C ) dry weight (DW) and ( D ) ratio of FW/DW in Dodonaea Viscosa Fresh weight Fresh weight measurements further confirmed significant differences among treatments. The HA0.75NPK1 treatment recorded the greatest effect with a 72.59% increase, closely followed by HA1.5NPK0.5 with 71.32%. Substantial increases were also observed under HA2NPK1 (62.94%), HA1.5NPK1 (56.21%), and HA2NPK0.5 (57.23%). By contrast, the HA0NPK0.5 treatment exerted the lowest influence, with only a 17% increase compared with the control (Fig. 2 B). Dry weight The interaction effects of HA and NPK on dry weight% also revealed significant differences. Consistent with fresh weight, the highest increase in dry weight was observed in HA0.75NPK1 (47.52%), followed by HA1.5NPK1 (43.98%), HA1.5NPK0.5 (34.83%), HA0.75NPK0 (34.18%), and HA2NPK1 (32.29%). The lowest effect was recorded in HA0NPK0.5, with only a 10.27% increase relative to the control (Fig. 2 C). Ratio of fresh weight / dry weight the HA1.5NPK1 treatment recorded the lowest ratio (1.866). These findings highlight the differential responses of Dodonaea viscosa to various HA and NPK combinations in regulating the fresh-to-dry biomass ratio, underscoring their distinct roles in biomass allocation and physiological balance (Fig. 2 D). Nitrogen, phosphor and potassium The highest increase was observed in HA2NPK1, with a 30.84% rise, while HA1.5NPK0.5 (26.81%) and both HA0.75NPK0 and HA0.75NPK0.5 (26.06% each) also showed substantial enhancements. In contrast, the lowest effect was recorded under HA0NPK1 (Fig. 3 A). For phosphorus (P), the results indicated significant differences between the interaction of HA and NPK treatments at different concentrations. The HA2NPK1 treatment exhibited the greatest increase, with a 28.43% rise relative to the control. Considerable improvements were also observed in HA0.75NPK0 (25.38%) and HA1.5NPK0.5 (24.17%). Conversely, HA0.75NPK0.5 showed the least improvement, with only a 4.93% increase (Fig. 3 B). The highest increase was recorded in HA2NPK1, with a 39.17% rise compared with the control. Notably, HA2NPK0.5 (37.98%), HA1.5NPK0.5 (31.82%), and HA1.5NPK1 (30.79%) also demonstrated remarkable enhancements throughout the experiment. On the other hand, the lowest increase in potassium was observed under HA0.75NPK0.5, with only a 3.24% rise (Fig. 3 C). Fig. 3. Open in a new tab Effect of Humic acid at four levels (0, 0.75, 1.5, 2) ml L − 1 and chemical (NPK 20:20:20) at three l levels (0, 0.5, 1) g pot − 1 in ( A ) nitrogen (N), ( B ) phosphor (P) and ( C ) potassium (K) of Dodonaea Viscosa Protein and total phenol compounds The present results revealed that the interaction between humic acid (HA) and NPK fertilizer had a significant increase on the percentage of crude protein. The greatest increase was observed in HA2NPK1, which exhibited a 43.27% rise compared with the control. In addition, HA1.5NPK0.5 (39.96%) and HA0.75NPK0 (38.86%) also demonstrated considerable improvements. The lowest increase, however, was recorded in HA0.75NPK0.5, with only a 14.19% rise (Fig. 4 A). The highest increase in phenols was registered in HA2NPK1 (29.86%), while the lowest was observed in HA0NPK0.5 (4.58%). (Fig. 4 B). Fig. 4. Open in a new tab Effect of Humic acid at four levels (0, 0.75, 1.5, 2) ml L − 1 and chemical (NPK 20:20:20) at three levels (0, 0.5, 1) g pot − 1 in ( A ) protein and ( B ) total phenolic compounds (TPC) of Dodonaea Viscosa Correlation matrix analysis The correlation matrix analysis revealed significant relationships among the evaluated physiological, biochemical, and growth traits (Fig. 5 ). The results indicated that Phe exhibited strong positive correlations with LN ( r = 0.75), BN ( r = 0.61), FW ( r = 0.48), and TChl ( r = 0.77). Regarding growth parameters, FW was highly correlated with DW ( r = 0.82), BN ( r = 0.63), and LN ( r = 0.56). Additionally, FW showed significant positive correlations with N ( r = 0.55), P ( r = 0.56), and K ( r = 0.60). Also, TPC exhibited strong positive correlations with protein content ( r = 0.68), K ( r = 0.76), and P ( r = 0.44). Protein content was also strongly and positively correlated with K ( r = 0.76), P ( r = 0.74), and N ( r = 0.54). Similarly, TChl showed strong positive correlations with LN ( r = 0.76), BN ( r = 0.61), and protein content ( r = 0.36). Fig. 5. Open in a new tab Correlation coefficients between the morphological and physiological traits of grapevine cultivars and rootstocks under SS and AA treatment. The color spectrum, from Salmon to dark cyan, represents highly negative to highly positive correlations. The numbers in the figure are the Pearson correlation coefficients between the indicators. See Figs. 1 , 2 , 3 and 4 for the definitions of the measured traits. Correlation matrix ( n = 36 replicate) shows Pearson correlation coefficients with significance levels indicated as (* P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. See Figs. 1 , 2 , 3 and 4 for the definitions of the measured traits Analysis of trait relationships In this study, we investigated the relationships among measured traits under different humic acid and NPK treatments in D. viscosa (Fig. 6 ). Linear regression results indicated a significant positive linear relationship between Phe with LN ( r = 0.56) and BN ( r = 0.36). Additionally, a notable positive relationship was observed between phosphorus content and protein content ( r = 0.54). Moreover, K showed strong positive correlations with TPC ( r = 0.58), P ( r = 0.40), and protein content ( r = 0.57). Fig. 6. Open in a new tab Reltionship between plant height (Phe) with ( A ) leaf number (LN) and ( B ) branch number (BN), and ( C ) phosphor (P) with protein, and potussiom (K) with ( D ) total phenolic compounds (TPC), ( E ) P and ( F ) protein. The correlation coefficients (r) of the relationships are given ( P < 0.05) Antioxidant activity of D. viscosa extracts To assess antioxidant activity, extracts were prepared from dried leaves of D. viscosa using various solvents, including hot water, ethanol, petroleum ether, ethyl acetate, and chloroform (Table 2 ). The results showed that the highest solid residues were obtained with ethanol (4.952 g) and hot water (3.850 g), indicating a higher extraction yield of bioactive compounds from dried leaves using these two solvents compared to the others. Table 2. The solid residue after extraction and evaporation from 25 g dry leaves Extract Residue (g) Hot water 3.856 Ethanol 4.952 Petroleum ether 0.312 Ethyl acetate 1.225 Chloroform 2.850 Open in a new tab The results, expressed as mean values, showed a concentration-dependent increase in activity for all solvents and the standard. At 50 µg·mL⁻¹, the standard exhibited the highest activity (82.3%), followed by ethyl acetate (76.8%), ethanol (73.2%), hot water (70.1%), chloroform (65.2%), and petroleum ether (71.2%). At 100 µg·mL⁻¹, the activity of the standard was 83.5%, with ethyl acetate (80.7%), ethanol (75.5%), hot water (73.3%), chloroform (70.7%), and petroleum ether (74.3%) ranked subsequently. At 150 µg·mL⁻¹, the standard reached 85.7%, followed by ethyl acetate (83.3%), ethanol (78.3%), hot water (75.2%), chloroform (73.9%), and petroleum ether (77.5%). At the highest concentration (200 µg·mL⁻¹), the standard exhibited 87.6% activity, with ethyl acetate (85.5%), ethanol (81.5%), hot water (77.5%), chloroform (78.7%), and petroleum ether (81.7%) in descending order (Table 3 ). Ethyl acetate and ethanol extracts performed better than the other solvents but were less effective than the standard, indicating the superior potency of the standard across all tested concentrations (Fig. 7 ). Table 3. The concentration and percentage of standard sample and crude extracts of D. Viscosa leaves as antioxidants (µ ml − 1 ) The concentration µ ml − 1 Petroleum ether Chloroform Ethyl acetate Ethanol Hot water Standard sample Crude Extracts 50 100 150 200 71.2 d 74.3 c 77.5 b 81.7 a 65.2 d 70.7 c 73.9 b 78.7 a 76.8 d 80.7 c 83.3 b 85.5 a 73.2 d 75.5 c 78.3 b 81.5 a 70.1 d 73.3 c 75.2 b 77.5 a 82.3 d 83.5 c 85.7 b 87.6 a Open in a new tab Values are means ± SE ( n = 3). Different letters within the same column indicate significant differences according to Duncan’s multiple range test ( p ≤ 0.05) Fig. 7. Open in a new tab Chromatograms HPLC of standerd phenolic compounds. ( A ) Salicylic acid, ( B ) P-Hydroxy benzoic acid, ( C ) Cinnamic acid, ( D ) Gallic acid and ( E ) Quercetin acid Table 4 ; Fig. 8 demonstrated that the individual application of NPK fertilizers and humic acid (HA) enhanced the concentration of phenolic compounds. Specifically, at the NPK level of 1 g per pot, quercetin reached 0.093, gallic acid 0.227, salicylic acid 0.213, and para-hydroxybenzoic acid 2.220 mg·g⁻¹. Application of humic acid at all levels gradually increased phenolic compound concentrations, with the highest bioactive compound levels observed at 2 mL·L⁻¹ HA, including quercetin (0.042 mg·g⁻¹), gallic acid (0.322 mg·g⁻¹), salicylic acid (0.062 mg·g⁻¹), para-hydroxybenzoic acid (0.153 mg·g⁻¹), and cinnamic acid (1.231 mg·g⁻¹). When the interaction between NPK at 0.5 g per pot and HA at 2 mL·L⁻¹ was examined, a significant increase in the concentrations of quercetin (0.091 mg·g⁻¹), salicylic acid (1.022 mg·g⁻¹), para-hydroxybenzoic acid (1.311 mg·g⁻¹), and cinnamic acid (3.711 mg·g⁻¹) was observed. Table 4. Effect of NPK and humic acid on the concentration of many phenolic compounds (mg g-1 dry weight) of D. viscosa Treatment Quercetin (mg g -1 ) Gallic acid (mg g -1 ) Salicylic acid (mg g -1 ) p-Hydroxy benzoic acid (mg g -1 ) Cinamic acid(mg/g) HA × NPK 0 × 0 0.001 0.010 0.014 0.011 0.021 0 × 0.75 0.003 0.008 0.01 0.013 0.115 0 × 1.5 0.031 0.023 0.045 0.025 0.126 0 × 2 0.041 0.322 0.062 0.153 1.231 0.5 × 0 0.052 0.143 0.112 1.231 1.303 0.5 × 0.75 0.019 0.003 0.132 0.105 1.421 0.5 × 1.5 0.037 0.389 0.203 0.321 2.621 0.5 × 2 0.091 0.005 1.022 1.311 3.711 1 × 0 0.093 0.227 0.113 2.220 1.251 1 × 0.75 0.0014 0.017 0.145 0.103 ** 1 × 1.5 0.003 0.073 0.080 ** 0.192 1 × 2 0.001 0.004 0.066 ** 0.204 Open in a new tab Values are means ± SE (n = 3). Different letters within the same column indicate significant differences according to Duncan’s multiple range test (p ≤ 0.05) ** the absence of compound Fig. 8. Open in a new tab HPLC results showing the effects of treatments with humic acid (HA) and NPK: ( A ) 0.5 g pot-1 NPK, ( B ) 0.75 ml L-1 HA and 0.5 g pot-1 NPK, ( C ) 1.5 ml L-1 HA and 1 g pot-1 NPK, ( D ) 1.5 ml L-1 HA and 1 g pot-1 NPK, ( E ) 1.5 ml L-1 HA and 1 g pot-1 NPK, ( F ) 1.5 ml L-1 HA and 1 g pot-1 NPK Essential oil of D. viscosa The essential oil extracted from D. viscosa was analyzed using gas chromatography–mass spectrometry (GC-MS), revealing multiple volatile compounds with varying relative percentages (Fig. 9 ; Table 5 ). Myrcene was identified as the major and most abundant component, accounting for 43.431% of the oil. Eugenol (20.122%) and α-terpineol (15.501%) were the next most abundant compounds, and together, these three accounted for approximately 79% of the oil composition. Other notable constituents included 1,8-cineole (5.975%), benzaldehyde (4.077%), linalool (3.330%), and α-calacorene (3.015%). Minor components identified included nerol (1.221%), benzoic acid (1.214%), n-octanol (1.102%), and geraniol (1.012%). These minor compounds, such as 1,8-cineole, benzaldehyde, linalool, and α-calacorene, contribute to the chemical complexity of the oil, while trace amounts of nerol, benzoic acid, n-octanol, and geraniol were also detected. Fig. 9. Open in a new tab The percentage of area and retention time fer compounds shown by using GC-Ms device Table 5. Permission of oil by GC-MS technology from D. viscosa Peak Name of compound Area% Retention time (min) 1 n-octanol 1.102 3.699 2 benzoic acid 1.214 4.678 3 linalool 3.330 5.878 4 α-terpineol 15.501 6.025 5 nerol 1.221 7.049 6 geraniol 1.012 8.296 7 α-calacorene 3.015 9.527 8 benzyldehyde 4.077 10.717 9 eugenol 20.122 13.295 10 myrcene 43.431 15.111 11 1.8-cinol 5.975 15.715 Total 100.00 Open in a new tab Discussion The results of the present study support the proposed hypothesis that integrated humic acid and NPK fertilizations induce coordinated improvements in vegetative growth, nutrient accumulation, and secondary metabolite biosynthesis in Dodonaea viscosa , rather than isolated or purely descriptive treatment responses. While some statistically significant differences were observed among treatments, their biological relevance was interpreted cautiously, particularly where the magnitude of change was relatively small. The foliar application of humic acid (HA) with NPK fertilizers appraised greater enhancement to vegetative growth performance in D. viscosa , demonstrating a synergistic effect between organic biostimulants and inorganic macronutrients. The greatest improvement was observed in plant height when moderate concentrations of HA was utilised with NPK fertilizer treatments, where balanced levels improved nutrient mobilization and uptake efficiencies [ 16 , 20 , 27 ]. The synergistic interactions validate prior studies, indicating that HA increases nutrient movement and assimilation through chelation processes and stimulating physiological functions, in contrast to higher concentrations of HA which can upset nutrient balance or alter soil chemistry and subsequently suppress plant growth responses. Alternative explanations, such as indirect effects of improved nutrient availability on overall plant vigor or cumulative growth responses over time, may also contribute to the observed treatment effects and cannot be excluded. Another growth response demonstrating similar trends was the number of leaves, which is indicative of photosynthesis potential and biomass productivity. The evidence of increased leaf production for NPK - HA treatments pointed to increased nitrogen assimilation, chlorophyll production and increased rates of photosynthesis per planting day [ 28 ]. Although several physiological and biochemical mechanisms have been proposed in the literature to explain the observed responses, it should be emphasized that such mechanisms were not directly measured in the present study. Therefore, the mechanistic interpretations discussed here should be regarded as plausible explanations inferred from previous studies rather than definitive conclusions. The important relationships between leaf development and internal concentrations of N, P, and K strengthen the explanation that HA improves nutrient uptake efficiency and translocation within plant tissues [ 16 , 29 ].The method of foliar application has likely contributed to this response by allowing for nutrients to be absorbed directly through leaf tissues, avoiding soil related limitations [ 29 , 30 ]. Branch number and stem diameter likewise respond positively to the combination of HA and NPK treatments to improve shoot architecture and structural integrity. The additional branches demonstrate an ability for lateral buds to expand which may be sustained with added nutrients and hormones, such as auxin and gibberellin modulation [ 31 – 33 ].The increase in stem diameter also substantiates the expectation of improvements in cell division and elongation processes as result of the combination. Treatment dependent variation indicates that effects are determined not only by presence of nutrients; They are also dependent on concentration ratios and physiological thresholds. Total chlorophyll content was higher under most HA-NPK treatments, which emphasizes the conclusion that the benefits of combined fertilization is due to enhancement of photosynthetic metabolism and pigment biosynthesis [ 29 – 32 ].The reduction in chlorophyll content with high HA concentrations indicates that dose optimization is necessary because of potentially high levels of temporary nutrient antagonism, osmotic stress, or ion exchange blockage with organic materials. With this increase in chlorophyll, we also saw greater increases in fresh and dry biomass, which reflects the integrated effect of HA and NPK to enhance nutrient use, water retention, photosynthetic productivity, and carbon partitioning [ 29 , 32 – 34 ]. The strong correlation between biomass traits and nutrient composition traits indicates that the treatments likely enhanced the growth rate of the plants and quality of assimilate allocation. The enhanced uptake of nitrogen, phosphorus, and potassium corroborates the functional role of HA in enhancing CEC, reducing nutrient loss, and forming slow-release complexes that balance nutrient supply to crop need [ 10 , 35 – 37 ]. Additionally, the positive relationship of nutrient with biomass development implies that improvements in nutrient conditions were in fact responsive to growth performance instead of merely being stored in plant tissues. In other words, increased protein content is viewed in biochemical terms to represent improved nitrogen uptake and incorporated the stimulating aspect of HA on the enzymes associated with nitrogen metabolism pathways [ 9 , 10 , 38 ]. In terms of phenolic content and phenolic acid exudation profiles the enhanced profiles observed means treatments did have an influence on secondary metabolic pathways and the phenylpropanoid pathway, to be specific, that responds to improved nutrient concentrations and metabolic activities [ 18 , 39 – 42 ]. Supporting this notion, GC–MS results provides clarity in the major volatiles being myrcene, eugenol, and α-terpineol in collected samples, supporting hypotheses regarding fertilization improving both quantitative and qualitative aspects of secondary metabolites [ 8 , 43 ]. Antioxidant activity results demonstrated strong DPPH radical scavenging ability, particularly in ethyl acetate extracts, revealing that extracted metabolites are rich in phenolic and flavonoid compounds with hydrogen-donating capacity [ 26 , 44 – 47 ]. Collectively, these findings demonstrate that balanced HA–NPK fertilization enhances not only growth and nutrient profile but also secondary metabolism and antioxidant potential in D. viscosa , emphasizing its value as a medicinal plant under optimized cultivation. It should be noted that some treatment-induced changes, although statistically significant, were of limited magnitude, and further studies incorporating additional physiological or molecular measurements would be required to confirm their functional significance. A limitation of the present study is the absence of direct physiological, biochemical, or molecular measurements that would allow confirmation of the proposed mechanisms underlying the observed responses. Future studies integrating enzyme activity assays, hormone profiling, or gene expression analyses would be valuable to validate the mechanistic pathways suggested here. Conclusion This study investigated the effects of different concentrations of humic acid (HA) and NPK fertilizer on the growth, nitrogen, phosphorus, potassium content, and phenolic acids of the medicinal plant Dodonaea viscosa . Seedlings treated with HA (2 ml·L⁻¹) and NPK (0.5 g per pot) exhibited significantly enhancement in growth and higher phytochemical content. Moreover, the combined HA and NPK treatment produced the greatest increases in plant height, leaf and branch number, as well as fresh and dry biomass, clearly demonstrating a synergistic effect between the two fertilizers. Phenolic content and antioxidant activity reached their highest recorded levels under this treatment, reflecting the high biological and medicinal potential of D. viscosa . These results might indicate by both qualitative and quantitative assays, including total phenolic content and DPPH analysis. The study also highlighted the importance of ethyl acetate extract as a source of native phytochemicals and complementary antioxidants for use in traditional medicinal systems. Overall, these findings indicate that the optimized application of HA and NPK can simultaneously enhance plant growth and the production of valuable secondary metabolites, supporting its application in medicinal agriculture and traditional therapeutic practices. In conclusion, the combined foliar application of HA and NPK effectively enhanced growth performance, nutrient accumulation, and phytochemical synthesis in D. viscosa . These findings suggest potential for integrated fertilization strategies in medicinal plant cultivation. Future work should investigate molecular mechanisms underlying these synergistic effects and their field-level applicability. Acknowledgements The authors were very grateful to the University of Mosul, Collage of Agriculture and Forestry for their providing facilities, which helped to improve the quality of this work. Authors' contributions Fanar hashum Al- hashemi performed the experiments, methodology and wrote the primary manuscript, Heidar Meftahizade analysis the data, wrote the final manuscript and corrected the reviewer’s comment. All authors certified the final manuscript version. Clinical trial number Not applicable. Funding No specific financial credit was used in this experiment. Data availability All data generated during this study are included in the article. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Rani MS, Pippalla RS, Mohan K. Dodonaea viscosa Linn.–An overview. 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