Australian Journal of Crop Science

Article | https://doi.org/10.21475/ajcs.26.20.10.pne283

Submitted: 20 March 2026 | Revised: 20 July 2026 | Accepted: 06 August 2026

Pages 834–845

Assessing the effects of two biostimulants on yield, calyx mineral content, and antioxidant constituents of roselle (Hibiscus sabdariffa L.)

Sahba M Kadhem¹, Majeed K Abbas*², Hasanain Riyadh Al-Isawi¹, Ali Sabah Al-Hasan³

¹College of Nursing, University of Al-Qadisiyah, Iraq

²College of Agriculture Technique, Al-Mustaqbal University, Babylon, Iraq

³College of Agriculture, University of Al-Qadisiyah, Iraq

*Corresponding author: [email protected]

*ORCID: https://orcid.org/0009-0006-2557-0554

Abstract: Roselle (Hibiscus sabdariffa L.) is a significant economic crop owing to its various nutritional and therapeutic applications. This study aimed to evaluate the impact of two potential biostimulants, ginger and saffron extracts, at different dosages on the production of Roselle cv. Red, as well as its NPK and antioxidant content. A randomized complete block design (RCBD) with two factors (4x3) was established. The first factor is the use of saffron stigma extract (S) at four concentrations (0, 1, 2, and 3 g.L⁻¹). Ginger rhizome extract (G) was applied at three concentrations (0, 5, and 10 g. L-¹) as the secondary variable. Three replications of the treatments were performed. The results indicated a substantial impact of the two biostimulant concentrations used. The maximum values for the yield parameters examined were achieved when the greatest concentrations of both extracts were amalgamated. The treatments resulted in an augmentation of NPK's mineral composition. Moreover, the antioxidant constituents in Roselle calyxes, including anthocyanins, flavonoids, total phenols, ascorbic acid, and citric acid, were elevated for the extracts and their combinations. However, the carotenoid content either increased solely in the S2 treatment, remained constant in the S2G1 and S2G2 treatments, or declined in all other treatments. The antioxidant activity in the leaves and calyxes escalated with higher concentrations of saffron and ginger extracts. The greatest free radical-scavenging activity was noted at the highest concentrations examined. Consequently, our findings indicate that the combination treatments at the maximum dosages of both stimulants had a superior response.

Keywords: Anthocyanin, Antioxidants, Free radical, NPK, Total Phenols.

Abbreviations: FWC: Fresh weight of calyxes, DWC: Dry weight of calyxes, FWF: Fresh weight of fruit, DWF: Dry weight of fruit, NF: Number of fruits, TY: Total yield, Car: Carotenoids, Anth: Anthocyanins, Flav: Flavonoids, AsA: Ascorbic acid, TPh: Total phenols, DPPHL: Free radical DPPH in leaves, DPPHC: Free radical DPPH in calyxes, CV: Coefficient of variation, SE: Standard error of the mean.

Introduction

Roselle (Hibiscus sabdariffa L.) belongs to the Malvaceae family. It is a shrub indigenous to the tropical and subtropical areas of Asia, Africa, and both North and South America. It is extensively grown in nearly all warm-climate nations as a domestic garden crop (Yusoff et al., 2024). Roselle is mostly grown for its calyxes, which represent the most commercially significant component. The plant is rich in nutrients such as anthocyanins, organic acids, pectin, phenolic compounds, and essential vitamins that help prevent several chronic diseases (Wu et al., 2018). The findings indicated the existence of alkaloids, flavonoids, saponins, steroids, sterols, and tannins in Roselle plants (Pacôme et al., 2014). Moreover, H. sabdariffa calyxes were identified to possess antioxidants (Bule et al., 2020; Mohamed et al., 2022).

Contemporary agriculture seeks to investigate and implement innovative methods to enhance agricultural yields as a measure towards sustainability (Carvalho et al., 2022). Numerous techniques can enhance plant development, including the application of nutrients and growth regulators. Due to the adverse environmental impacts of these materials, focus has shifted towards using natural extracts (plant extracts) that are environmentally safe and promote growth, owing to their abundance of minerals, organic acids, vitamins, and growth regulators (Al-Samaraee & Abdul-Kadhm, 2020). They also encompass a variety of antioxidant chemicals (Ngcobo et al., 2024). Numerous natural plant extracts, such as seaweed, ginger, garlic, and saffron, have been utilized to enhance plant growth (Alsalhy & Aljabary, 2020; Kozłowska et al., 2022; Aryan et al., 2023; Moosavipoor et al., 2024).

Saffron (Crocus sativus L.) is a perennial species within the Iridaceae family. The growth-promoting properties of saffron are primarily attributed to its numerous bioactive constituents. Research has demonstrated the presence of nitrogenous compounds, anthocyanins, monoterpenes, glycosides, flavonoids, vitamins (notably riboflavin and thiamin), aldehydes, volatile oils, carbohydrates, proteins, and minerals including sodium, potassium, phosphorus, calcium, manganese, magnesium, zinc, iron, and certain amino acids (Anaigodari et al., 2023; Yildiz & Hulya, 2025). Saffron stigmas are abundant in crocins (water-soluble carotenoid glycosides), picrocin, crocetin, and safranal, which exhibit significant antioxidant capabilities and enhance plant resilience to abiotic stressors. These compounds can stimulate physiological activities by augmenting antioxidant defenses, enhancing nutrient absorption and utilization, facilitating chlorophyll formation, and fostering plant growth and productivity (Kyriakoudi et al., 2015; Avila-Sosa et al., 2022). An aqueous extract of saffron stigmas has been identified as a biostimulant that enhances tomato growth (Solanum lycopersicum L.), resulting in notable improvements in plant height and antioxidant levels (Khoulati et al., 2019). Saffron leaves and tubers have been shown to produce bioactive compounds, some of which significantly contribute to plant growth (Feizi et al., 2018; Tavakoli et al., 2023).

Ginger (Zingiber officinale Roscoe) is a member of the Zingiberaceae family. Ginger extract is considered a natural plant biostimulant because of its abundance of bioactive chemicals that stimulate plant metabolism, improve nutrient absorption, fortify antioxidant defenses, and increase tolerance to biotic and abiotic challenges. The biostimulant properties of ginger are chiefly attributed to its phenolic compounds (Gingerol, Zingerone, Shogaol), flavonoids (Quercetin), volatile terpenes, vitamins C and B-complex, antioxidants, minerals (P, K, Ca, Mg, Fe, Zn), organic acids, amino acids, and carbohydrates, all of which augment physiological processes, thereby promoting vegetative growth and overall crop yield. (Ali et al., 2022; Ayustaningwarno et al., 2024). Consequently, ginger extract may serve as an effective biostimulant and an alternative approach for promoting plant development and augmenting output. Research indicates that applying ginger rhizome extract to seedlings enhances vegetative growth and improves chemical composition (Al-Samaraee & Abdul-Kadhm, 2020; Hardan & Ahmed, 2024).

Nonetheless, research investigating the synergistic impact of these two extracts on crop growth is limited, if not absent. This study aims to examine the synergistic effect of saffron and ginger extracts on roselle yield and antioxidant content in the Al-Qadisiyah Governorate of Iraq.

Results

Yield components

Analysis of variance indicated that saffron and ginger concentrations significantly influenced both fresh and dry calyx weight (P < 0.001) (Table 3). Both characteristics increased steadily with increasing saffron concentrations. The highest values were recorded in S4 treatment (221.10 g for fresh weight and 79.47 g for dry weight), with all four values showing substantial differences. The G3 treatment yielded the highest average fresh and dry weight for ginger. The two biostimulants significantly influenced both fresh and dry fruit weight (P < 0.01), with the highest concentrations yielding greater mean values for both metrics. Similarly, both S and G variables exerted highly significant effects on fruit numbernd overall yield (P < 0.001) (Table 2).

Table 2. Impact of primary variables, saffron (S) extracts at four concentrations and ginger extracts (G) at three concentrations on six yield characteristics of Roselle (Mean ± SE)

Note: FWC: Fresh weight of calyxes, DWC: Dry weight of calyxes, FWF: Fresh weight of fruit, DWF: Dry weight of fruit, NF: Number of fruits, TY: Total yield, Con: Concentration, SE: Standard error of the mean. Means denoted by identical letters (s) inside the same column for each factor are not statistically different as per Duncan's Multiple Range Test at the 5% significance level

Table 3. Analysis of variance (mean squares) regarding the impacts of saffron concentrations (S), ginger concentrations (G), and their interaction (S × G) on the yield, mineral composition, and antioxidant elements of roselle.

Note: FWC: Fresh weight of calyxes, DWC: Dry weight of calyxes, FFW: Fresh fruit weight, DWF: Dry weight of fruit, NF: Number of fruit, TY: Total yield, Car: Carotenoids, Anth: Anthocyanins, Flav: Flavonoids, AsA: Ascorbic acid, CtA: Citric acid, TPh: total phenols, CV: coefficient of variation, with * and ** signifying significance at P ≤ 0.05 and P ≤ 0.01, respectively, ns: not significant

The interaction between the two factors S and G; significantly influenced both calyx fresh and dry weights (P < 0.0001) (Table 4). The maximum calyx fresh weight was observed at S4G2 combination treatment, at 243.36 g, greatly surpassing all other treatment combinations except S4G3, while the minimum was observed at S1G2, at 134.79 g. The highest calyx dry weight values were recorded for S4G2, S4G3, and S3G2, ranging from 74.80 to 75.20 g.

In contrast, S1G1 (45.50 g) and S1G2 (45.90 g) treatments yielded the lowest dry weights and were statistically comparable. The interaction between S and G factors was significant for both fresh and dry weight of fruit (P < 0.05). The S4G3 combination treatment yielded the largest fruit fresh weight at 495.40 g, while the S3G3 treatment resulted in the greatest fruit dry weight at 175.65 g. The interaction concerning fruit number and total yield was not significant (P = 0.057); thus, the comparison of interaction means is merely descriptive.

Table 4. Impact of the interaction between saffron (S) extracts at four concentrations and ginger extracts (G) at three concentrations on six features of Roselle (Mean ± SE)

Note: FWC: Fresh weight of calyxes, DWC: Dry weight of calyxes, FFW: Fruit fresh weight, DWF: Dry weight of fruit, NF: Number of fruit, TY: Total yield, SE: Standard error for the mean; * and ** denote significance at P ≤ 0.05 and P ≤ 0.01, respectively, ns: not significant. Means denoted by identical letters (s) within the same column are not statistically different as per Duncan's Multiple Range Test at the 5% significance level

The S4G3 combination treatment exhibited the highest values for the characteristics, recording 89.88 for fruit number and 3.650 (Mg ha-1) for total yield, whereas the S1G1 treatment yielded the lowest values.

NPK content

Saffron and ginger extracts significantly influenced the level of nitrogen, phosphorus, and potassium (P < 0.001) (Table 5). Nitrogen rose from 1.92% at S1 to 2.37% at S4 treatment, and from 1.78% at G1 to 2.76% at G3 treatment. S3, S4, and G3 treatments yielded the highest phosphorus concentrations, which differed significantly from those of the other treatments. Furthermore, potassium levels increased progressively as the concentrations of the two biostimulants rose (Table 5). The interaction between the two factors (S x G) for nitrogen was not significant (P = 0.128); however, it was significant (P = 0.003) for potassium content. The highest interaction mean was seen in S3G3 treatment at 0.372%, whilst S1G1 recorded the lowest value at 0.264%. The interaction for potassium was similarly significant (P = 0.022). The greatest means were recorded for S4G3 and S2G3 treatments, while S2G1 exhibited the lowest potassium concentration (Table 6).

Table 5. Impact of primary variables, saffron (S) extracts at four concentrations and ginger (G) extracts at three concentrations, on the NPK content of Roselle (Mean ± SE)

Means denoted by identical letters (s) within the same column are not statistically different as per Duncan's Multiple Range Test at the 5% significance level

Table 6. Impact of the interaction between saffron (S) extracts at four concentrations and ginger extracts (G) at three concentrations on the NPK content of Roselle (Mean ± SE)

Note: ns: not significant

The two variables significantly influenced carotenoid content (P < 0.001). S2 treatment yielded the highest carotenoid content (4.38 mg.100g^-1 FW), followed by S1. G1 treatment exhibited the maximum carotenoid content among the G treatments (Table 7). The ANOVA table indicated that S and G had a substantial impact on anthocyanin content (P ≤ 0.01) (Table 3). S4 and G3 treatments yielded the highest average anthocyanin concentrations, measuring 0.283 and 0.266 mg.100g^-1 FW, respectively. The saffron extracts markedly influenced flavonoid content (P = 0.00026), with the maximum level recorded in S4 treatment (35.52 mg QE.g^-1), whilst S1 yielded the lowest value (25.65 mg QE.g^-1). The G factor has no significant impact on flavonoid content.

Table 7. Impact of saffron (S) and ginger (G) extract concentrations on the levels of carotenoids, anthocyanins, flavonoids, ascorbic acid, citric acid and total phenols in the calyxes of Roselle.

Note: Car: carotenoids, Anth: anthocyanin, Flav: flavonoids, AsA: ascorbic acid, CtA: citric acid, TPh: total phenols, ns: not significant. Means denoted by identical letters (s) inside the same column for each factor are not statistically different as per Duncan's Multiple Range Test at the 5% significance level

Antioxidant constituents

The variance analysis revealed that S treatments had a substantial impact on ascorbic and citric acid content (P < 0.001). The maximum average ascorbic acid concentration was recorded under S4 (57.81) and G3 (54.03) for the two factors, whereas S1 and G1 had considerably lower values. Citric acid exhibited a progressive increase from 1.09 at S1 to 1.91 at S4 treatment. Additionally, G3 yielded the greatest average citric acid concentration (1.64). Saffron extracts had a considerable influence on the total phenol level. The mean total phenols rose from 137.00 (mg GAE g⁻¹ DW) in S1 treatment to 155.33 (mg GAE g⁻¹ DW) in S4. The G factor exhibited a substantial impact (P = 0.000610). G3 had the highest mean total phenol content (151.75 mg GAE g⁻¹ DW)), significantly higher than G1 and G2, which were statistically comparable. The S × G interaction was statistically significant (P < 0.001) for carotenoid content. The combinations S1G1, S2G1, and S2G2 yielded the highest content (4.97, 4.90, and 4.90 mg.100g^-1 FW, respectively) and were statistically comparable. The minimal carotenoid concentration was observed in S3G3 (2.75 mg.100g^-1 FW) (Table 8).

Table 8. Impact of the interaction between saffron (S) extracts at four concentrations and ginger (G) extracts at three concentrations on the amount of carotene, anthocyanin, flavonoids, ascorbic acid, citric acid, and total phenols (TPh) in Roselle (Mean ± S)

Note: Car: carotenoids, Anth: anthocyanins, Flav: flavonoids, AsA: ascorbic acid, CtA: citric acid, TPh: total phenols, ns: not significant. Means denoted by identical letters (s) within the same column are not statistically different as per Duncan's Multiple Range Test at the 5% significance level..

The interaction means for anthocyanin and flavonoid indicated that S4G3 yielded the highest content, with values of 0.315 and 39.80, respectively, while the lowest values were 0.200 for anthocyanin at S1G1 and 24.24 for flavonoid at S1G2. The S × G interaction was not significant (P > 0.05) for ascorbic acid, while it was significant (P = 0.0489) for citric acid. The highest citric acid concentrations were recorded in S4G1 and S4G3, which were statistically comparable and considerably higher than those in the lowest treatment, S1G1. The interaction was significant (P = 0.006082) regarding the total phenols. Among the interaction treatments, S4G3 combination treatment exhibited the highest total phenol content (161 mg GAE g⁻¹ DW), while S1G1 displayed the lowest value (124 mg GAE g⁻¹ DW). Table 9 indicates that the application of saffron and ginger extracts significantly influenced DPPH free radical activity (P < 0.001). An increase in activity was observed in the leaves and calyxes of roselle with higher amounts of saffron and ginger extracts. The activity varied from 76.33% to 84.96% with the application of saffron at 3 g.L^-1 (S4) and from 76.95% to 83.07% with the application of ginger at 10 g.L^-1 (G3). Nonetheless, the interaction was insignificant for leaves but substantial for calyxes, with the S4G3 combo treatment exhibiting the maximum activity.

Table 9 Impact of saffron (S) and ginger (G) extract concentrations and their interactions on DPPH free radical activity (%) in Roselle.

Note: DPPH: 2,2-diphenyl-1-picrylhydrazyl.

Means denoted by identical letters (s) within the same column are not statistically different as per Duncan's Multiple Range Test at the 5% significance level

Discussion

The global interest in organic farming practices is increasing due to customer demand for safe and healthy agricultural products. The use of natural, safe materials in agriculture, including plant extracts (Rouphael & colla, 2020; Deepak Kumar & Alok, 2020), has become prevalent. Plant extracts are currently employed as a sustainable agricultural approach to enhance crop yields and reduce chemical use.

The current findings demonstrate beneficial improvements in both extracts for production features linked to the fresh and dry weights of calyxes and fruits, fruit number, and total yield (Tables 2 and 4). The beneficial enhancements were increasingly apparent at elevated concentrations of both extracts, exhibiting a concentration-dependent biostimulatory effect, with peak values for the assessed production indicators reached at the highest concentrations of saffron (3 g. L^-1) and ginger (10 g. L^-1).

Many researchers have noted the advantageous effects of saffron and ginger extracts on the vegetative growth of treated plants, leading to improved yield characteristics due to the growth-promoting compounds present in these extracts (Ali et al., 2022; Tavakoli et al., 2023; Hardan & Ahmad, 2024). Saffron extract has been shown to be vital for tomato plants and can act as a biostimulant (Khoulati et al., 2019). Mbuisa et al. (2025) demonstrated improvements in the vegetative growth, physiological characteristics, and yield attributes of squash (Cucurbita pepo L.) with the application of ginger extracts. Saffron consists of nitrogenous molecules, vitamins (particularly riboflavin and thiamin), carbohydrates, and minerals such as sodium, K, P, Mg, Ca, Zn, Mn and Fe, in addition to certain amino acids (Anaigodari et al., 2023; Yildiz & Hulya, 2025). The saffron stigma comprises crocines, crocetin, picrocin, safranal, flavonoids, carotenoids, and phenolic chemicals, all of which demonstrate considerable antioxidant properties and may augment metabolism (Kyriakoudi et al., 2015; Avila-Sosa et al., 2022). Ginger rhizomes possess elevated levels of gingerols, shogaols, zingerone, flavonoids, amino acids, and minerals, which facilitate physiological processes including cell division, chlorophyll synthesis, and enzyme activation (Ali et al., 2022; Ayustaningwarno et al., 2024), thus enhancing plant performance.

The significant increase in the weight of fresh and dried calyx is remarkable, as the calyx serves as the primary commercial product of roselle. The increased calyx growth noted with saffron and ginger spray may suggest enhanced photosynthetic translocation and nutrient absorption. Similar results have been noted for medicinal and aromatic plants subjected to plant extracts, where greater dry matter accumulation is associated with improved nutrient uptake and elevated photosynthetic efficiency (Rouphael & Colla, 2020). The weights of fresh and dried fruit displayed a similar pattern, suggesting that both biostimulants promoted reproductive growth instead of solely increasing vegetative biomass. Furthermore, the increased dry weight of the fruit denotes improved efficacy in the allocation of photosynthetic products to storage compounds, reflecting better carbon fixation. The significant increase in fruit number and total yield can be ascribed to enhanced flowering, namely an augmented fruit set and diminished flower drop, stemming from improved physiological circumstances in the treated plants. Ei et al. (2024) noted that the advantageous effects of these extracts correlate with increased photosynthesis and nutrient uptake, leading to enhanced flowering and fruit characteristics.

The interaction between saffron and ginger notably influenced most crop components; however, it was not statistically significant for fruit number or overall yield (Table 4). The S4G3 treatment consistently yielded the highest numerical values, indicating synergistic physiological effects between the two extracts, attributed to compounds in both extracts that function as effective natural biostimulants, thereby enhancing roselle productivity through various physiological mechanisms. Our present findings align with those of previous researchers. Al-Humaidi (2023) demonstrated that the application of seaweed extract on roselle markedly enhanced crop attributes, including fruit number and overall calyx yield, in comparison to the control treatment. In marigolds, Al-Samarrai and Abdul-Kadhim (2020) noted that the application of an aqueous ginger rhizome extract led to greater vegetative growth and improved flowering.

Our results demonstrated that the two biostimulants significantly elevated nitrogen, phosphorus, and potassium concentrations (Tables 5 and 6). The gradual enhancement of nutritional content at elevated doses of both extracts indicates that these natural biostimulants enhance nutrient absorption, transport efficacy, and digestion. The amounts of all three elements consistently escalated with increasing concentrations of saffron and ginger, attaining maximum values in treatments S4 and G3. Some researchers have suggested that plant extracts improve various physiological and biological processes, potentially leading to increased photosynthetic efficiency, enhanced vegetative growth, and a larger root system area, thereby promoting the absorption and accumulation of minerals such as nitrogen, phosphorus, and potassium (Al-Sultani & Amir, 2021; Chen et al., 2021; Ghabour et al., 2021; Ei et al., 2024). Our improved mineral supplementation offers physiological understanding of the noted enhancements in production and quality in our research. Increased nitrogen promotes protein synthesis and chlorophyll formation, whereas phosphorus provides energy for metabolic processes, and potassium regulates photosynthesis, metabolite transport, and enzyme activity (Taiz et al., 2015). Consequently, the simultaneous enhancement of these three macronutrients creates ideal conditions for vigorous growth, efficient biomass production, and the accumulation of beneficial phytochemicals in roselle. Our findings correspond with those of Bulgari et al. (2017), who demonstrated that plant biostimulants improve nutrient uptake by fostering root development and stimulating membrane transport processes. The greatest potassium concentration recorded in S4G3 (Table 6) indicates that the combined application of saffron and ginger markedly enhanced potassium absorption relative to the individual usage of either extract. The results demonstrate that saffron and ginger extracts are effective natural biostimulants that improve nutrient utilization efficiency and diminish dependence on synthetic fertilizers.

Our research demonstrated that roselle plants subjected to saffron calyx and ginger rhizome extracts displayed increased antioxidant levels in comparison to untreated plants. Elevated levels of flavonoids, anthocyanins, ascorbic acid, citric acid, and polyphenols were observed, although carotenoid accumulation exhibited a unique response pattern (Tables 7 and 8). The findings indicate that both plant extracts augmented crop productivity and improved the nutritional and functional quality of roselle calyx by stimulating secondary metabolic pathways. The levels of these antioxidants frequently rose with elevated concentrations of the utilized extracts. The concentration of anthocyanins escalated significantly with elevated amounts of saffron and ginger, attaining its peak in the S4G3 treatment. The increased buildup of anthocyanins noted in this study suggests that saffron and ginger extracts stimulated essential biosynthetic enzymes, such as phenylalanine ammonia lyase (PAL) and others (Sharma et al., 2019; Rouphael & Colla, 2020). The addition of saffron significantly increased flavonoid content, while ginger alone did not yield a statistically significant effect. This study reveals that saffron extract possesses more effective stimulators of flavonoid production compared to ginger extract. Nevertheless, its amalgamation with a high concentration of saffron enhanced the metabolic milieu, thereby facilitating flavonoid production (Table 8). The content of ascorbic acid increased markedly with higher doses of both extracts, however the interaction between saffron and ginger was not statistically significant. The lack of a statistically significant interaction indicates that both extracts separately promoted ascorbic biosynthesis via analogous physiological pathways, rather than generating synergistic benefits. The total phenolic content steadily rose with higher quantities of saffron and ginger, peaking in the S4G3 treatment. Similar levels of phenolic compounds have been recorded following the application of plant biostimulants in many medicinal plants, including basil, thyme, oregano, and hibiscus (Ertani et al., 2018; Bulgari et al., 2019). The findings correspond with those of Khoulati et al. (2019), who indicated that an aqueous saffron extract increases the synthesis of total polyphenols, flavonoids, anthocyanins, and condensed tannins. Flavonoids, tannins, and anthocyanins are acknowledged as secondary metabolites of polyphenols and possess equivalent biosynthetic routes, elucidating their similar reactions to saffron and ginger extracts in our current study (Yaghi et al., 2023). Research has shown that polyphenolic chemicals, such as flavonoids, phenolic acids, and anthocyanins, commonly found in plants, display many biological effects, particularly antioxidant activity. Increased antioxidant activity in pepper leaves and fruits is associated with heightened concentrations of total flavonoids, total polyphenols, and anthocyanins (Alejandro et al., 2016).

Multiple studies indicate that applying plant extracts to vegetation markedly enhances their capacity to generate antioxidant chemicals, a conclusion corroborated by the present research (Muhammad et al., 2019; Hamza et al., 2024). Research conducted by Hayat et al. (2018) showed notable alterations in the antioxidant activity of eggplant and pepper seedlings subjected to garlic clove extract. Comparable outcomes were achieved utilizing Geum urbanum extract (Kozlovska et al., 2022).

Carotenoids exhibited a unique response relative to other antioxidant components, with the greatest concentrations detected in the S2 treatment and the untreated ginger sample (G1). Conversely, increased levels of saffron and ginger led to reduced carotenoid accumulation. This inverse relationship may suggest metabolic competition among various branches of the isoprenoid and phenylpropanoid pathways. Under ideal physiological conditions, the carbon structures and reducing capacity of primary pigments, such as carotenoids, can be redirected towards the formation of phenolic and anthocyanin antioxidants, which demonstrate enhanced antioxidant effectiveness. Similar changes in the distribution of secondary metabolites have been observed in medicinal plants exposed to biostimulants and natural stimuli, marked by an increase in phenolic compound synthesis and a decrease in carotenoid accumulation (Li et al., 2020; Ertani et al., 2018). The strong S-G interaction in carotenoids suggests that the balance between pigment synthesis and antioxidant metabolism depends on the collaborative effects of both extracts.

Our findings indicate that saffron and ginger extracts significantly improved the ability of roselle to suppress DPPH free radicals in both the leaves and calyx (Table 9). The rise of extract concentrations resulted in a gradual enhancement of antioxidant activity, with peak values recorded at the highest concentrations of saffron (S4) and ginger (G3). The observed interaction in DPPH activity indicated that the combined use of the two extracts enhances antioxidant capacity beyond that of each extract individually, particularly at the highest dosage of each stimulant (S4G3). The findings confirm that saffron and ginger extracts serve as plant growth enhancers and potent natural stimulators that improve the antioxidant defense mechanism in hibiscus. The increased DPPH free radical scavenging ability is closely linked to the concurrent accumulation of anthocyanins, flavonoids, phenolic compounds, and ascorbic acid, as evidenced in this study. Many researches have identified a correlation between polyphenols, flavonoids, and DPPH free radical scavenging ability. Khoulati et al. (2019) established a link between DPPH free radical scavenging activity and the concentrations of polyphenols, flavonoids, lycopene, concentrated tannins, and anthocyanins. Akullo et al. (2023) noted a comparable result, establishing that the antioxidant activity of the extracts, assessed via the DPPH assay, was significantly correlated with their total phenolic and flavonoid content. The notable increase in antioxidant activity after the administration of saffron or ginger can be chiefly ascribed to their chemical makeup and the existence of compounds with marked antioxidant characteristics (Ali et al., 2018; El-Naggar et al., 2022). The improved antioxidant activity associated with the combined S4G3 treatment is likely due to the synergistic effects of bioactive substances from both extracts.

Therefore, these findings endorse the proliferation of plant-derived biostimulants as sustainable substitutes for synthetic chemical growth enhancers. Plant extracts are biodegradable, ecologically safe, and enhance agricultural output.

Materials and Methods

Plant materials

A field experiment was conducted from May 9 to November 20, 2023, to investigate the impact of foliar application of varying concentrations of saffron (Crocus sativus L.) stigma extract and ginger (Zingiber officinale Roscoe) rhizome extract, along with their interactions, on the yield, NPK content, and antioxidant constituents of Roselle (Hibiscus sabdariffa L.) in Al-Qadisiyah Governorate, Iraq. Table 1 displays specific chemical and physical properties of the cultivated soil at a depth of 0-30 cm. Roselle seeds, saffron stigmas, and ginger rhizomes were obtained from local marketplaces.

Table 1. Physical and chemical properties of soil.

Field preparation

The cultivation area was partitioned into three blocks, with each block comprising 12 experimental units, each unit size 6 square meters (2 × 3 m). The seeds were planted in rows within the experimental units on May 9, 2023. Seeds were sown at a depth of 3 cm on one side of the row, with a spacing of 50 cm between plants. Three weeks post-planting, seedlings were reduced to a single plant per hole. The experimental plot received 90 kg.ha^-1 of nitrogen fertilizer in the form of urea (46% nitrogen), half of which was applied during soil preparation, and 80 kg.ha^-1 of phosphate fertilizer in the form of phosphorus pentoxide during soil preparation also. After six weeks of sowing, another dose of nitrogen fertilizer at a rate of 90 kg.ha^-1 was applied (Nasrallah, 2012). Surface irrigation of the plants was conducted 2-3 times weekly during the initial growth stages, subsequently reducing to twice weekly. Additional agricultural practices, including weeding, trimming, and pest and disease management, were conducted as necessary.

Treatments

Preparation of the aqueous extract of saffron and ginger

Dried saffron stigmas were utilized, finely milled with a laboratory grinder, and the resultant powder was preserved at -20°C until required. One day prior to spraying, 1, 2, and 3 grams of the powder were measured and individually dissolved in 1L of distilled water to achieve concentrations of 1, 2, and 3 g.L^-1, along with a control at 0 g.L^-1. The mixture was agitated for one hour and subsequently allowed to rest for 24 hours at ambient temperature in the absence of light to avert photo-oxidation. The mixture was filtered with Whatman No. 1 filter paper, and the filtrate was promptly utilized for spraying.

For ginger, after cleaning, the ginger rhizomes were segmented into small pieces and dehydrated at 40-45°C to avert the destruction of phenolic chemicals and maintain their weight. The materials were further finely milled with a laboratory grinder and sieved through a 40-50 mesh screen to achieve a uniform powder. One day prior to application, 5 g and 10 g of the powder were measured and individually dissolved in 1 liter of distilled water to achieve concentrations of 5 g.L^-1 and 10 g.L^-1, respectively, along with a control group at 0 g.L^-1. The mixture was agitated for one hour and subsequently allowed to rest at ambient temperature in darkness for twenty-four hours. The mixture was filtered using Whatman No. 1 filter paper, and the filtrate was promptly utilized for spraying (Stoilova et al., 2007).

Duration of the spray

Four concentrations of saffron stigma extract, designated as S1, S2, S3, and S4, were produced at 0, 1, 2, and 3 g.L^-1, respectively. Ginger rhizome extract was produced at three concentrations: 0, 5, and 10 g.L^-1, labeled as G1, G2, and G3. Application of these concentrations occurred twice during the season: the first spray was administered 45 days post-germination, when the plants reached a height of 30-40 cm and were in the early branching phase. The second spray was one month later, when the plant reached around 80 cm in height and entered the flower bud production phase. Spraying occurred in the early morning, with a one-day delay between the application of saffron and ginger to enhance the absorption of bioactive compounds. A few drops of Tween-20 was employed as a wetting agent in the spray solution.

Experiment design

The experiment was conducted in a randomized complete block design with two factors. The first factor reflected four concentrations of saffron stigma extracts. The second factor indicated ginger rhizome extract at three concentrations. There were 12 combination treatments with three replicates.

Measurements

Approximately 140 days post-planting, the plants were harvested, and the subsequent measurements were recorded. Yield component measurements and chemical tests were conducted on individual plants from each replicate..

First: Yield assessments

Three plants were randomly chosen from each planting row (nine plants per experimental unit) for yield assessment. The measurements comprised the fresh and dry weights of calyxes per plant (g), the fresh and dry weights of fruits per plant (g), the fruit count (nuts), and the total yield (Mg ha-1). The total yield was determined based on a plant density of 19,047 plants per hectare (Dibeen, 2022)

Second: Assessment of NPK in Roselle calyxes

A sample of calyxes was collected and meticulously cleansed to eliminate contaminants. The material was further dried in an electric oven at 70°C until a consistent weight was attained. The calyxes were pulverized utilizing a Thomas-Wiley Model 4 laboratory grinder fitted with a 30-mesh screen, subsequently enclosed in hermetically sealed plastic bags, and preserved in a desiccated environment. A wet digestion was conducted. The nitrogen content in the digested calyxes was assessed following Chapman & Pratt (1961), the phosphorus content was evaluated according to Jackson (1970), and the potassium content was measured as per Schuffelen et al. (1961).

Third: Assessment of antioxidants

Carotenoid content (mg.100g^-1 FW) were quantified utilizing the spectrophotometric technique (Lichtenthaler, 1987) using the formula

Carotenoids = (1000A₄₇₀​−1.82C_a​−85.02C_b​​ )/ 198

Where A₄₇₀ absorbance at 470nm, C_a Chlorophyll a concentration, C_b Chlorophyll b concentration. Then, the result converted to mg.100g ^-1 FW

The total anthocyanin content was quantified via UV-Vis spectroscopy, and the concentration (mg. 100 g^-1 fresh weight) was determined following the methodology of Wan Zaliha (2009). The total flavonoid content was quantified using a spectrometer, with the TFC value derived from the calibration curve and expressed as quercetin equivalents (mg QE.g^-1) of the extract (Pacôme et al., 2014). The ascorbic acid content was quantified utilizing the spectrometric method established by Jagota and Dani (1982). Citric acid was quantified following Kyllingsbaek (1984), while phenolic content (mg GAE g⁻¹ DW) was assessed using the Folin-Ciocalteu test as per Zhen et al. (2016), with results expressed in mg g^-1 of gallic acid.

Fourth: Assesment of antioxidant activity

The antioxidant ability of 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a free radical scavenger was assessed in leaves and calyxes using the methodology of Liu and Yao (2007).

Statistical analysis

The data obtained were statistically tested using analysis of variance (ANOVA) at the 5% significance level. When a significant effect was observed, differences among means were compared using DMRT at the 0.05 level.

Conclusion

The current study shown that the utilization of saffron stigma and ginger rhizome extracts on roselle significantly improved the plant's yield, NPK content, and antioxidant components. The enhancement was most significant at the maximum concentration of both extracts—3 g. L^-1 (S4) demonstrated the highest overall efficacy—while a concentration of 10 g. L^-1 of ginger (G3) led to a marked improvement in crop components, mineral nutrition, phenolic compounds, and DPPH free radical scavenging activity. The collaboration between the two research agents produced enhanced results, particularly at increased dosages of both biostimulants, with the S4G3 mixture achieving the highest values for the majority of yield and quality parameters. Antioxidants, such as polyphenols, anthocyanins, flavonoids, ascorbic acid (vitamin C), and citric acid, were increased in plants subjected to the extracts, and this increase was associated with improved antioxidant activity as measured by the DPPH assay. The results indicate that the combined use of saffron and ginger extracts is an effective and environmentally sustainable method for improving roselle yield and phytochemical quality.

Acknowledgments

The authors express gratitude to the faculty and staff of Al-Mustaqbal University/College of Agriculture Techniques and Al-Qadisiyah University/College of Nursing for their unwavering support.

Statement of contributions

The authors work as one team in doing every stage of the experiment and preparing the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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