Hybrid Percolation–Ultrasound Method for Extracting Bioactive Compounds from Urtica dioica and Salvia officinalis

Abstract

Enhancing the efficacy of biofertilizers and biopesticides for horticultural applications presents numerous challenges, given the need to balance effectiveness with environmental and economic factors. Achieving these goals requires rigorous research into advanced technologies and formulations capable of effectively replacing or complementing traditional chemical inputs, without compromising crop yield or quality. The present study aimed to develop a versatile and yet accessible hybrid percolation–sonication system and method, designed to optimize polyphenol extraction from nettle and sage plants. The resulting extracts were combined and applied on organic tomato crops, to evaluate their biofertilizer and biopesticide effectiveness, in comparison to conventional chemical inputs. Operating the equipment in a hybrid percolation–sonication system led to a 36% increase in polyphenols extraction for nettle and a 29% increase from sage. Regarding the effect on tomatoes, data showed that plants treated with biofertilizer extracts were over 42.88% more productive than control samples and 17.67% more productive than tomatoes treated with chemical fertilizers. Tomato plants treated with biofertilizers grew approximately 10% taller and developed stems that were 5% thicker compared to those treated with chemical fertilizers.

1. Introduction

Widespread use of chemical inputs in agriculture has become a common practice due to major productivity benefits. However, prolonged use of these substances has shown considerable adverse effects on human health and the environment. These chemicals disrupt natural biological balances, negatively affecting biodiversity and contaminating agricultural soils and water resources [1,2,3]. They also contribute to the intensification of harmful phenomena such as desertification, which have the primary effect of degrading fertile lands. To reduce the dependence on synthetic chemicals in agriculture, it is important to adopt alternative treatment solutions such as plant extracts, which are rich in bioactive compounds. These biological alternatives provide natural protection against pests and diseases and improve soil fertility, promoting sustainable agriculture [4]. This endeavor requires innovative approaches that can optimize nutrient uptake, secure pest control, and provide overall plant health, while adhering to sustainable practices.

Specialized literature has shown that extracts from nettle (Urtica dioica) and sage (Salvia officinalis) can significantly enhance the growth of vegetables cultivated in open spaces, while also providing protection against diseases and pests. A study carried out on tomato crops [5], highlighted the beneficial effects of nettle extracts on tomato seedlings, improving both tomato growth and metabolism. Nettle extracts significantly increased chlorophyll levels and enzyme activity. These extracts also activated genes related to cytokinin production, contributing to the more vigorous and healthy development of tomato seedlings. The 300 ppm aqueous extract of nettle notably increased growth rate, chlorophyll and phenol content, and antioxidant enzyme activity. This research showed that nettle extracts can serve as an effective growth stimulant for tomato crops, offering an organic alternative to traditional chemical compounds.

A study compared the effectiveness of nettle extract with conventional chemical pesticides in controlling aphid development [6]. The results indicated that bioactive compounds from nettle can effectively maintain pest levels below the economic damage threshold, while supporting satisfactory tomato yields. The solution was prepared by soaking 1 kg of nettle leaves in 10 L of water. Another study [7] examined the acaricidal and ovicidal effects of sage extracts on Tetranychus urticae mites, a common pest affecting fruits and vegetables. The extracts were obtained from dried leaves and tested at four different concentrations: 1%, 3%, 6%, and 12%. The results indicated that sage extract at a 12% concentration exhibited the highest efficacy, achieving a mortality rate of 79% for nymphs, 62.2% for adults, and an ovicidal effect rate of 30.2%.

Evaluations of sage extract’s impact on tomato pests showed that concentrations of 2000 and 4000 ppm can significantly reduce the hatching of Meloidogyne incognita nematode eggs and reduce the severity of transmitted viral diseases [8,9]. A research study [10] evaluated the yield of obtaining phenolic compounds from medicinal plants, using ultrasonic-assisted extraction (UAE). Compared to the traditional extraction method by boiling at a temperature of 90 °C for 2 h, ultrasonically assisted extraction proved to be much more efficient in obtaining polyphenols, offering higher yields and a reduced extraction time, especially at temperatures of 60–70 °C. Another study [11], comparing conventional extraction with UAE techniques for obtaining phenolic compounds from sage found that ultrasound processing is more efficient, offering shorter extraction times and higher yields of bioactive compounds. The optimal conditions for UAE according to the proposed method were 11 min extraction time, ethanol concentration of 30%, and 400 W output power of the ultrasonic device. The yield for UAE for obtaining total phenols was 6.77552 g RA/100 g (RA—rosmarinic acid equivalents). Bioactive compounds such as 1,8-cineole, camphor, and cis-thujone were extracted from Salvia officinalis L. using an ultrasonic bath with a total nominal power of 3 × 50 W and a frequency of 40 kHz, while maintaining the temperature at 40 ± 1 °C [12]. The optimal results were achieved using water as the solvent, an S/L ratio of 1:10, and an extraction time of 20 min.

A comparative evaluation of different methods for extracting polyphenols from nettle demonstrated that UAE increased efficiency when using 50% ethanol in water as solvent [13]. The methods included conventional extraction at room temperature for 24 h and UAE with two systems: a probe system (200 W, 5–15 min) and a bath system (35 kHz, 10–30 min). The UAE method required a shorter extraction time and yielded higher results compared to conventional extraction. However, the presence of alcohols may complicate the application process of the biofertilizer for plant treatment. In another study [14] UAE was conducted on nettle leaves using water as the solvent in a sonication bath. The process parameters included an ultrasonic power of 156 W, a S/L ratio of 30:1, and an extraction time of 30 min. The total amount of polyphenolic compounds extracted via UAE was 71.44 mg/L.

The extracts obtained in the mentioned studies are beneficial because they contain polyphenols, compounds known for their health-promoting phenolic groups. They play an important role in plant defense against diverse stresses and provide numerous benefits for plant health, such as antioxidant, antibacterial, and pesticidal effects [15,16,17,18,19,20]. These compounds safeguard cells and inhibit the growth of harmful microorganisms, making them valuable in functional nutrition and the pharmaceutical industry [21,22,23,24,25,26]. Polyphenols have also shown effective bio-stimulative properties in organic agriculture. They improve the metabolism of cultivated plants and significantly contribute to increasing production yields [27,28]. Several studies [5,29] have found that polyphenols extracted from nettle and sage possess biofertilizer properties that enhance plant growth rate, chlorophyll and phenol content, and antioxidant enzyme activity. Additionally, these extracts reduce the incidence of fruit rot disease and decrease the severity of late blight attacks. Polyphenols have demonstrated effective repellent properties against insects, aphids, mites, and nematodes. Studies [7,30] showed that these compounds disrupt the neurotransmitters and sensory receptors of pests, making plants less attractive for feeding and egg-laying. Additionally, polyphenols can inhibit the growth and development of larvae and nymphs, helping reduce pest populations. This provides a natural alternative to chemical pesticides for crop protection.

The aim of the present research was to assess different operational regimes of a novel ultrasound–percolation hybrid technology for extracting polyphenols from nettle and sage plants. The development of a hybrid percolation–ultrasound extraction method using water as the solvent has not been adequately explored in previous research. This approach is designed to maximize polyphenol content for usage as an organic treatment in horticulture. The synergy created between these techniques offers significant extraction advantages, enhancing efficiency while ensuring cost-effectiveness and ease of implementation. The extracts with the highest polyphenol content were then combined into mixtures and applied to tomato crops, both as soil and foliar treatments, in order to assess their protective effects on plants. The bioactive extracts testing on tomato plants aimed to identify their effects, in comparison with control samples and chemical treatments. This was achieved by evaluating four key indicators: plant thickness, plant length, chlorophyll content, and tomato productivity per plant.

2. Materials and Methods

2.1. Research Design

Figure 1 illustrates the experimental design, which includes plant cultivation, mechanical processing, hybrid ultrasound–percolation extraction, and polyphenol content identification. The potential of the extract was assessed by applying the formulations on tomato crops.

2.2. Plant Harvesting and Preparing for Processing

Nettle (Urtica dioica) and sage (Salvia officinalis) plants were harvested in April–May from two experimental crops established in Ilfov county, Romania (Figure 2 and Figure 3).

The experimental crops measured 40 m in length and 1 m in width each and were established in 2022. The plants were cut 5 cm above the ground, dried in an oven for 12 h at 105 °C, and then shredded into pieces no larger than 3 cm using automatic plant-cutting equipment. The shredded plants were weighed and placed into permeable mesh bags.

2.3. Hybrid Percolation–Sonication Technology and Extraction Parameters

For efficient extraction of polyphenols from nettle and sage, several operational methods aimed to maximize the efficiency of percolation and sonication, both individually and in combination. The percolation process utilized a modified TIMATIC Duo percolator (Tecnolab srl, Spello, Italy), capable of operating under both low and high pressures, in conjunction with the Hielscher UP400St ultrasound generator (Hielscher Ultrasonics GmbH, Teltow, Germany), which was connected to the extraction vessel. The designed system consisted of an extraction chamber made for processing dry and shredded plant samples; a control panel that allows setting the time and pressure; and an expansion tank having a dual role: removing the air from the installation before the actual start of the extraction process as well as supplying the installation with the amount of solvent necessary to carry out the process at optimal conditions; a compressor that allows pressure regulation, 2 hydropneumatic cylinders, 1 pump, and 1 ultrasound generator that induces the phenomenon of cavitation in the solvent and helps to release the compounds from the vegetable matrix (Figure 4). Distilled water was selected as a solvent due to its non-toxicity and cost-effectiveness.

The dried plants were shredded and placed into permeable mesh bags (Figure 5), and then loaded into the extraction chamber of the percolator. The solvent was then added, and the operating parameters (pressure, time, power) were set.

The extraction process was conducted either solely by percolation (under low—5 bar or high—7 bar pressures), solely by sonication (using ultrasonic waves), or by combining the two extraction methods—hybrid percolation and sonication method.

Therefore, five methods of processing the plant material were tested (the degree of shredding of the plants being 3 cm for all samples), while the extraction parameters established for each option were according to

Table 1. Parameters of the extraction processes of polyphenols from nettle and sage.

	Pressure	Time	Cycles	Percolation	Power	Amplitude
unit	bar	min	-	-	W	%
E1	5	60	6	8	-	-
E2	7	60	6	8	-	-
E3	5	60	6	8	140	20
E4	7	60	6	8	140	20
E5	-	60	-	-	140	20

E1—percolation at low pressure; E2—percolation at high pressure; E3—percolation at low pressure and ultrasound; E4—high-pressure percolation and ultrasound; E5—sonication.

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The selected processing modes were designed to evaluate if hybrid ultrasound–percolation processing produces superior results compared to methods that use only percolation or ultrasound individually. The experimental parameters outlined in Table 1 were chosen to test comparable processing variations, with each extraction time limited to one hour. The values were selected based on previous research and other studies that utilized water as the solvent for the extraction of polyphenols. Ultrasonic power of 140 W and an amplitude of 20% were selected to ensure efficient and uniform cavitation, optimizing the desired effects on the plants.

For all the tested methods, the temperature in the extraction chamber was monitored with a temperature sensor to ensure it did not exceed 60 °C (since some studies found that polyphenols begin to degrade at temperatures above 60 °C) [31].

Samples processed under both low- and high-pressure regimes involve a cycle that includes a 4 min compression phase (TP1) followed by a 6 min decompression phase (TP0). The pressures alternated, the cyclic phases being illustrated in Figure 6 and Figure 7.

2.4. Total Polyphenols Content

The physico-chemical analyses of the produced extracts were carried out using the Folin–Ciocalteu spectrophotometric method, which consisted of the extraction of total polyphenols in a methanol–water mixture (1:1), treatment with the Folin–Ciocalteu reagent and measurement of absorbance at λ = 755 nm, with the quantification of phenolic compounds being carried out based on the gallic acid calibration curve and expressed in mg GAE/100 g.

2.5. Antioxidant Capacity

The antioxidant capacity of nettle and sage extracts was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) method at λ = 517 nm, using the Trolox calibration curve in the concentration range 0–0.4375 mmol/L.

2.6. Procedure for Evaluating Bioactive Extracts Influence on Tomato Crops Grown in Pots

The biofertilizer extracts influence evaluation was carried out in pots (for a more efficient control) on the tomato seedlings Vitara F1 type. Three types of tests were considered: ecological treatment with biofertilizing extracts, treatment with commercial phytosanitary substances, and control samples (no treatment applied). The same amount and type of soil was added to each pot: 1468 g of Agro garden substrate—obtained from quality peat and humus with a pH between 6 and 7 and the following chemical composition: N: 150–400 mg/L, P₂O₅: 80–250 mg/L, and K₂O: 250–600 mg/L. Therefore, 5 pots were treated with a mix of extracts obtained from nettle and sage, 5 pots were chemically treated with NutriCig—fertilizer, Nissorun—acaricide, Movento—insecticide, and Score—fungicide, and 5 pots were left untreated as a control sample. For all methods, the experiment was replicated 3 times. The Vitara F1 hybrid was chosen because it offers some advantages such as high productivity, superior fruit quality, and resistance to common diseases (fusarium and verticillosis). This hybrid has a short growth cycle, making it ideal for early production and multiple annual harvests.

After evaluating five extraction methods, the most effective in terms of polyphenol content was chosen for processing the plants. The biofertilizer was then produced by combining equal parts of sage and nettle extracts. The organic extract was prepared by combining 100 mL of nettle and sage extract in one liter of water. All tomato pots were monitored for 17 weeks, from seedling planting until reaching maximum tomato maturity. The biofertilizer extract and chemical treatment were applied as depicted in Figure 8, in the time interval 6:00–7:00 a.m.

The plants treated with chemical substances were sprayed with the recommended doses according to the manufacturer’s guidelines during weeks 8 and 12. All plants were irrigated daily between 6:00 and 7:00 a.m., each pot receiving an amount of 0.5–1.5 L of water. The average ambient temperature during the experiment was about 33 °C. The amount of precipitation recorded was very low and of low intensity, and the relative humidity of the air varied between 20% and 23%.

Tomato leaf chlorophyll was monitored twice a week, conducting a total of 34 measurements during the monitoring period. The measurements were made with the Hansatech model CL-01 equipment. The monitoring of tomato stem thickness was carried out with GEAKISO electronic measuring equipment.

2.7. Statistical Analysis

Statistical analysis helps in identifying the probability that the observed differences between treatment groups (treatment using the bioactive extract, chemical input, and control sample) are not attributable to random variation. The comparison of the main quality parameters (stem thickness, plant length, chlorophyll content, and fruit productivity), between tomato plants treated with biofertilizer extracts and chemical inputs versus untreated control samples was conducted using t-tests, one-way ANOVA, and Tukey’s HSD test. A t-test (two-tailed, independent two-sample) was conducted to compare plant thickness and length evolution in the 17 weeks of crop development. The two-tailed test was chosen to evaluate the possibility of a difference in either direction—whether the treatment could either increase or decrease compared to the control, while the two groups (treated vs. control samples) were considered independent. A one-way ANOVA was employed to compare the effects of biofertilizer, chemical treatment, and control on tomato crop productivity and chlorophyll evolution over time. This approach was chosen to efficiently evaluate significant differences among the means of all three groups in a single test. For the productivity indicator, an additional Tukey’s HSD test was conducted, in order to determine which of the three treatments significantly differs from the control, thereby identifying which treatment produces a statistically different effect. The evaluations were made in Microsoft Excel 2019.

3. Results

3.1. Total Polyphenol Content and Antioxidant Capacity of Nettle and Sage Extracts

The total polyphenol content and antioxidant capacity for nettle and sage across the five extraction modes (E1–E5) are presented in

Table 2. Total polyphenol content and antioxidant capacity of nettle and sage extracts.

Extraction Mode	Total Polyphenol Content from Nettle Extracts (mg GAE/100 g)	Antioxidant Capacity for Nettle Extracts (mg Trolox/100 g)	Total Polyphenol Content from Sage Extracts (mg GAE/100 g)	Antioxidant Capacity of Sage Extracts (mg Trolox/100 g)
E1	5.19 ± 0.12	11.78 ± 0.28	17.19 ± 0.25	61.79 ± 1.78
E2	6.24 ± 0.17	11.16 ± 0.28	20.75 ± 0.38	66.11 ± 1.47
E3	7.58 ± 0.14	11.83 ± 0.20	23.1 ± 0.28	67.47 ± 1.62
E4	8.16 ± 0.20	12.66 ± 0.30	24.24 ± 0.51	70.35 ± 1.67
E5	7.09 ± 0.17	12.03 ± 0.30	21.51 ± 0.38	72.74 ± 1.71

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For nettle, method E4 recorded the highest antioxidant activity. In the case of sage, while method E5 showed a slightly higher antioxidant activity than E4, the difference was not significant. Since method E4 also yielded the highest concentration of polyphenols, it was decided that the experiment would be conducted exclusively using extracts obtained by the E4 method for the tomato treatment. The evaluations were conducted to obtain relevant data on the growth rate of tomato plants, pest resistance, and fruit productivity, using several key indicators. Furthermore, by weighing the dry plants before processing, and after the experiment, the percentage of organic matter transferred from the plants to the solvent (distilled water) was determined. For nettle, the result was between 29.88 and 33.24%, and for sage between 32.21 and 36.08%.

3.2. Chlorophyll Variation

Figure 9 illustrates the impact of two fertilizer treatments on the chlorophyll content of tomato leaves, an essential pigment for photosynthesis and plant health. The study aimed to determine which type of fertilizer—biofertilizers, chemical fertilizers, or no treatment (control)—most effectively increases chlorophyll content, given its critical role in optimal plant development.

The increase in chlorophyll was closely correlated with the application of the fertilizing solution. However, additional analysis is required to assess the statistical significance of these results. Chlorophyll analysis using ANOVA showed an F-value of 4.26. This value is larger than the critical F-value (F crit = 3.19), suggesting that there is a statistically significant difference between the means of the groups. In addition, the p-value was 0.0198, which is less than the common alpha level chosen, of 0.05. This means that there is strong evidence against the null hypothesis, and there is a statistically significant effect on tomato productivity.

3.3. Tomato Plant Stem Height and Stem Thickness

Given the close values observed for tomato plant stem heights and stem thicknesses across the monitoring weeks 1–6, Figure 10 and Figure 11 illustrate only the measurements taken between weeks 6 and 17. These measurements were conducted to investigate whether there were significant variations between the three experimental groups over a longer period.

Regarding the statistical evaluation of plant height, although there were notable variations of 7–21% between treated and untreated plants during the assessment period, the analysis revealed no significant correlation between the treated plants (both biofertilizer or chemical input) and the control group. It is likely that the growth of the plant stems was influenced by random factors that were not considered in the study, such as the positioning of the pots. The t-test conducted to compare the stem thickness indicated a p-value of 0.0402, which is below the conventional threshold of 0.05. This suggests that the difference in stem thickness between the treated and control plants is statistically significant. The obtained t-statistic of 2.18 suggests a moderate difference between the treated and control groups. Although is not very high, it is sufficient to reject the null hypothesis, suggesting that the biofertilizer treatment had a statistically significant effect on stem thickness. The t-test comparing the stem thickness of tomato plants treated with chemical substances to that of the control group yielded a p-value of 0.166. This indicates that the difference in stem thickness between the chemically treated plants and the control group is not statistically significant.

3.4. Tomato Plant Productivity

Tomato productivity measured as fruit weight on each monitored plant can be found depicted in Figure 12.

The quantities of tomatoes produced indicate that plants treated with biofertilizers achieved the highest productivity, demonstrating superior effectiveness in enhancing tomato yield. The increase in productivity was primarily driven by an increase in fruit mass rather than by an increase in fruit number, which could suggest a foliar fertilizing effect. The ANOVA results indicate that there are significant differences among the means of the three groups (tomato plants treated with biofertilizer extracts, tomato plants treated with chemical inputs, and control sample). The high F-value (approximately 12.6) suggests that the differences between the group means are not due to random chance. Since the p-value is very small, the null hypothesis, that all group means are equal, was rejected. This means that the type of treatment (biofertilizer, chemical inputs, or control) has a significant effect on the measured outcome. Since ANOVA does not reveal which of the treatments have a significant effect, Tukey’s HSD Test Calculation was performed to identify which specific treatments are most effective, or different from others.

Tukey’s HSD Test Calculation showed that the mean difference between biofertilizer vs. control is significant, indicating that the biofertilizer treatment and control samples have different effects on the outcome measure. In contrast, the analysis revealed no significant difference between chemical inputs and control samples, indicating that chemical inputs do not produce a statistically different effect compared to untreated control.

3.5. Monitoring of Tomato Growth

Figure 13 presents a comparison of tomato growth during week 15 of monitoring, highlighting that tomatoes treated with biofertilizers recorded approximately 5% more growth compared to the other crops.

The control tomato samples, (untreated), showed several pest issues (aphids: Myzus persicae “black peach aphid”, mites: Tetranychus urticae “common red spider mite”) throughout the observation period. The absence of preventive or curative measures made these plants more susceptible to insect infestations and other harmful organisms, thereby affecting their overall health and productivity. Figure 14 shows some images of the pest attack on tomatoes.

4. Discussion

The hybrid ultrasound–percolation method achieved excellent results in the extraction of polyphenols from nettle and sage, by combining the strengths of both techniques. Ultrasound generates cavitation bubbles in the solvent, which effectively penetrate plant cell walls, while percolation ensures a continuous solvent flow through the plant material, maintaining high solvent-to-sample contact. Additionally, the process was kept at low temperatures, preventing the degradation of sensitive polyphenols and preserving the bioactivity of the extracted compounds. By incorporating ultrasound into the extraction processes, improved yields of polyphenols were achieved, as confirmed by other studies [32,33].

The most efficient extraction method among those analyzed was percolation combined with sonication at high pressures, which produced the highest concentration of polyphenols (8.16 ± 0.20 mg GAE/100 g). The extraction time for the method was only 1 h, which is notably shorter compared to the study [32], where the ultrasound-assisted extraction method required 4 h of processing. Additionally, the results were superior to those reported in study [34], where extracts were obtained from dried nettle using a mixture of 20% ethanol and 80% water and left at room temperature for 72 h, resulting in a total polyphenol content of 18.46 ± 0.50 GAE/g. In another study [35], a concentration of 19.67 mg GAE/g was reported for polyphenols extracted from sage using the conventional method. This is lower than the total polyphenol content achieved with the hybrid percolation–sonication extraction, which reached 24.24 ± 0.51 mg GAE/100 g.

Regarding the effect of the extracts on the treatment of tomatoes, the results were satisfactory both in terms of bio-stimulation and pesticidal effects. The polyphenol-rich extracts promoted the development of the tomato plants, enhancing their growth and overall health. This bio-stimulatory effect was evident in increasing chlorophyll content, and a better resistance to environmental stressors. Additionally, the extracts demonstrated effective pesticidal properties, reducing the incidence of common pests such as aphids and mites. Although several extracts have been tested on vegetables in other studies [36,37,38], the current research demonstrated the effect of the combination of nettle–sage extracts on tomatoes.

In the experiment, it was found that the treatments applied to the tomatoes had a positive effect on the amount of chlorophyll, the height of the plant, and the thickness of their stems. Untreated plants had the lowest growth, highlighting the importance of applying treatments to optimize plant development. An unexpected outcome was that plants treated with organic fertilizer exhibited slightly higher productivity compared to those treated with chemical inputs. This suggests that, under the ideal growing conditions established in the experiment, organic fertilizers can rival chemical ones in terms of efficiency. Although all parameters were higher with biofertilizer extract application, the results showed the strongest statistical significance for chlorophyll and productivity.

In terms of pest control, both the plant extracts and the chemical inputs demonstrated high performance. However, since the control samples also showed low pest damage, this suggests that the weather conditions during the analyzed period were unfavorable for pest development.

The extraction method presented significant advantages, including cost-effectiveness and ease of implementation. The use of water as a solvent makes the extracts ideal for horticultural applications. The intensive application of biofertilizers boosted productivity by 42.88% compared to the control group (as evidenced by Figure 12). As many companies in Romania are shifting toward organic practices due to the higher profitability of tomatoes, effective biofertilizing solutions are increasingly valuable.

5. Conclusions

The hybrid extraction method ultrasound–percolation (utilizing both high and low pressures) demonstrated significant benefits in enhancing polyphenolic content in a cost-effective and efficient manner. Consequently, the advancement of these technologies could lead to the production of eco-friendly alternatives to chemical treatments, providing a sustainable solution for improving tomato crop yield and quality. The combination of nettle and sage extracts can serve as a biofertilization treatment for tomato crops. However, further research is needed to validate their effectiveness in pest control and determine pest sensitivity to these biofertilizing solutions.

Compared to the control samples, tomatoes treated with biofertilizers exhibited a 31.53% increase in chlorophyll content, reflecting more efficient photosynthesis and better plant health. Additionally, productivity improved by over 42.88%, demonstrating the potential of biofertilizers to enhance agricultural performance and plant development. The main limitation of the present experiments was the use of water as the solvent. Previous research indicates that other solvents can more effectively extract polyphenols from plants, although the subsequent synthesis can be more complex.