Foliar Application of a Methanolic Extract of Ricinus communis L. Modulates Growth, Yield, Photosynthetic Pigments, and Antioxidant Capacity of Jalapeño Pepper (Capsicum annuum L.) Under Open Field Conditions

Abstract

The jalapeño pepper (Capsicum annuum L.) is a crop of great economic and nutritional importance worldwide; however, increasing yield and quality under conditions of reduced synthetic inputs remains a significant challenge, mainly due to restrictions in plant nutrition and stress response capacity; in this context, plant-based biostimulants, such as Ricinus communis extracts, are of particular interest due to their potential to modulate plant metabolism, promote growth, and favor the accumulation of bioactive compounds. In this study, the effect of a foliar-applied biostimulant derived from a methanolic extract of Ricinus communis L. on the physiological, agronomic, and biochemical parameters of jalapeño peppers was evaluated under open field conditions. A randomized complete design with five treatments was established: three extract concentrations (T50: 50 mg L⁻¹, T75: 75 mg L⁻¹, and T100: 100 mg L⁻¹), a commercial biostimulant (Pepton 85/16 ^®), and an absolute control. Significant differences (α ≤ 0.05) were observed between treatments T50, T75, and T100 with the application of castor bean and the absolute control in stem diameter, fruit number, yield, and polar and equatorial fruit diameter, as well as phenols, flavonoids, and antioxidant capacity (ABTS and DPPH). The application of R. communis extract (T50, T75, and T100) significantly improved plant performance compared to the control, particularly in yield (up to 270%), fruit number (73%), shoot biomass (up to 38%), and root development (up to 32%). Furthermore, increases in chlorophyll content and in antioxidant-related compounds were observed, including phenols, flavonoids, ABTS, and DPPH (up to 17%). Spearman correlation analysis revealed strong associations between structural and metabolic variables, highlighting the relationship between stem diameter, fruit traits, and bioactive compound accumulation, as well as the link between chlorophyll content and reproductive performance. The ¹H NMR analysis indicated the presence of secondary metabolites such as ricin, unsaturated fatty acids, and phenolic compounds; however, their isolation and relationship with the biostimulant activity of the extract require further specific studies. Overall, foliar application of R. communis extract improved the growth, productivity, and biochemical attributes of jalapeño pepper, highlighting its potential as a sustainable alternative for crop management.

1. Introduction

The jalapeño pepper (Capsicum annuum L.) is one of the most popular and widely cultivated chili pepper varieties worldwide. In addition to its culinary value, it stands out for its nutritional richness, containing vitamins A, C, E, and K, as well as the B complex, and essential minerals [1]. Global production of this crop reached approximately 4.9 million tons in 2024, solidifying its position as one of the leading horticultural crops worldwide [2]. However, one of the main current challenges in its production lies in increasing the yield and quality of the crop under schemes that reduce dependence on synthetic inputs, in accordance with the principles of sustainable agriculture [3].

In this context, biostimulants are emerging as a promising alternative. They are defined as substances or microorganisms that, when applied in low doses, promote physiological processes in plants such as increased nutrient absorption, photosynthetic efficiency, resistance to biotic and abiotic stress, and stimulation of root and foliar growth, without replacing traditional fertilizers or fungicides [4]. Biostimulants act by regulating hormone production, activating antioxidant defenses, and improving the efficiency of water and nutrient use [5]. Plant biostimulants have been documented to influence key physiological processes such as chlorophyll accumulation, photosynthetic efficiency, and regulation of cellular redox state, resulting in improvements in crop growth, yield, and quality. These effects have been associated with the presence of secondary metabolites, which can modulate plant metabolism and induce adaptive responses [6].

Among natural sources of biostimulants, wild plants such as castor bean (Ricinus communis L.) stand out for their richness in functional compounds, including unsaturated fatty acids, phenols, flavonoids, phytosterols, and bioactive peptides [7]. These compounds have been linked to antioxidant and plant growth-regulating activities, suggesting their possible involvement in modulating physiological responses [8]. R. communis is also well known for its allelopathic potential, as it produces secondary metabolites that inhibit germination, growth, or physiological processes in neighboring plants [9]. This dual nature underscores the need for careful evaluation of its extracts, as their biological activity may shift from inhibitory to stimulatory depending on the concentration, extraction method, and mode of application [10]. In this sense, the biological response of plant extracts depends not only on their chemical composition but also on the dose and the physiological context of the crop.

In contrast, several studies have evaluated other biostimulants in jalapeño peppers, with promising results. For example, Majkowska-Gadomska et al. [11] evaluated microbial biostimulants in a greenhouse, applied via irrigation and foliar sprays; although they did not observe consistent improvements in morphological parameters or yield, they did note increases in nitrate concentration in the fruit. In another study, Mezeyová et al. [12] applied humic hydrolysates to different chili pepper varieties, including jalapeño, and observed significant increases in total yield and average fruit weight. Golian et al. [13] observed improvements in fruit weight and bioactive compound content when applying commercial biostimulants. Nevertheless, most of these studies have focused on commercial products or microorganisms, with limited information on the use of chemically characterized wild plant extracts, particularly under open field conditions.

Therefore, the objective of this research was to develop a castor bean-based biostimulant and evaluate its effects on physiological and biochemical parameters in jalapeño chili pepper plants under field conditions. Additionally, the chemical characterization of the extract was performed using ¹H nuclear magnetic resonance (NMR) spectroscopy to establish possible relationships between its metabolic profile and the crop’s physiological response. The novelty of this study lies in integrating agronomic evaluation under field conditions with the chemical characterization of the extract, providing evidence for the possible mechanisms underlying the biostimulant effect of a plant species with recognized allelopathic potential. The hypothesis of this study is that the foliar application of the methanolic extract of Ricinus communis L. at defined concentrations (50, 75 and 100 mg L⁻¹) generates differential and statistically significant effects (α ≤ 0.05) on agronomic (yield, number of fruits, stem diameter and fruit size), physiological (chlorophyll content) and biochemical (phenols, flavonoids and antioxidant capacity) variables in jalapeño pepper plants, compared to the absolute control and the commercial biostimulant.

2. Materials and Methods

2.1. Study Area and Crop Establishment

This study was conducted in an agricultural field located in the town of Banzha, municipality of Tecozautla, Hidalgo, Mexico (20°31′41.1″ N, 99°39′53.8″ W), at an approximate altitude of 2000 m above sea level. The experiment was conducted during a single production cycle corresponding to the spring–summer season 2025. The area is characterized by a warm, semi-arid climate, with an average annual temperature of 18 °C, an average rainfall of 600 mm, and a relative humidity of about 45% [14]. Prior to crop establishment, a soil analysis was conducted to characterize its physicochemical properties. The soil was classified as Phaeozem, with a clay texture, a pH of 6.59, a cation exchange capacity (CEC) of 35 cmol kg⁻¹, and an organic matter content of 3.5%. It also exhibited a saturation point of 73%, a field capacity of 39.2%, a bulk density of 1.19 g cm⁻³, and an electrical conductivity of 0.67 dS m⁻¹, indicating non-saline conditions. These values were compared with those reported in the literature for soils from the same geographic area [15]. The experimental plot had a total area of 1000 m². Raised beds measuring 90 cm wide and 40 cm high, with a length of 50 m and 80 cm wide walkways in between, were prepared. Plants were established in two rows per bed, with 30 cm between plants within each row. Based on this planting arrangement, plant density was approximately 39,215 plants ha⁻¹. A drip irrigation system was used, employing drip tape with emitters spaced every 30 cm (Figure 1A). Irrigation water was supplied from an on-site groundwater capture well, a common practice in the region for agricultural production. Different irrigation levels were applied at each growth stage, taking into account management practices, environmental conditions, and crop development, namely, 100 mL for germination, 500 mL for vegetative growth, 1500 mL for flowering and fruit set, and 2500 mL for fruiting, as described by Flores [16]. Black plastic mulch was used in all beds for weed control. Throughout the experiment, environmental conditions were monitored in an open field. Soil moisture was controlled by scheduled irrigation to avoid prolonged water stress or waterlogging; in addition, a tensiometer (Kelway 94302, Palo Alto, California, USA) was used for continuous monitoring of soil moisture, in order to maintain it above 35% of field capacity (10–20 kPa). Climatic conditions during the experimental period were monitored using a digital hygrometer (Delmhorst HTX-30, New York, NY, USA) to record air temperature and relative humidity. The maximum temperature (25 °C), minimum temperature (14 °C), and relative humidity (37%) values were verified and supplemented with information from the National Water Commission (CONAGUA) to ensure the accuracy and representativeness of the environmental conditions [17].

Two hundred seeds of jalapeño pepper (Capsicum annuum L.) variety “Mixteco” (Harris Moran^®, Modesto, CA, USA) were sown in germination trays, using a substrate composed of peat moss and perlite in a 1:1 (v/v) ratio. The seedlings were kept under nursery conditions and, 30 days after sowing, when they reached an average height of approximately 15–20 cm, they were transplanted to the experimental field. This variety was selected for its adaptability to the region’s agroclimatic conditions and the growing season’s temperatures. Transplanting was carried out in a staggered planting pattern, with a spacing of 35 cm between plants. Fertilizer application was managed through the drip irrigation system using a fertigation strategy adjusted to the crop phenological stage. Specifically, 6 mM NH₄NO₃, 1.6 mM K₂HPO₄, 0.3 mM K₂SO₄, 4 mM CaCl₂, 1.4 mM MgSO₄, 5 µM Fe-EDDHA, 2 µM MnSO₄, 0.25 µM CuSO₄, and 0.5 µM H₃BO₃ were used, in accordance with agronomic recommendations commonly reported for jalapeño pepper cultivation under field conditions [18]. Nutrient supply was fractionated during the crop cycle, namely, approximately 25% during the vegetative stage, 50% during flowering, 75% during fruit set, and 100% during fruit filling and harvest, following the nutrient solution approach described by Sanchez [19]. This fertilization regime was applied uniformly across all treatments to ensure that observed differences were attributable to the biostimulant application rather than variations in mineral nutrition. Throughout the crop’s phenological cycle, cultural practices such as manual weeding and disease monitoring were implemented to maintain optimal growing conditions. Additionally, phytosanitary management was implemented through the targeted application of chemical products to control key pests. For whitefly (Bemisia tabaci), Confidor 350 SC was applied at transplanting (2 mL L⁻¹ of water), while Dimethoate 400 EC (1.0–1.5 L ha⁻¹) was used during crop development. For aphid (Aphis spp.) control, Malathion 1000 was used at a dose of 0.5 to 1.0 L ha⁻¹. The presence of defoliating larvae, such as the false looper, was managed by applying Ambush 34 (0.4–0.6 L ha⁻¹). Applications were based on field monitoring and pest incidence levels.

2.2. Preparation of Ricinus Communis Leaf Extract

Healthy leaves of Ricinus communis L. were collected in the town of Banzha, municipality of Tecozautla, Hidalgo, Mexico (20°31′41.1″ N, 99°39′53.8″ W). The samples were placed in clean plastic bags and transported in an ice box to the Agricultural and Environmental Chemistry Laboratory of the Autonomous University of the State of Hidalgo (UAEH). Immediately upon arrival at the laboratory, the leaves were manually separated from the stems and washed with a 5% sodium hypochlorite solution to remove surface impurities. Subsequently, the samples were stored at −70 °C (Thermo Scientific 703 Ultra-Low Freezer; Thermo Fisher Scientific, Waltham, MA, USA), then lyophilized (Freeze Dryer, model 79480; Labconco Corporation, Kansas City, MO, USA), and macerated to a fine powder. A whole plant was preserved for species identification, which was carried out in the botany laboratory of the Institute of Biological Sciences at the Autonomous University of the State of Hidalgo. For the preparation of the plant extract, a 1:10 (100 g dried material per liter of methanol) ratio was used, and the mixture was macerated for 24 h with stirring every 40 min. The extract was then filtered twice through a Whatman filter 1 and concentrated in a rotary evaporator (Büchi R-215, Flawil, Switzerland) for 4 h at 40 °C and 60 mbar, with the pressure adjusted to prevent thermal degradation of the secondary metabolites. The final concentrate was stored in amber glass bottles and refrigerated at 4 °C until experimental use, following Sasidharan et al. [20]. This procedure was repeated throughout the experiment to ensure an adequate supply of the plant extract for the different research applications. On average, the extract was 40% (40 g of solid material).

2.3. Biostimulant Application

The experiment was conducted in a completely randomized design, with five treatments assigned to five raised beds (one bed per treatment). Plants were established following the defined planting arrangement, ensuring a representative field population. For data collection, 10 plants per treatment were randomly selected and used as experimental units for agronomic evaluation (Figure 1A). This sampling approach was defined to simulate conditions similar to those of small-scale producers operating under sustainable production schemes with limited resources. The treatments consisted of three concentrations of castor oil, T50 (50 mg L⁻¹), T75 (75 mg L⁻¹), and T100 (100 mg L⁻¹), all combined with 1.5 mL L⁻¹ of Bionex^® (San Jose, CA, USA) adjuvant; a commercial treatment (Pepton 85/16^®) a biostimulant composed of compounds of natural origin obtained through enzymatic hydrolysis, with a high content of soluble organic nitrogen in the form of free amino acids (16%) and peptides, and a total amino acid content of 85%, in addition to essential nutrients such as potassium, phosphorus and trace elements, with the same adjuvant concentration; and an absolute control, to which only water was applied (Figure 1C). The concentrations evaluated (50, 75, and 100 mg L⁻¹) correspond to a significantly lower range than that reported in allelopathy studies for Ricinus communis, where extracts are commonly used in concentrations of the order of 1–10% (10,000–100,000 mg L⁻¹), which have shown inhibitory effects on germination and plant growth [21,22]. Applications were made via foliar spray using a multipurpose manual vacuum pump to ensure uniform coverage of each plant’s leaf surface. In each application, 50 mL of the corresponding solution was supplied per plant. The treatments were applied at three phenological stages of the crop: vegetative growth, flowering, and fruiting. These applications were made 7, 30, and 51 days after transplanting, respectively (Figure 1C,D). The selection of these stages was based on their physiological relevance, as they represent critical developmental periods associated with biomass accumulation, reproductive transition, and fruit formation, during which plants exhibit a greater capacity to respond to biostimulant applications [23]. It is important to mention that the use of adjuvants modifies the properties of the sprayed solution, such as surface tension, contact angle, and droplet wetting, favoring its dispersion, adhesion, and retention on the leaf surface, which can increase the deposition and absorption of the active compounds. Their main function is technological, as they improve the efficiency of application and delivery of the active ingredient, rather than acting as biostimulants [24,25,26]. Unlike biostimulants, whose function is to stimulate natural processes of nutrition, stress tolerance, or crop quality, adjuvants are not used for this primary physiological purpose; therefore, they were not used as an additional treatment [23].

2.4. Agronomic Variables

The measurements were taken 90 days after transplanting (DAT), as this stage corresponds to a point at which the crop has reached an advanced vegetative and reproductive development, allowing for an integrated evaluation of the effect of the treatments. In each growing bed, ten plants were selected for evaluation, which were considered as individual experimental units for the measurement of agronomic parameters [27] (Figure 1E,F). The vegetative variables considered included plant height, stem diameter, shoot fresh and dry weight, root length, root fresh and dry weight, number of fruits, fruit weight, yield, and polar and equatorial diameter. Height was measured from the base of the stem at ground level to the growth apex using a measuring tape (Model PRO-8ME-R, TRUPER, Mexico City, Mexico) according to the evaluation criteria proposed by González-Lemus [28]. Stem diameter was measured with a digital caliper (Model CALDI-6MP, TRUPER, Mexico City, Mexico), allowing observation of the biostimulant’s effect on the plant’s structural robustness. Ninety days after transplanting, the plants were removed from the soil and taken to the Environmental Agricultural Chemistry Laboratory at the Institute of Agricultural Sciences (UAEH). For the assessment of root length and fresh root weight, plants were carefully extracted from the soil by manual excavation around the root zone to minimize root loss, preserving the main root and as many adventitious roots as possible. Soil residue was then removed by washing with running water for 10 min, and each plant was sectioned into its aerial parts (stem and leaves) and its underground parts, which were weighed on an analytical balance (PW124^®, Adam Equipment, Milton Keynes MK9 1EJ, UK) to obtain the fresh weight. A measuring tape (Model PRO-8ME-R, TRUPER, Mexico City, Mexico) was used to measure the length of the main root, starting at the stem neck and extending to the cap. Once weighed, the plant parts were placed in paper bags and placed in a drying oven (LW-201C^®, GRIEVE, Round Lake, IL, USA) at 60 °C for 72 h until a steady weight was observed. After the allotted time, the fruits were removed and reweighed on an analytical balance (PW124^®; Adam Equipment, Milton Keynes MK9 1EJ, UK) to obtain the dry biomass, as described by Sánchez-Granados [29]. Diameter measurements were taken with a digital vernier caliper, recording the longitudinal (polar) and transverse (equatorial) axes. The weight of each fruit was determined using a precision digital scale (OHAUS Compass™ model, ULINE, Mexico City, Mexico), and the total number of fruits per plant was counted manually at harvest. Finally, yield was measured using ten representative plants per treatment and subsequently extrapolated to t ha⁻¹ based on the calculated plant density, to compare productive efficiency across treatments, following the methodology proposed by Tapia-Zayago [30] (Figure 1D).

2.5. Leaf Sampling

Leaf samples comprised three plants per treatment per growing bed, and four fully expanded young leaves from each plant (2nd and 3rd leaves). The samples were stored at −70 °C (Thermo Scientific 703 Ultra-Low Freezer; Thermo Fisher Scientific, Waltham, MA, USA), then lyophilized (Freeze Dryer, model 79480; Labconco Corporation, Kansas City, MO, USA), and macerated into a fine powder. This sample was then used to determine photosynthetic pigments, compounds, and total antioxidant capacity, according to the methodology proposed by Hernández-Soto [31].

2.6. Measurements of Photosynthetic Pigments

Chlorophyll a and b concentrations, as well as total chlorophyll, were analyzed in freeze-dried leaves. A mixture of 10 mg of freeze-dried leaves and 2 mL of hexane–acetone (3:2) was centrifuged at 12,000 rpm for 10 min at 4 °C. The resulting extract was read in a spectrophotometer at wavelengths of 645 and 663 nm. The resulting absorbances were used for further calculations using the equations proposed by Nagata [32].

2.7. Extraction of Bioactive Compounds

The bioactive compounds in jalapeño pepper leaves were extracted using the method described by [33]. Five grams of leaves were placed in a centrifuge tube, and 15 mL of an ethanol/water (1:1) solution was added. The mixture was homogenized for 1 min at 4 °C and subsequently centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was used to determine the presence of bioactive compounds and total antioxidant activity.

2.8. Determination of Total Phenols

Total phenolic content was determined using the Folin–Ciocalteau assay described by Cenobio-Galindo et al. [34]. One milliliter of the extract was mixed with 5 mL of diluted Folin–Ciocalteau reagent (1:10). After 6 min, 4 mL of Na₂CO₃ (20%) was added to the mixture, left for 2 h at room temperature, and the absorbance against the reagent blank was determined at 760 nm using a UV-Visible spectrophotometer. Total phenolic content was expressed as gallic acid equivalents −g of jalapeño pepper leaves. All assays were performed in triplicate.

2.9. Determination of Total Flavonoids

Total flavonoid content was measured using the aluminum chloride colorimetric assay used by [35] with some modifications. An aliquot (1 mL) of quercetin extract or standard solution was added to 4 mL of deionized water in a 10 mL flask. To the flask, 300 µL of 5% NaNO₂ was added after 5 min, followed by 300 µL of 10% AICI₃. After another 5 min, 2 mL of 1 M NaOH was added, and the volume was brought to 10 mL with deionized water. A blank was prepared in the same manner using distilled water. The solution was mixed, and the absorbance against the blank was measured at 415 nm. Total flavonoid content was expressed as quercetin equivalents −g of plant sample.

2.10. Determination of Total Antioxidant Capacity by Inhibition of the ABTS Radical

The determination of total antioxidant capacity by inhibition of the 2,2-Azino-bis (3- ethylbenzthiazolinone) radical (ABTS) was carried out as described by Vargas-Ortiz et al. [33]. A 10 mL solution of 7 mM ABTS was prepared and reacted with 10 mL of 2.45 mM K₂S₂0₈. The mixture was stirred for 16 h in a container in complete darkness. Subsequently, the absorbance was measured at 734 nm using a spectrophotometer. The absorbance was adjusted with 20% ethanol to reach 0.7 ± 0.1. Two hundred µL of the sample were added to 2 mL of ABTS solution, and the mixture was incubated for 6 min, after which the absorbance was measured at 734 nm. The results are expressed in mg of ascorbic acid equivalents per g of sample vegetables.

2.11. Determination of Total Antioxidant Capacity by Inhibition of the DPPH Radical

A 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent was used to illustrate compounds with antioxidant capacity. A 6.5 × 10⁵ M DPPH solution in 80% methanol was prepared, and the mixture was stirred for 2 h in complete darkness. Then, 0.5 mL of the sample was mixed with 2.5 mL of the DPPH solution, and the mixture was stirred. The absorbance of the mixture was immediately measured at 515 nm. The mixture was left to react in the dark for 1 h. 80% methanol was used as a blank. The results obtained are expressed in mg of ascorbic acid equivalents −g of plant sample [34].

2.12. Preliminary Structural Analysis of Metabolites

Column chromatography (CC) and thin-layer chromatography (TLC) were used to separate extracts from Ricinus communis L. A 5 cm internal diameter glass column and silica gel plates with a particle size of 0.063–0.200 mm were used. The mobile phases consisted of the following mixtures: hexane:acetone (7:3), hexane:acetone (1:1), hexane:acetone (3:7), acetone, and methanol. From the methanolic extract of Ricinus communis, 20 fractions of 100 mL each were obtained, labeled F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14, F15, F16, F17, F18, F19, and F20. Two additional fractions of 500 mL each, designated F21 and F22, were also collected. The fractions were regrouped based on the retention factor (Rf) values obtained on the chromatographic plates, forming the following subfractions: F2, F3–F5, F6–F10, F11–F14, F15–F17, and F18–F22. The ¹H NMR spectroscopic analysis was performed on a Bruker Ascend 400 spectrometer operating at 400 MHz. Samples were dissolved in deuterated chloroform (CDCl₃). Chemical shifts (δ) were expressed in parts per million (ppm) relative to the tetramethylsilane (TMS) signal as an internal reference. Proton signals are described using the following abbreviations: (s) single, (d) double, (t) triple, (q) quadruple, and (m) multiple, while coupling constants (J) were expressed in Hertz (Hz). During the chromatographic separation of the methanolic extract of Ricinus communis L., a total of 22 fractions were obtained using the methodology proposed by Hernández-Pérez [36].

2.13. Statistical Analysis

For the analysis of agronomic variables, the Shapiro–Wilk normality test and Levene’s test for homogeneity of variance (TS1) were applied. Subsequently, statistical analysis was performed using a one-way analysis of variance (ANOVA) with the Fisher–Snedecor F-test to determine the significance of treatment effects. When significant differences were detected, mean comparisons were performed using Fisher’s least significant difference (LSD) test at the α ≤ 0.05 significance level. All statistical procedures were performed using Infostat 2020 software. Additionally, a Spearman correlation analysis was conducted using the statistical software R 4.1.2. Graphs were generated using SigmaPlot version 14.5.

3. Results

3.1. Agronomic and Biochemical Variables

Analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test (α = 0.05) revealed significant effects of the treatments on plant height, stem diameter, fresh and dry shoot biomass, root length, and fresh and dry root weight (Figure 2). However, no significant differences were detected between the extract concentrations (T50, T75, and T100) for any of these variables, indicating that the concentration of Ricinus communis extract did not significantly influence the vegetative responses. For plant height, treatments CP, T50, T75, and T100 showed an average increase of approximately 5% compared to the control; however, no significant differences were observed among them (Figure 2A). For stem diameter, all treatments increased the values compared to the control, with CP showing the greatest increase (46%), followed by T75 (38%), T100 (32%), and T50 (28%). Despite these numerical differences, no statistically significant differences were detected among CP, T50, T75, and T100 (Figure 2B). For fresh shoot biomass, CP showed the greatest increase (88%) compared to the control, followed by T50 (44%), T100 (37%), and T75 (35%). However, no significant differences were observed between the extract concentrations, indicating a uniform response regardless of the dose (Figure 2C). In dry shoot biomass, CP showed the greatest increase (53%), followed by T50 (30%), T100 (23%), and T75 (10%), compared to the control. No significant differences were detected between T50, T75 and T100 (Figure 2D). Regarding root length, no significant differences were observed between treatments (Figure 2E). For fresh and dry root weight, CP showed the greatest increases compared to the control, 65% and 63% respectively; however, no significant differences were detected between the extract concentrations (Figure 2F,G).

Analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test (α = 0.05), indicated that no significant differences were observed between treatments in chlorophyll a, chlorophyll b, and total chlorophyll content (Figure 3). For chlorophyll a, treatment T75 showed the greatest increase (18%) compared to the control (Figure 3A). For chlorophyll b content, treatment CP showed the greatest increase (14%) compared to the control, while T50 and T75 showed similar increases (9%) (Figure 3B). For total chlorophyll, treatment T75 showed the greatest increase (16%) compared to the control, followed by T50 (4%) and T100 (3%) (Figure 3C).

Analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test (α = 0.05), showed significant effects of the treatments on the number of fruits, average fruit weight, yield, and polar and equatorial fruit diameters (Figure 4). For the number of fruits, treatment T75 showed a 90% increase compared to the control, followed by treatment CP (82%), while T50 and T100 showed average increases of 56% (Figure 4A). For average fruit weight, treatment CP showed the greatest increase (17%) compared to the control, followed by T100 (12%) and T50 (3%) (Figure 4B). For yield, treatment CP showed the greatest increase (401%) compared to the control, followed by T50 (290%), while T75 and T100 showed smaller increases (249%) (Figure 4C). In the polar diameter of the fruit, treatments CP and T75 showed the greatest increases (30%) compared to the control (Figure 4D). For the equatorial diameter of the fruit, all treatments increased this variable compared to the control, with CP showing the greatest increase (16%), followed by T50 (7%) and T100 (2%) (Figure 4E and Figure 5). However, no significant differences were found between the CP, T50, T75, and T100 treatments.

Analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test (α = 0.05), showed significant effects of the treatments on phenol and flavonoid content and on antioxidant capacity determined by the ABTS and DPPH methods (Figure 6). For phenol content, the CP treatment showed the greatest increase (21%) compared to the control, followed by T100 and T75, which showed similar increases (20%), while T50 showed the smallest increase (10%) (Figure 6A). For flavonoid content, the CP treatment showed the greatest increase (22%) compared to the control, while the T50, T75, and T100 treatments showed similar increases (17%) (Figure 6B). For antioxidant capacity determined by ABTS, treatment T100 showed the greatest increase (22%) compared to the control, followed by CP (19%), while T75 and T50 showed smaller increases (11%) (Figure 6C). Finally, for antioxidant capacity assessed by DPPH, treatment CP showed the greatest increase (25%) compared to the control, followed by T100 (22%), T75 (20%), and T50 (9%) (Figure 6D).

3.2. Correlations Between Agronomic Variables, Bioactive Compounds, and Antioxidant Activity

The Spearman correlation coefficients calculated for the evaluated variables (Figure 7) revealed significant associations between agronomic, physiological, and biochemical variables. Fruit weight showed a strong positive correlation with stem diameter (ρ = 0.58) and the number of fruits (ρ = 0.68). Similarly, crop yield was highly correlated with the number of fruits (ρ = 0.85) and fruit weight (ρ = 0.92) and showed a moderate correlation with stem diameter (ρ = 0.41). Chlorophyll a and b content showed a strong association with the number of fruits (ρ = 0.59), while total chlorophyll was positively correlated with chlorophyll b (ρ = 0.85) and the number of fruits (ρ = 0.73). Fresh and dry shoot weight were positively associated with the number of fruits (ρ = 0.86 and ρ = 0.85) and with yield (ρ = 0.75 and ρ = 0.78), respectively. Root length showed a moderate correlation with stem diameter (ρ = 0.37), while fresh and dry root weights were positively correlated with fresh shoot weight (ρ = 0.88 and ρ = 0.79) and dry shoot weight (ρ = 0.84 and ρ = 0.76), respectively. Regarding fruit dimensions, polar and equatorial diameters showed a strong association with stem diameter (ρ = 0.64 and ρ = 0.60) and a weak relationship with plant height (ρ = −0.08 and ρ = −0.02). Finally, the phenol and flavonoid content showed a strong correlation with stem diameter (ρ = 0.59 and ρ = 0.63), a behavior also observed in the total antioxidant activity determined by ABTS and DPPH radicals (ρ = 0.64 and ρ = 0.67). In turn, the antioxidant activity by ABTS was strongly correlated with the phenol content (ρ = 0.75), while the antioxidant activity by DPPH was strongly associated with the flavonoid content (ρ = 0.78).

3.3. Preliminary Structural Analysis of Metabolites

Table 1. Tentative assignment of metabolites from the methanolic extract of Ricinus communis L. based on ¹H NMR spectra.

δ (ppm)/Range	Multiplicity	Proton Type/Chemical Environment	Tentative Assignment	Metabolite Class	Fraction(s) Where Observed
0.7–1.0	m	Terminal –CH3 of long aliphatic chains	Terminal methyl groups of fatty acids	Fatty acids (saturated/unsaturated)	F2, F6–10, F11–14
1.0–1.5	m	–(CH2)n– of aliphatic chains	Acyl chain methylenes	Fatty acids, terpenoids	F2, F3–5, F6–10, F11–14
1.8–2.3	m	CH2 adjacent to unsaturation (–CH2–CH=CH–)	Allylic methylenes	Unsaturated fatty acids	F2, F11–14
2.5–3.0	m	CH2 adjacent to electronegative groups (–CH2–O–/–CH2–N–)	Functionalized side chains	Modified fatty acids/phenolics	F2, F3–5, F6–10
3.0–3.5	m	Oxygenated CH and CH2 (–CHOH–, –CH2–O–)	Sugar and polyol protons	Sugars/carbohydrates	MeOH fraction
3.5–4.1	s, m	–OCH3 (methoxyl groups), oxygenated CH	Methoxyl groups of aromatic compounds	Methoxylated phenolics/flavonoids	F3–5, F6–10, F15–17, F18–22
3.55	s	–OCH3	Methoxyl group on pyridinic ring	Ricinine	Crystals (acetone)
3.99	s	–OCH3	Segundo grupo metoxilo	Ricinine	Crystals (acetone)
4.0–5.5	m	Anomeric and vinylic protons (sugars/double bonds)	Anomeric sugar protons and olefinic H	Sugars, unsaturated fatty acids	F2, F3–5, F6–10, MeOH
5.0–5.5	m	–CH=CH–	Olefinic protons	Unsaturated fatty acids, terpenoids	F2, F3–5, F6–10, F11–14
6.0–7.0	m	Substituted aromatic protons	Aromatic nuclei of phenolics and flavonoids	Phenolic compounds	F3–5, F6–10, F11–14, F15–17, F18–22
6.06	d (J ~7–10 Hz)	Vinylic/aromatic proton in conjugated system	Heteroaromatic proton of ricinine	Ricinine	Crystals (acetone)
7.0–8.0	m	Aromatic protons (benzenoid and heteroaromatic rings)	Phenolics, flavonoids, aromatic alkaloids	Phenolics, flavonoids, ricinine	F3–5, F6–10, F11–14, F15–17, F18–22
7.51	d (J ~7–10 Hz)	Aromatic proton in pyridinic ring	Aromatic proton of ricinine	Ricine	Crystals (acetone)
8.0–8.5	m	Aromatic protons	Highly conjugated phenolics/heteroaromatics	Phenolics, alkaloids	F15–17, F18–22, MeOH

shows high metabolomic complexity and a differential distribution of metabolites throughout the fractionation process, determined primarily by the compounds’ polarity and structural properties. In general, the initial and intermediate fractions are characterized by a predominance of aliphatic and olefinic proton environments, suggesting a high contribution of lipid compounds, particularly saturated and unsaturated fatty acids. The coexistence of different degrees of unsaturation indicates structural diversity within this class of primary metabolites, consistent with the known chemical composition of R. communis. These components tend to concentrate in less-polar fractions, reflecting effective separation based on their physicochemical properties. As fractionation progresses, a gradual increase in signals from aromatic systems and methoxyl groups is observed, suggesting an enrichment in phenolic compounds and other aromatic secondary metabolites. This change in the spectral profile suggests a transition from predominantly nonpolar metabolites to more complex, functionally modified structures typical of plant secondary metabolism, highlighting the extract’s chemical heterogeneity. A key finding is the detection of highly diagnostic ricinine signals, concentrated in specific fractions. This selective distribution indicates that the fractionation procedure effectively isolated this alkaloid, clearly differentiating it from the broader set of phenolic and lipid compounds present in the extract. The identification of ricinine is a key element for the phytochemical characterization of R. communis and supports the proposed structural assignment.

In contrast, the final methanolic fraction exhibits a profile dominated by oxygenated proton environments, consistent with highly polar metabolites such as sugars and polyols. The accumulation of these compounds in the most polar fraction confirms the efficiency of the fractionation scheme and demonstrates a clear segregation of metabolites by chemical affinity. Taken together, the information presented in the table provides a robust qualitative overview of the distribution of metabolite classes across the fractions of the methanolic extract of Ricinus communis. Although the assignments are considered tentative, the ¹H NMR analysis establishes clear relationships between fractions and metabolite types, laying a solid foundation for interpreting biological results and for designing subsequent studies aimed at structural confirmation or quantitative analysis of specific compounds.

4. Discussion

This study demonstrated that extracts of Ricinus communis L. exhibit biostimulant activity (Figure 2 and Figure 6) and contain phenolic compounds, terpenes, unsaturated fatty acids, and ricin (Table 1). The results indicate that the hypothesis of this research is accepted, namely, the foliar application of the methanolic extract of Ricinus communis L. in defined concentrations (50, 75 and 100 mg L⁻¹) generates differential and statistically significant effects (α ≤ 0.05) on agronomic variables (yield, number of fruits, stem diameter and fruit size) and biochemical variables (phenols, flavonoids and antioxidant capacity), although physiological variables such as chlorophyll content did not show statistically significant differences, consistent response trends were observed in jalapeño pepper plants, compared to the absolute control and the commercial biostimulant. The differential response observed among treatments, particularly between T50 and T75, can be attributed to dose-dependent physiological regulation, where intermediate concentrations optimize metabolic activation without inducing stress. In this sense, T50 promoted improvements in growth-related variables, while T75 and T100 enhanced biochemical and trends associated with photosynthetic activity, suggesting a functional specialization of responses depending on extract concentration. Castor bean (Ricinus communis L.) is a rich source of secondary metabolites with bioactive properties [37] (Table 1). These compounds modulate metabolic pathways in plant cells, particularly those related to growth and defense [4]. A relevant aspect of this study is that Ricinus communis is widely recognized for its allelopathic potential, which is attributed to secondary metabolites that inhibit germination and plant growth. However, allelopathy is not exclusively a negative phenomenon, as numerous allelopathic compounds can induce stimulatory responses when applied at low concentrations and via application routes that limit direct exposure of sensitive tissues. In this context, foliar application of the extract at relatively low concentrations could have reduced the risk of direct phytotoxicity and promoted adaptive physiological responses in the treated plants. Chemical characterization of the extract using proton nuclear magnetic resonance spectroscopy (¹H NMR) revealed the presence of secondary metabolites, including ricinine, unsaturated fatty acids, and phenolic compounds. Although this analysis was qualitative rather than quantitative, it provides relevant structural information that supports the extract’s chemical complexity and its potential biological activity. It is important to note that the presence of these metabolites does not necessarily imply a direct causal relationship with the observed effects, but it does suggest that the extract contains compounds that interact with key physiological processes in plants.

The increase in plant height, stem diameter, shoot fresh and dry weight, and root variables observed in treatments T50, T75, and T100 (Figure 2) can be explained by greater cell division and elongation. This effect is associated with the activation of the auxin and gibberellin hormonal pathways, which in turn induce the expression of growth-regulating genes such as ARF7, ARF19, PIN1, and CYCD3;1, involved in cell expansion and differentiation [28]. These results are supported by the correlation analysis (Figure 7), which showed that stem diameter was strongly positively associated with fruit weight (ρ = 0.58), fruit size, and bioactive compounds, indicating that structural growth is closely linked to productive and metabolic performance [38]. Similarly, the strong correlation between the number of fruits and yield (ρ = 0.85), as well as fruit weight (ρ = 0.92), confirms that reproductive traits are the main determinants of productivity in jalapeño pepper. Plant extracts, such as castor bean extract, can also enhance photosynthetic capacity by increasing leaf development and meristematic activity [31]. In this context, the strong association between chlorophyll content and number of fruits (ρ = 0.59), as well as between chlorophyll b and total chlorophyll (ρ = 0.85), suggests that improvements in photosynthetic pigments directly contribute to enhanced reproductive performance, particularly in treatments such as T75.

Since leaves are the main source of carbohydrates, an increase in photosynthetic efficiency directly affects the accumulation of photoassimilates, which are subsequently translocated to fruits [39]. Furthermore, R. communis has been reported to modulate essential processes, including nitrogen assimilation, photosynthetic activity, and cellular redox balance, through the combined action of fatty acids, flavonoids, and saponins. These enzymes increase the activity of enzymes such as nitrate reductase, Rubisco, membrane ATPase, and phosphoenolpyruvate carboxylase (PEPC), improving primary metabolism and active nutrient transport [40]. At the molecular level, these bioactive compounds act as elicitors, activating intracellular signaling cascades via plasma membrane kinase-like receptors. These receptors recognize molecules such as saponins and flavonoids, triggering a response that includes transient increases in reactive oxygen species (ROS), nitric oxide (NO), and cytosolic calcium [32]. These signals activate transcription factors such as WRKY, MYB, and DREB, which regulate the expression of genes involved in protein synthesis, flavonoid biosynthesis, and cellular defense mechanisms [41]. These mechanisms are consistent with the modulation of biochemical responses and antioxidant capacity observed in this study (Figure 6) and may also contribute to the trends detected in chlorophyll content, even in the absence of statistically significant differences (Figure 3).

The absence of statistically significant differences in chlorophyll content between treatments can be explained by the highly regulated and conserved nature of the photosynthetic apparatus. At the physiological level, chlorophyll synthesis and stability are strictly controlled by internal factors such as nutritional status, nitrogen and magnesium availability, and photosystem activity, which limit abrupt changes in response to biostimulant application [42]. In this study, although no significant differences were detected, numerical increases in chlorophyll a and total chlorophyll were observed in treatment T75, suggesting a trend toward greater photosynthetic efficiency at intermediate extract concentrations. Conversely, the increase in chlorophyll b observed in the commercial treatment may be associated with a more immediate availability of organic nitrogen, which favors the synthesis of accessory pigments [43]. Furthermore, the inherent variability of physiological measurements under open-field conditions, along with the plasticity of photosynthetic metabolism, may have contributed to the lack of statistical differentiation between treatments despite the observed trends. This suggests that chlorophyll content remained within a physiologically stable range, despite metabolic activation induced by the biostimulant [44].

The physiological response to plant-derived biostimulants follows a non-linear dose–response curve, with intermediate doses typically producing the greatest benefits. This behavior explains the differential response between T50 and T75, where T50 was sufficient to stimulate vegetative growth and biomass accumulation, while T75 more effectively activated secondary metabolism and antioxidant systems, as supported by the observed correlations between biomass, fruit production, and biochemical variables and the correlations observed between phenols, flavonoids, and antioxidant activity (ABTS ρ = 0.75; DPPH ρ = 0.78) (Figure 2 and Figure 5), indicating that higher metabolic activation, as observed in T75, promotes the synthesis of secondary metabolites involved in oxidative stress regulation. Similar results were reported by the authors of [45], who observed that medium concentrations of bioproducts increased chlorophyll and phenolic compounds, whereas low doses did not significantly stimulate these pathways. Conversely, treatment T100, the highest concentration of castor bean extract evaluated, reduced fruit number and total crop yield (Figure 3). This behavior may be attributed to a nutritional imbalance or to a phytotoxic effect caused by the high concentration of active compounds, leading to alterations in the absorption of essential nutrients or to metabolic stress [46,47]. Another possible cause is vegetative overstimulation, where energy resources are redirected to leaf growth at the expense of the reproductive phase. This phenomenon has been documented by Guo [48], who observed that excessive fertilization decreases photosynthetic efficiency and the partitioning of photoassimilates to the fruit. Consequently, treatment T100 could have resulted in abundant vegetative growth but lower reproductive productivity, indicating that the optimal dose of castor bean extract lies at intermediate levels, such as treatment T75 (Figure 4). Additionally, the positive correlation between root and shoot biomass (ρ = 0.75) suggests coordinated resource allocation between belowground and aboveground structures, which was more balanced in T50 and T75 than in T100.

Finally, biostimulant compounds appear to induce greater resource partitioning and nutrient redistribution toward reproductive organs [5], reflected in the greater number and weight of fruits per plant in treatments with optimal agronomic response. This is further supported by the correlation between fruit dimensions and stem diameter (ρ = 0.62), indicating that structural robustness contributes to fruit development. In this regard, treatments T50 and T75 established an adequate physiological balance between metabolic stimulation and homeostasis, whereas the overdosage observed in T100 led to signal saturation, reducing reproductive efficiency (Figure 4). The results of this research are consistent with those reported by Márquez-Mendoza [49], who applied microbial biostimulants to jalapeño peppers and observed increases in fruit weight, polar and equatorial diameters, and yield. In another study conducted by Adame-García [50], commercial biostimulants were evaluated in jalapeño peppers, and an increase in seedling dry weight, root weight, and fruit weight was reported.

The synthesis and accumulation of secondary metabolites in Ricinus communis L. are strongly influenced by environmental, physiological, and geographic factors [51]. Several authors have documented that variables such as latitude, altitude, average temperature, soil type, precipitation, and solar radiation can significantly modify the phytochemical profile of these species [52]. Furthermore, the phenological stage at which plant material is collected determines the relative proportions of fatty acids, alkaloids, phenolic compounds, and terpenes in the tissues. This is due to the differential regulation of metabolic pathways such as the shikimate, mevalonate, and malonate pathways, which respond dynamically to environmental and physiological conditions [53].

Previous studies have reported that R. communis extracts contain ricin, ricinolein, linoleic acid, oleic acid, and palmitic acid, as well as flavonoids (quercetin and kaempferol), condensed tannins, triterpenes, and phytosterols [54,55]. In studies focused on methanolic extracts, the dominant presence of pyridone-type alkaloids, such as ricinine, has been described, along with a significant fraction of unsaturated fatty acids and phenolic compounds, especially when the samples come from leaves collected in warm-dry climates, similar to those of Tecozautla, Hidalgo [56]. The results of this research align with reports by Sotelo-Leyva et al. [57] and Mamy et al. [58], confirming the presence of ricinine, unsaturated fatty acids, and phenols via ¹H-NMR analysis. While in scientific research it is common to first establish the phytochemical characterization of the bioproduct and subsequently evaluate its biological effect, in agroecological and rural innovation systems, the validation of empirical practices is also fundamental. In many agricultural contexts, especially in the management of plants with ethnobotanical traditions, the initial use of plant extracts stems from practical observations and local knowledge that enable the identification of positive effects before conducting formal analytical studies [59].

From a sustainability perspective, the use of plant-based extracts, such as Ricinus communis, represents a promising strategy to reduce dependence on synthetic agrochemicals and promote more environmentally friendly agricultural practices [60]. These biostimulants can contribute to greater nutrient-use efficiency, improve plant resilience, and reduce environmental impact, in line with agroecological approaches and sustainable intensification [61]. In this regard, the positive responses observed at intermediate doses highlight the potential for optimizing natural inputs to achieve a balance between productivity and sustainability. For producers, these results are particularly relevant, as they offer a viable alternative for increasing crop yield and quality without significantly increasing production costs, especially in small- and medium-scale systems where access to synthetic inputs may be limited. Furthermore, the use of plant extracts can promote productive stability under stress conditions, reducing risks associated with environmental variability and improving the profitability of the agricultural system [62].

From an agronomic perspective, the observed improvements in plant productivity and performance support the potential of Ricinus communis extract as a functional input in sustainable production systems, contributing to the development of more resilient and resource-efficient agricultural strategies. However, while the presence of bioactive metabolites such as ricin, phenolic compounds, and unsaturated fatty acids provides a plausible biochemical basis for the observed effects, their specific contribution was not directly quantified in this study and should be interpreted with caution. In this context, future research should focus on identifying and quantifying these compounds, as well as their interaction with plant metabolic pathways, to better elucidate their mode of action. Furthermore, it will be necessary to evaluate the behavior of these extracts under different environmental conditions, cropping systems, and production scales to validate their applicability in diverse agricultural contexts. Finally, it is essential to analyze the phytotoxicity, residual effects, and persistence of these bioactive compounds in jalapeño pepper fruits, with the aim of determining possible accumulation risks, establishing safe use criteria, and ensuring the safety and quality of the product for consumption, thus strengthening the agronomic viability and regulatory acceptance of plant-based biostimulants in horticultural systems.

5. Conclusions

Foliar application of Ricinus communis L. extract is a promising, sustainable strategy for improving the yield and quality of jalapeño peppers under open-field conditions. Treatments, particularly T50 and T75, increased fruit number and yield compared to the control, indicating a more efficient balance between vegetative and reproductive growth. The observed responses suggest a dose-dependent effect: intermediate concentrations optimize plant productivity and metabolic activation, while higher concentrations (T100) reduce yield, possibly due to physiological imbalance or overstimulation. The extract also increased biochemical parameters, such as phenols, flavonoids, and antioxidant capacity, reflecting greater metabolic activity. Although no statistically significant differences in chlorophyll content were detected, consistent trends were observed, suggesting that photosynthetic processes remained within a stable physiological range. Taken together, these findings support the potential of Ricinus communis extract as a sustainable bio-input for improving crop productivity, highlighting the importance of dose optimization for its practical application.