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Article

Use of Sorbitan to Extract Capsaicinoids and Bioactive Compounds: Condition Optimization Study

by
Andrea de J. Campos-Badillo
1,
Edén A. Luna-Zapién
2,
Juan A. Ascacio-Valdés
1,
Jolanta E. Marszalek
3,
Rafael Minjares-Fuentes
2,
Armando Quintero-Ramos
4 and
Jorge A. Meza-Velázquez
2,*
1
Facultad de Ciencias Químicas, Departamento de Investigación en Alimentos, Universidad Autónoma de Coahuila, Boulevard Venustiano Carranza, Col. República Oriente 935, Saltillo C. P. 25280, Coahuila, Mexico
2
Facultad de Ciencias Químicas, Universidad Juárez del Estado de Durango, Avenida Artículo 123 S/N, Fraccionamiento Filadelfia, Gómez Palacio C. P. 35010, Durango, Mexico
3
Facultad de Ciencias Biológicas, Universidad Autónoma de Coahuila, Carretera Torreón-Matamoros km 7.5, Torreón C. P. 27276, Coahuila, Mexico
4
Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito Universitario S/N, Campus Universitario # 2, Chihuahua C. P. 31125, Chihuahua, Mexico
*
Author to whom correspondence should be addressed.
AppliedChem 2025, 5(2), 7; https://doi.org/10.3390/appliedchem5020007
Submission received: 28 January 2025 / Revised: 27 February 2025 / Accepted: 10 March 2025 / Published: 24 March 2025
Browse Figures
Versions Notes

Abstract

:
Capsaicinoids obtained from lyophilized serrano chili by sorbitan monooleate solutions were investigated. Sorbitan monooleate was as effective as methanol in extracting capsaicin and dihydrocapsaicin (DHC). Subsequently, a Box–Behnken design was used to optimize capsaicin, DHC, and polyphenol extraction, as well as to evaluate the antioxidant capacity of dehydrated serrano chili. Particle size (PS) (20–60 mesh), processing temperature (55–75 °C), and sorbitan concentration (1.5–2.5%) were selected as independent variables. The statistical analysis showed that the quadratic models adequately describe the response of the concentration of capsaicin and DHC, but not with polyphenols and antioxidant capacity. The highest extraction of capsaicin (~620 mg/100 g dw) and DHC (~520 mg/100 g dw) was achieved with the combination of sorbitan at 2%, temperature at 65 °C, and PS from 40 mesh. Experimental and predicted values were closely consistent. Meanwhile, extracts with the highest antioxidant potential (~7510 and ~5820 µM of Trolox Eq/100 g dw for ABTS and FRAP, respectively) were those extracted in sorbitan and PS from 40 mesh. In contrast, the highest values of polyphenols (~171 mg gallic acid Eq/100 g dw) were found in the extracts prepared at 75 °C. These results suggest that sorbitan monooleate solutions can be an effective, non-toxic, and environmentally responsible way to obtain capsaicinoids and bioactive compounds from dehydrated serrano chili.

Graphical Abstract

1. Introduction

Chili (Capsicum spp.) is a fruit appreciated for its pungence, flavor, and aroma. In addition, pigments and capsaicinoids can be extracted from chili fruit to be used as additives and dyes in the food and pharmaceutical industry. The capsaicinoids in the Capsicum varieties are mainly capsaicin and dihydrocapsaicin (DHC). These compounds represent approximately 80–90% of the capsaicinoids in the chili and are the main ones responsible for their pungence [1]. Capsaicinoids have been extensively studied due to their antimicrobial, antioxidant, and pharmaceutical properties [2,3]. Regular chili pepper consumption is associated with lower mortality from cardiovascular diseases [4], which is likely due to the presence of capsaicinoids. These promote functional recovery from neurological deficiencies [5], provide neuroprotection [6,7,8], and can attenuate the progression of atherosclerotic plaques [9], among others. Therefore, obtaining and purifying capsaicinoids from plants is of great interest.
Generally, capsaicinoids are extracted employing organic solvents using different processes such as maceration [10], Soxhlet extraction [11], ultrasound-assisted extraction [12], and microwave-assisted extraction [13], among others. However, organic solvents are often expensive, toxic to personnel, and environmental pollutants. With nanoemulsions, which are hydrophobic compounds, such as capsaicinoids, dispersion in aqueous solutions employing emulsifiers has been reported [14,15]. The nanoemulsions provide superior stability and high contact area, thus consequently increasing the solubilization of the extracted lipid compound. Oil-in-water nanoemulsions prepared with surfactants, such as sorbitan monooleate, are stable, translucent, and bright solutions with small oil-domain size and color [16]. Sorbitan monooleate is a non-ionic, low molecular-weight emulsifier capable of generating emulsions with high- or low-energy homogenization methods [17] as well as generating spontaneous emulsions that facilitate the dispersion of hydrophobic compounds. In addition, sorbitan monooleate is a non-toxic surfactant with proven efficacy in extracting bioactive compounds from plants [18,19]. Due to these characteristics, in recent years, the extraction of bioactive compounds through the use of emulsions and nanoemulsions has gained interest, since these types of solutions support hydrophilic and hydrophobic molecules more efficiently than pure common solvents and overcome the nanoemulsion limit or remove the use of organic solvents [20,21,22]. It has been suggested that surfactants can extract bioactive compounds from plant cells by breaking their cell membranes and walls, releasing these molecules into the medium [23]. In addition, several studies have demonstrated the importance of sample temperature and particle size, (PS), in extracting lipophilic compounds; however, these have been made with organic solvents [24,25]. The disadvantages of extracting capsaicinoids with organic solvents (expensive, toxic, and polluting) make it necessary to have new alternatives. Using emulsifiers to extract lipophilic compounds is an excellent option to replace these substances. The resulting process would proceed without organic solvents or costly equipment and be a low-cost, non-toxic, and environmentally responsible technology.
Therefore, the objective of this research was to demonstrate the extracting efficacy of capsaicinoids and bioactive compounds from dehydrated serrano chili peppers utilizing sorbitan monooleate solutions, as well as to optimize the extraction process of these compounds considering the particle size of the sample, process temperature, and sorbitan concentration as variables.

2. Materials and Methods

2.1. Chemical Reagents

Sorbitan monooleate (SM), Folin–Ciocalteu reagent, gallic acid standard, ABTS (2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), DPPH (1,1-diphenyl-2-picrilhydrazil), potassium persulfate, Trolox, TPTZ (2,4,6-Tris(2-pyridyl)-striazine), sodium carbonate, and capsaicin and DHC standards were acquired from Sigma-Aldrich (St. Louis, MO, USA). Hexane, acetone, acetonitrile, methanol, and HPLC-grade water were purchased from J.T. Baker (Phillipsburg, NJ, USA).

2.2. Plant Material

Approximately 5 kg of green serrano chili of commercial maturity (Capsicum annuum ‘Serrano’) was acquired at a local shopping center. The chili fruits were pre-selected, washed, and, after seed removal, cut into slices. Subsequently, they were freeze-dried, ground, and separated into 3 particle sizes using mesh 20 (850 µm), 40 (425 µm), and 60 (250 µm). The chili powders were vacuum-packed, refrigerated, and stored to be used in the experiments [26].

2.3. Experimental Process

The study was divided into two stages. Firstly, the efficacy of SM as an extractor of capsaicinoids was demonstrated. Subsequently, the optimization of the capsaicinoids and bioactive compounds extraction process from freeze-dried serrano chili with SM under certain process conditions was evaluated and confirmed.
1.
Use of sorbitan as a capsaicinoid-extracting agent
The extraction of capsaicinoids was carried out using different extracting agents: 1.5% SM in water, acetone, acetonitrile, methanol, and hexane. Specifically, 20 mL of each extracting agent, heated to 55 °C, was mixed with 200 mg of freeze-dried chili powder (40 mesh) using a vortex at 3200 rpm (Scientific Industries, Bohemia, NY, USA) for 3 min. Next, the mixture was centrifuged at 1650× g for 10 min (K241R, Centurion Scientific Ltd., Stoughton, UK); the supernatant was recovered, filtered (Millipore, Billerica, MA, USA, 0.45 µm), and analyzed for capsaicin and DHC presence by HPLC-DAD [27]. Likewise, a control extraction was performed using the Soxhlet method with acetone as a solvent (2 g of freeze-dried chili powder, 40 mesh, for 4 h). The extractions were carried out in triplicate. Preliminary tests and bibliographic references determined extraction logistics, sample quantity, and solvent [28,29]. Likewise, capsaicin and DHC calibration curves in SM at 1.5% were carried out to determine accuracy, detection, and quantification limits. In addition, a sample of chili fortified with standards of these capsaicinoids was analyzed to determine the relative recoveries.
2.
Optimization of capsaicinoid extraction in sorbitan
The experiment variables were particle size, SM concentration, and extraction temperature. Exactly 200 mg of freeze-dried serrano chili powder was mixed with 20 mL of SM solution at different temperatures, and the experiment was carried out according to the proposed design. The sample was then agitated in a vortex for 3 min and centrifuged at 1650× g for 10 min. The supernatant was recovered, filtered, and analyzed for capsaicin and DHC by HPLC [30]. In addition, analyses of total polyphenolic concentration (TPC) and antioxidant capacity (AC) by ABTS and FRAP were performed.

2.4. Experimental Design for Optimization

A Box–Behnken design (BBD) with three independent variables was used to optimize capsaicinoid extraction conditions. The variables used were: PS (X1, x1), SM concentration (X2, x2), and solution temperature (X3, x3). The uncoded (Xi) and encoded (xi) values are shown in Table 3. The design comprised 17 experiments, including twelve factorial experiments and five replicates at the center point (Table 3) [31]. Three replicates of each condition were carried out, and the mean values were stated as observed responses. All the experiments were conducted at random.
The mathematical model corresponding to the composite design is:
Y = β o + ∑ i = 1 3 β i X i + ∑ i = 1 3 β i i X i i 2 + ∑ i = 1 2   ∑ j = i + 1 3 β i j X i X j + ε
where Y represents the dependent variable for each of the functional properties evaluated (capsaicin and DHC), β o is the model constant, β i , β i i , and β i j are the model coefficients, and ε is the error. They represent the linear, quadratic, and interaction effects of the variables.

2.5. Capsaicinoid Quantification

Capsaicin and DHC were analyzed according to the method described by Chinn et al. [29] with slight modifications. The analysis was carried out using an HPLC Agilent 1200 (Agilent Technology, Palo Alto, CA, USA) equipped with a diode array detector and a 150 mm × 4.6 mm 5 μm Zorbax XBD-C8 column. Temperature, flow rate, and injection volume were 25 °C, 1 mL/min, and 20 μL, respectively. The mobile phase comprised (A) acetonitrile and (B) water. The mobile phase gradient was 40% A and 60% B at 3 min, 70% A and 30% B at 9 min, 0% A and 100% B at 11 min, and 40% A and 60 B at 3 min. Capsaicinoids were analyzed at 280 nm. These compounds were identified and quantified using pure standards (capsaicin and dihydrocapsaicin) and calibration curves.

2.6. Evaluation of Antioxidant Capacity (ABTS and FRAP) and Total Polyphenolic Content

To determine the antioxidant activity of the serrano chili extracts, the 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric-reducing antioxidant power (FRAP) were used. For the first assay, a 7 mM ABTS solution was prepared and dissolved in a 2.6 mM sodium persulfate solution. It was left to react for 24 h in the dark. Then, 50 µL of the extract and 1950 µL of the ABTS solution were mixed. The absorbance reduction was measured in a spectrophotometer at 734 nm [32]. For the ferric-reducing ability of plasma (FRAP) method, a solution of 0.01 M 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 0.02 M ferric chloride hexahydrate, 0.01 M dissolved in HCl, and 300 mM sodium acetate was prepared, pH 3.6. A total of 50 µL of the extract and 1950 µL of the FRAP reagent were mixed, incubated for 30 min, and the absorbance was read at 593 nm [32].
Total phenolic content was measured using a modification of the Folin-Ciocalteau method. Solutions of 200 mg of lyophilized serrano chili in 1 mL of SM were used for the assay. Exactly 100 μL of the serrano chili extract was mixed by vortex; 950 μL of SM and 50 μL of Folin-Ciocalteau reagent were mixed by vortexing. After 5 min, 800 μL of Na2CO3 (7.5% w/v) was added and vortexed. The mixture was incubated for 30 min in the darkness at 25 °C. The absorbance of the solution was read at 745 nm in a UV–Vis spectrophotometer (HACH DR 5000, Loveland, CO, USA). The phenolic content was calculated by means of a standard curve using gallic acid as a standard (R2 = 0.99). The results were expressed as mg/g GAE of dry weight (dw). The analyses were performed in triplicate [32].

2.7. Statistical Analysis

Analysis of variance and Fisher’s least significant difference (LSD) (p ≤ 0.05) were carried out to determine differences between extracts of various solvents (n = 3). Also, the optimization design data and calculation of predicted responses were analyzed by regression analysis using Statistica software (version 7.0, Statsoft, Tulsa, OK, USA).

3. Results and Discussion

3.1. Sorbitan as a Capsaicinoid Extractor

The validation for capsaicinoid quantification from lyophilized serrano chili presented acceptably high values of relative recoveries and quality attributes, such as accuracy, detection limit, and quantification limit (Table 1).
Table 2 shows the amount of capsaicin and DHC obtained with different solvents and extraction by Soxhlet as a control. Overall, the ratio between the amount of capsaicin and DHC found in our study ranged from 56 to 44 and was very similar to that reported in various studies [34,35,36]. Several reports have concluded that methanol has the highest capsaicinoid extraction power [25,37,38], which was also observed in this study. Interestingly, the concentrations of DHC obtained with methanol and the SM solution were similar (p > 0.05) but higher than those found in the other organic solvents used (p ≤ 0.05). Also, the amount of capsaicin extracted with sorbitan was slightly lower than with methanol but higher than with acetone and hexane. There is no evidence of studies on capsaicinoid extraction by surfactants. However, Akbas et al. [16] showed that oleoresin capsicum can be dispersed in SM, resulting in stable nanoemulsions. A representative chromatogram depicting the two distinct peaks corresponding to capsaicin and dihydrocapsaicin from a chili extract obtained with sorbitan monooleate is shown in Figure 1.
The extraction of capsaicinoids may be due to the location of these compounds in plant tissue. Sugiyama [39] described that they are found in the subcuticular space between the cell wall of the epidermis and the cuticle. The cell wall of the epidermis consists mainly of pectin, cellulose, and hemicellulose. These polysaccharides can be partially dissolved or swollen by water, improving the entry of the aqueous solution and making the extraction efficient. The present study found that using SM as a surfactant could promote the extraction of capsaicinoids present in the tissue of serrano chili. Using an SM solution facilitates the capsaicinoid extraction process in a manner similar to that of Soxhlet’s extraction (p > 0.05).

3.2. Optimization of Capsaicinoid Extraction in Sorbitan

3.2.1. Analysis of the Design and Model Fitting

The effect of PS (X1), concentration of SM (X2), and temperature of the solution (X3) were investigated. The factors of interest were capsaicin and DHC, antioxidant capacity (ABTS and FRAP), and total polyphenol content (TPC).
Table 3 presents the results of 17 runs using BBD, which includes the design, observed responses, and the predicted values for capsaicin and DHC quantity, which were adjusted to the proposed model.
The results of antioxidant capacity and TPC did not fit the model (The data are shown in Table 4). There is little difference between experimental and predicted values. The amount of capsaicin and DHC obtained was between 206.46 and 732.69 mg/100 g dw and 175.22 and 665.98 mg/100 g dw, respectively. The highest extraction yield of these capsaicinoids was obtained under the experimental conditions of X1 = 40 mesh, X2 = 2%, and X3 = 65 °C.
Different statistical parameters, such as lack of fit (LOF), R2, and adequate precision, have been used to determine the fit of an optimization model. The analysis of variance, corresponding to the concentration of capsaicinoids of lyophilized chili extracts, showed a non-significant lack of fit (p > 0.05), R2 values of 0.97 and 0.95, and an adequate precision of 27.2 and 41.7 for capsaicin and DHC, respectively (Table 5). Therefore, based on the previous results, surface models were formulated for the concentration of capsaicin and DHC (Equations (1) and (2)):
C a p s a i c i n m g / 100   g   d w   =   671.6   +   69.1 x 1   −   22.5 x 2   +   0.9 x 3   −   301.2 x 1 2   − 82.7 x 2 2   −   62.1 x 3 2   −   8.0 x 1 x 2   +   2.1 x 1 x 3   +   17.8 x 2 x 3
D H C   m g / 100   g   d w   =   596.5   +   64.4 x 1   −   22.9   −   3.0 x 3   −   276.5 x 1 2   −   66.9 x 2 2   − 51.2 x 3 2   −   6.2 x 1 x 2   +   2.1 x 1 x 3   +   24.3 x 2 x 3
The obtained values of LOF, R2, and adequate precision suggest that the regression models obtained showed an appropriate fit to predict the effect of PS, temperature, and concentration of SM on the concentration of capsaicin and DHC in extracts of dehydrated serrano chili.

3.2.2. Capsaicin and Dihydrocapsaicin

As can be seen in Pareto’s plots (Figure 2a), the variables with the most significant effect on capsaicin extraction were the quadratic and linear terms of PS ( x 1 2 , x 1 ). The quadratic and linear terms of temperature ( x 2 2 , x 2 ), as well as the quadratic terms of SM concentration ( x 3 2 ) were also significant. However, the linear term of SM concentration ( x 3 ) and factor interactions had no significant effect on the concentration of capsaicin extracted from lyophilized serrano chili.
In the case of DHC concentration, the quadratic and linear terms of PS ( x 1 2 , x 1 ) were the most predominant (p ≤ 0.05), followed by the thermal quadratic of temperature ( x 2 2 ). On the contrary, linear and quadratic terms of temperature and the interactions of the factors were not significant for the concentration of this compound extracted from freeze-dried serrano chili (Figure 2b). The relationship between independent and dependent variables was illustrated in three-dimensional (3D) response surface charts and two-dimensional (2D) contour graphs generated by the model for capsaicin and DHC concentration (Figure 3 and Figure 4, respectively). Two variables were depicted in one 3D surface plot, while the other variables were kept at level zero.
The effect of PS and temperature on capsaicin concentration is presented in a 3D graph and contours (Figure 3a), showing the quadratic impacts of these factors. It is observed that an increase in PS, up to 40 mesh, with an extraction temperature of 65 °C (for a concentration of SM of 2%) promoted a significant increase in capsaicin extraction from ~220 to ~560 mg/100 g dw. Likewise, the continuous increase in the PS and temperature decreases the concentration of the compound. Also, Figure 3b shows that the variables PS and concentration of MS presented quadratic effects when increasing the PS to 40 mesh; using solutions of SM at 1.8–2.2% (at 65 °C), capsaicin extraction was increased from ~290 to ~620 mg/100 g dw. Here, the quadratic effect of PS is noteworthy, since a decrease or increase in this variable causes significant changes in capsaicin values.
Also, Figure 4a clearly shows the quadratic effects of PS and temperature on DHC extraction from lyophilized serrano chili. Here, an increase in PS and temperature levels (up to 40 mesh and 65 °C, respectively), with 2% SM shows the highest DHC extraction values of ~210 to ~520 mg/100 g dw. However, the continuous increase in factors decreases response. When analyzing the quadratic effect of PS, it is observed that the concentration of DHC rises as the PS increases until it reaches maximum extraction at 40 mesh (~500 mg/100 g dw), and further increasing PS results in a lower DHC concentration.
Most of the information on capsaicinoid extraction is based on the impact of factors such as temperature, time, solvent type (usually organic), and sample size on the compound yield [40,41]. Yet, there is little research on the effect of particle size on capsaicinoid production. Smaller particle sizes generate higher extraction performance by increasing the material contact area with the solvent [42,43]. Shah et al. [44], when extracting capsaicinoids from paprika using supercritical fluid (65 °C; 200.62 bar pressure and 90 min), found that particle sizes of 0.5 mm (0.25–1.25 mm tested range) yielded higher compound content. On the contrary, Santos et al. [45] reported that samples of Capsicum frutescens L. treated with supercritical fluid and ultrasound (150 bar and 40 °C) produced more capsaicinoids when the particle size was smaller (≤0.23 ± 0.16 mm).
Other authors report results similar to those found in this study; however, they were performed with different lipophilic substances. For example, Putra et al. [46] obtained higher oil yields and antioxidant activity in peanut skin extracts while using PS of 425 μm (~40 mesh) from a size range of 250 to 500 μm. When PS is increased from 0.24 to 0.6 mm, the oleoresin yield extracted from Curcuma Longa L. increases (Haldar et al. [24]). Meanwhile, Eman and Muhamad [47] saw the highest yield with PS of 1 mm (0.5 to 2 mm range tested) in oil extraction from Moringa oleifera seeds. They observed that samples with smaller particle size did not produce greater uptake of fat-soluble compounds. However, these studies do not explain this behavior, so it is necessary to further research the diffusivity of capsaicinoids in complex matrices to understand this phenomenon.
The particle-size effect is also conditioned by other factors of the extraction process. However, interestingly, using larger particle sizes and obtaining higher capsaicinoid yield would have an additional advantage in the reducing excessive grinding processes of the raw material.
Figure 3c and Figure 4c illustrate the quadratic effects of temperature and concentration of MS on capsaicin and DHC concentration. Increasing the temperature to 65 °C, at 2% SM concentration, the capsaicin and DHC levels reached the highest values that oscillate around ~320 to ~490 mg/100 g dw and ~215 to ~460 mg/100 g dw, respectively. Yet, capsaicin and DHC extraction decreased as the temperature and concentration of MS increased.
Several studies agree that the 50–80 °C temperature range most effectively removes capsaicinoid compounds [38,48,49]. At these temperatures, the hydrogen bonds might start to break, and a greater swelling of cellulose and hemicellulose might occur, resulting in a more significant compound release from the cells [50]. However, higher temperatures may encourage greater hydrophobicity in cell-wall cellulose and decrease the extraction of these compounds [51].

3.3. Effect of Particle Size, Temperature, and Concentration of Sorbitan on the Antioxidant Capacity and Polyphenols in Freeze-Dried Serrano Pepper Extracts

It has been reported that the consumption of capsaicinoids can improve insulin resistance [52] and that the capsaicinoids have anti-cancerogenic [53] and anti-inflammatory activities [54]. These qualities may be associated with their ability to inhibit highly oxidizing compounds. The antioxidant capacity, by ABTS and FRAP, of lyophilized serrano chili extracts is shown in Table 3. The extracts obtained with PS 40 mesh at temperatures 55 and 65 °C presented the highest values of ABTS (p ≤ 0.05). For example, the treatment with 40 mesh, 55 °C, and 2.5% of SM had an ABTS value of ~7760 μM Trolox Eq/100 g dw), which is higher than that obtained in extracts with methanol (~6840 μM Trolox Eq/100 g dw). Also, a very evident decrease in antioxidant capacity is shown by increasing the PS to 20 mesh (~4450 μM Trolox Eq/g dw). The values of FRAP were the highest (~6100 μM Trolox Eq/100 g dw) in treatments with PS 40 mesh. There was no apparent influence from the temperature on FRAP values. Also, FRAP values decreased as the PS increased (20 mesh) (~3530 μM Trolox Eq/100 g dm). Interestingly, the concentrations of capsaicin and DHC showed good correlations with the values of ABTS (R2 = 0.65 and 0.64, respectively; p ≤ 0.05) and FRAP (R2 = 0.76 and 0.78, respectively; p ≤ 0.05). Thus, as the concentration of capsaicinoids increased, the antioxidant capacity increased.
It is well known that chili peppers have a high antioxidant capacity due to their high amount of vitamin C and chlorophylls [55,56]. Still, little is known whether capsaicinoids provide part of this characteristic. Materska and Perucka [57] found a strong correlation between capsaicinoid concentration and the antioxidant activity of different varieties of chili peppers. The presence of methoxy and hydroxyl groups of these compounds probably influences the radical-capturing property [1].
Likewise, total phenol content, TPC, values ranged from 790 to 1710 mg of gallic acid Eq/100 g dw (Table 4). A pronounced effect of the temperature of these compounds is observed so that the extracts obtained at 75 °C presented the highest concentration of TPC (p ≤ 0.05). In comparison, those obtained at 55 °C had the lowest values. In addition, extracts obtained with methanol showed lower levels of TPC (~1020 mg gallic acid Eq/100 g dw) compared to extracts obtained with SM in combination with processing temperatures of 65–75 °C (~1640 mg gallic acid Eq/100 g dw).
Tunchaiyaphum et al. [58] reported that the TPC of mango skin extracts increased as the temperature rose due to the increased solubility of polyphenols in the medium. Increasing the temperature and decreasing the solvent’s viscosity increases the solubility and polyphenol diffusion by making the plant’s cell walls more permeable. The largest amount of TPC obtained with SM may be due to surfactants breaking down the membranes and cell walls of plant tissue and releasing these molecules into the medium [23]. In addition, it is assumed that SM can bind polyphenols through its phenol groups by means of hydrogen bonds and improve their extraction [59].

4. Conclusions

This study investigated sorbitan monooleate as a capsaicin extractor, giving comparable results to the traditional extraction methods. The effectiveness of sorbitan solutions to obtain capsaicin was similar to or greater than that of conventional extraction with solvents such as methanol, acetone, acetonitrile, and hexane, without the drawbacks of the latter, such as toxicity, high costs, and environmental poisoning. Optimization was carried out using response surface methodology with Box–Behnken design to determine the most effective conditions for extracting capsaicin and dihydrocapsaicin from lyophilized serrano chili. Temperature, particle size, and sorbitan concentration were the optimization parameters, giving the highest capsaicinoid extraction yield under 65 °C, 40 mesh, and 2% SM conditions. Relying on the LOF, R2, and ADEQ precision values, the mathematical models predicted the extraction process of capsaicinoids from dehydrated serrano pepper. In addition, total polyphenols and antioxidant capacity were evaluated in the extracts, and their levels were related to the extraction conditions. Particle size significantly affected antioxidant capacity, with the extracts prepared from 40 mesh exhibiting the highest antioxidant capacity values. TPC increased with extraction temperature. Thus, sorbitan monooleate solutions can be an outstanding alternative to extract capsaicinoids and bioactive compounds from dehydrated chili. Consequently, the high levels of capsaicinoids and bioactive compounds in the extracts obtained with sorbitan monooleate could facilitate the utilization of this extraction method in food, pharmaceutical, and agronomic industries, removing the need for organic solvents. Future research can focus on investigating the effect of other optimization parameters on obtaining capsaicins. In addition, the developed models can be further validated with different types and concentrations of surfactants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/appliedchem5020007/s1, Table S1. Validation parameters in accordance with The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).

Author Contributions

Conceptualization: J.A.M.-V.; Data curation: A.d.J.C.-B., R.M.-F. and J.A.M.-V.; Formal analysis: A.d.J.C.-B. and J.A.M.-V.; Funding acquisition: J.A.M.-V.; Investigation: A.d.J.C.-B. and E.A.L.-Z.; Methodology: A.d.J.C.-B., E.A.L.-Z. and J.A.A.-V.; Project administration: J.A.M.-V.; Resources: J.A.M.-V.; Software: A.d.J.C.-B., E.A.L.-Z. and J.A.M.-V.; Supervision: J.A.M.-V.; Validation: J.E.M. and R.M.-F.; Visualization: A.d.J.C.-B. and J.A.M.-V.; Writing—original draft: A.d.J.C.-B.; Writing—review and editing: J.A.M.-V., J.E.M. and A.Q.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The author, Campos-Badillo, would like to thank the scholarship for master’s degree studies awarded of the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT).

Conflicts of Interest

The authors declare no conflicts of interest.

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Appliedchem 05 00007 g001
Figure 1. HPLC chromatogram of capsaicin (A) and dihydrocapsaicin (B) present in sorbitan extracts of freeze-dried serrano chili.
Figure 1. HPLC chromatogram of capsaicin (A) and dihydrocapsaicin (B) present in sorbitan extracts of freeze-dried serrano chili.
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Figure 2. The effects of particle size, temperature, and concentration of SM on the (a) capsaicin and (b) DHC concentration extracted from freeze-dried serrano chili.
Figure 2. The effects of particle size, temperature, and concentration of SM on the (a) capsaicin and (b) DHC concentration extracted from freeze-dried serrano chili.
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Figure 3. Response surface and contour plots showing particle size (mesh), temperature, and SM concentration effects on the capsaicin levels from lyophilized serrano chili. (a) Temperature vs. PS (sorbitan = 2%); (b) sorbitan vs. PS (temperature = 65 °C); (c) sorbitan vs. temperature (TP = mesh 40).
Figure 3. Response surface and contour plots showing particle size (mesh), temperature, and SM concentration effects on the capsaicin levels from lyophilized serrano chili. (a) Temperature vs. PS (sorbitan = 2%); (b) sorbitan vs. PS (temperature = 65 °C); (c) sorbitan vs. temperature (TP = mesh 40).
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Figure 4. Response surface and contour plots showing effects of the particle size (mesh), temperature, and SM concentration on the DHC levels from lyophilized serrano chili powder. (a) Temperature vs. PS (sorbitan = 2%); (b) sorbitan vs. PS (temperature = 65 °C); (c) sorbitan vs. temperature (TP = mesh 40).
Figure 4. Response surface and contour plots showing effects of the particle size (mesh), temperature, and SM concentration on the DHC levels from lyophilized serrano chili powder. (a) Temperature vs. PS (sorbitan = 2%); (b) sorbitan vs. PS (temperature = 65 °C); (c) sorbitan vs. temperature (TP = mesh 40).
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Table 1. Extraction validation data and quantification using 1.5% sorbitan solution.
Table 1. Extraction validation data and quantification using 1.5% sorbitan solution.
CapsaicinDihydrocapsaicin
Correlation coefficient0.9990.999
Precision8.09%7.81%
Relative recoveries95–105%93–102%
Detection limit *2.4 mg/L2.7 mg/L
Quantification limit *7.2 mg/L7.6 mg/L
* Limits of detection and quantification calculated in accordance with ICH [33]. See Supplementary Materials.
Table 2. Capsaicin and DHC concentration (mg/100 g dw) of freeze-dried serrano pepper extracted with different solvents and extraction by Soxhlet as a control.
Table 2. Capsaicin and DHC concentration (mg/100 g dw) of freeze-dried serrano pepper extracted with different solvents and extraction by Soxhlet as a control.
CapsaicinDihydrocapsaicin
Sorbitan *595.01 ± 30.12 b553.13 ± 20.50 a
Acetonitrile558.40 ± 16.37 bc494.99 ± 27.69 b
Methanol686.36 ± 5.010 a561.78 ± 23.21 a
Acetone531.59 ± 21.55 c466.13 ± 13.93 b
Hexane192.33 ± 26.25 d121.66 ± 10.39 c
Soxhlet **612.26 ± 17.83 b564.58 ± 21.67 a
* 1.5% Sorbitan monooleate solution in water. ** Extraction by Soxhlet using acetone as solvent §. Within the columns, different lowercase letters mean significant differences according to the LSD test (p ≤ 0.05).
Table 3. BBD with the observed responses and predicted values for capsaicin and dihydrocapsaicin (DHC) concentration (mg/100 g dw) in the serrano chili extracts.
Table 3. BBD with the observed responses and predicted values for capsaicin and dihydrocapsaicin (DHC) concentration (mg/100 g dw) in the serrano chili extracts.
Uncoded (Xi) and Coded (xi) ValueCapsaicin DHC
RunMeshTemperatureSorbitanObservedPredictedObservedPredicted
120 (−1)55 (−1)2.0 (0)243.24233.06215.23205.39
260 (1)55 (−1)2.0 (0)371.29387.26330.00346.61
340 (0)55 (−1)1.5 (−1)590.00566.25553.13528.71
440 (0)55 (−1)2.5 (1)514.34532.29456.32473.97
520 (−1)65 (0)1.5 (−1)206.46240.39175.22209.47
660 (1)65 (0)1.5 (−1)366.60374.38326.27334.08
720 (−1)65 (0)2.5 (1)245.74237.97206.96199.15
860 (1)65 (0)2.5 (1)414.17380.23366.56332.30
940 (0)75 (1)1.5 (−1)503.56485.61451.77434.12
1020 (−1)75 (1)2.0 (0)220.14204.16188.45171.84
1160 (1)75 (1)2.0 (0)316.04326.22278.53288.37
1240 (0)75 (1)2.5 (1)499.24523.00452.36476.77
1340 (0)65 (0)2.0 (0)651.25671.57580.45596.49
1440 (0)65 (0)2.0 (0)732.69671.57665.98596.49
1540 (0)65 (0)2.0 (0)674.72671.57606.10596.49
1640 (0)65 (0)2.0 (0)634.06671.57511.29596.49
1740 (0)65 (0)2.0 (0)665.14671.57618.63596.49
Table 4. Average antioxidant activity (µM of Trolox/100 g dw), estimated by ABTS and FRAP, and total polyphenols (TPC) (mg gallic acid Eq/100 g dw) of crude extracts of freeze-dried serrano pepper obtained with different combinations of various particle sizes, temperatures, and concentrations of sorbitan, as well as a methanol control.
Table 4. Average antioxidant activity (µM of Trolox/100 g dw), estimated by ABTS and FRAP, and total polyphenols (TPC) (mg gallic acid Eq/100 g dw) of crude extracts of freeze-dried serrano pepper obtained with different combinations of various particle sizes, temperatures, and concentrations of sorbitan, as well as a methanol control.
MeshTemperature
(° C)		Sorbitan
(%)		TPCABTSFRAP
20552877 ± 108.8 d5124 ± 175 f3906 ± 94 ef
20651.5926 ± 240.5 d4457 ± 296 g3641 ± 481 f
20652.51041 ± 233.4 cd5262 ± 225 ef3699 ± 110 f
207521706 ± 101.5 ab5715 ± 97 e3538 ± 371 f
40551.51322 ± 166.5 bc7355 ± 127 ab5929 ± 206 ab
40552.5797 ± 18.1 d7760 ± 87 a4793 ± 860 d
406521487 ± 30.0 ab7418 ± 103 ab5667 ± 524 bc
40751.51715 ± 94.2 a6427 ± 126 cd5577 ± 293 bc
40752.51501 ± 223.2 ab5712 ± 165 e6108 ± 566 ab
60552935 ± 40.2 d7039 ± 155 bc5140 ± 378 cd
60651.51348 ± 236.2 abc6230 ± 152 de4255 ± 795 df
60652.5835 ± 33.9 d5980 ± 181 de4537 ± 510 de
607521374 ± 84.2 abc5943 ± 719 de4814 ± 219 d
Methanol * 1029 ± 62 cd6847 ± 259 bc6511 ± 428 a
Lowercase letters (a, b, c, d, e, f, g) indicate significant differences in treatments according to the LSD test (p ≤ 0.05) (n = 3) * 40 mesh samples submitted at 55 °C.
Table 5. Analysis of variance for the response surface of capsaicin and DHC concentration extracted from freeze-dried serrano chili.
Table 5. Analysis of variance for the response surface of capsaicin and DHC concentration extracted from freeze-dried serrano chili.
SourceDFSSMSF-Valuep-ValueR2Adeq. Precision
Capsaicin
Regression9496,26855,14136.690.0010.97927.240
Linear342,21014,0709.360.008
Quadratic3452,510150,837100.350.000
Cross-point315485160.340.795
Lack of fit3491516381.170.426
DHC
Regression9410,97945,664.0017.800.0110.95841.365
Linear337,50312,5014.870.039
Quadratic3370,933123,64448.20.000
Cross-point325428470.330.804
Lack of fit3502916760.520.690
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Campos-Badillo, A.d.J.; Luna-Zapién, E.A.; Ascacio-Valdés, J.A.; Marszalek, J.E.; Minjares-Fuentes, R.; Quintero-Ramos, A.; Meza-Velázquez, J.A. Use of Sorbitan to Extract Capsaicinoids and Bioactive Compounds: Condition Optimization Study. AppliedChem 2025, 5, 7. https://doi.org/10.3390/appliedchem5020007

AMA Style

Campos-Badillo AdJ, Luna-Zapién EA, Ascacio-Valdés JA, Marszalek JE, Minjares-Fuentes R, Quintero-Ramos A, Meza-Velázquez JA. Use of Sorbitan to Extract Capsaicinoids and Bioactive Compounds: Condition Optimization Study. AppliedChem. 2025; 5(2):7. https://doi.org/10.3390/appliedchem5020007

Chicago/Turabian Style

Campos-Badillo, Andrea de J., Edén A. Luna-Zapién, Juan A. Ascacio-Valdés, Jolanta E. Marszalek, Rafael Minjares-Fuentes, Armando Quintero-Ramos, and Jorge A. Meza-Velázquez. 2025. "Use of Sorbitan to Extract Capsaicinoids and Bioactive Compounds: Condition Optimization Study" AppliedChem 5, no. 2: 7. https://doi.org/10.3390/appliedchem5020007

APA Style

Campos-Badillo, A. d. J., Luna-Zapién, E. A., Ascacio-Valdés, J. A., Marszalek, J. E., Minjares-Fuentes, R., Quintero-Ramos, A., & Meza-Velázquez, J. A. (2025). Use of Sorbitan to Extract Capsaicinoids and Bioactive Compounds: Condition Optimization Study. AppliedChem, 5(2), 7. https://doi.org/10.3390/appliedchem5020007

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