Chayote (Sechium edule) Peel Extracts: A Source of Bioactive Compounds for Cosmeceutical Design ^†

Abstract

This study explores the nutritional and economic potential of chayote (Sechium edule) peels (Cps) by extracting bioactive compounds using ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and maceration (ME) techniques. Among these methods, UAE was the most effective, yielding the highest levels of phenolics and carotenoids, along with superior in vitro antioxidant activity. The UAE-derived extract displayed a low cytotoxicity on keratinocyte (HaCaT) cells. Furthermore, a stable gel formulated with the UAE-Cp extract exhibited excellent stability and promising cosmeceutical properties, highlighting the potential for the sustainable utilization of chayote peel waste in skincare applications.

1. Introduction

Sechium edule (Jacq.), commonly known as chayote, is a member of the Cucurbitaceae family, which includes cucumbers, pumpkins, and squashes. This vegetable is notable for its remarkable nutritional profile and phytochemical composition, which are associated with various health-promoting properties [1,2]. Particular attention has been directed toward chayote peels (Cps), a significant by-product of chayote processing. To enhance the sustainable utilization and economic valorization of Cps, diverse green extraction techniques have been employed to recover bioactive compounds with potential applications in the food, cosmetic, and pharmaceutical industries.

Ultrasound-assisted extraction (UAE) is considered one of the most efficient tech-niques for extracting carotenoids and phenolic compounds from plant materials. This is due to its high effectiveness, the widespread availability of ultrasonic equipment, and its use of low temperatures, which helps preserve heat-sensitive compounds [3]. Recently, we implemented ultrasound-assisted extraction (UAE) to isolate phenolic compounds, carotenoids, and antioxidants from Cps. Under optimized conditions—37% ethanol, 55 °C, 224 W, and 30 min—UAE achieved high yields of total phenolics (406 mg GAE/100 g dw) and high antioxidant activity (FRAP value: 82.83 mg AAE/100 g dw; ABTS^•+ value: 319 mg AAE/100 g dw) [4]. High-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis revealed that phenolic acids constituted 71% of the total quantified phenolics, with flavonols, primarily myricetin, accounting for 21%. Among the identified phenolic compounds, the most abundant were 4-hydroxyphenylacetic acid (33.32 ± 1.67 mg/100 g dw), gallic acid (15.09 ± 0.75 mg/100 g dw), protocatechuic acid (14.99 ± 0.75 mg/100 g dw), ferulic acid (14.90 ± 0.75 mg/100 g dw), and p-coumaric acid (11.20 ± 0.56 mg/100 g dw), which collectively contributed to the antioxidant properties of the UAE-Cp extract [4]. Given its bioactive potential, Cp extract holds promise for the development of innovative gel-based drug delivery systems that enhance skin protection and beauty by combating oxidative stress and harmful radiation. To the best of our knowledge, this is the first study to explore the potential use of bioactive extracts from Cp in the formulation of a gel. Hence, a comprehensive analysis of the Cp extract is essential to fully assess its potential effects and ensure its safety for cosmetic use, in compliance with EU regulations [5].

In this study, we compared the efficiency of optimized ultrasound-assisted extraction (UAE) [3] with microwave-assisted extraction (MAE) and maceration extraction (ME), all performed under similar time and temperature conditions, to obtain antioxidant-rich extracts. The safety of the selected extract was evaluated in keratocyte cells, which serve as a barrier between the environment and the internal body. Subsequently, gels incorporating the promising Cp extract were formulated and tested for organoleptic properties and physical stability. The results of this study are relevant for advancing the selection of the most effective extraction method for phenolics and antioxidant compounds from chayote peel, with the aim of developing extracts suitable for cosmetic applications.

2. Material and Methods

2.1. Chayote Samples

Chayote samples (green variety) of 10 fruits collected at Cinfães (Douro, Portugal). at the mature stage were randomly sampled. After washing, fruits were peeled with the help of a manual peeling device, dried (Excalibur 9 Tray Dehydrator, Model 4926 T, Sacramento, CA, USA) at 52 °C for 18 h, ground, sieved (0.75 mm), mixed and stored at room temperature in dark conditions.

2.2. Preparation of Cp Extracts

Cp extracts were obtained (in triplicate) by three extraction procedures (UAE, MAE and ME) using a solid to sample ratio of 1:30 g/mL (37% ethanol/water), temperature and extraction time of 55 °C and 30 min.

• UAE was performed in the ultrasonic bath (Bandelin SON- OREXTM Digital 10 P Ultrasonic baths DK 102 P, Bandelin Electronic GmbH, Berlin, Germany) at 224 W.
• MAE was performed with a MARS-X 1500 W (Microwave Accelerated Reaction System for Extraction and Digestion, CEM, Mathews, NC, USA) at 224 W.
• ME was performed in the same equipment used for UAE without application of ultrasonic power during the extraction process.

The obtained Cp extracts were filtered, centrifuged (5000 rpm for 15 min at 4 °C), evaporated under reduced pressure at 45 °C to eliminate ethanol (Büchi rotavapor R-200, Flawil, Switzerland), and lyophilized (Telstar, model Cryodos-80, Barcelona, Spain) for 48 h and stored at 4 °C until further use. The percent yield of Cp extract was assessed by dividing the weight of the lyophilized extract with the sample weight and multiplying by 100.

2.3. Characterization of Cp Extracts

The total phenolic content (TPC), total carotenoid content (TCC), and antioxidant activity, evaluated by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH^•) radical scavenging, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS^•+) radical scavenging, and ferric reduction antioxidant power (FRAP) assays, were performed as previously described [3]. Results were expressed as milligrams of gallic acid equivalents (GAE) and ascorbic acid equivalents (AAEs) per gram of dry weight (DW) depending on the assay. Based on the previous results, the most interesting extract was selected for cell viability assays. The cytotoxicity of the Cp extracts was assessed (in triplicate) by the 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide (MTT) assay on human dermal cells (HaCaT), as described by [6]. keratinocytes cells were incubated for 24 h in fresh medium in the absence or presence of Cp extract (0.1, 1, 10, 100 and 1000 μg/mL) dissolved in a cell culture medium. The positive control used was DMEM and the negative control was 1% (w/v) Triton X-100 [6].

2.4. Formulation of Gels Containing UAE-Cp Extract

Gels were formulated in triplicate following the methodology described by [7] with slight modifications. Briefly, carbopol Ultrez-21^® (0.7% w/w) was dispersed in an aqueous medium containing propylene glycol (1.5% w/w) and glycerin (2.5% w/w), followed by the addition of UAE-Cp extract (1% w/w) and phenoxyethanol (1% w/w). Then, triethanolamine (0.8% w/w) was added dropwise until the gel was formed. Gels formulated without UAE-Cp extract were regarded as the control gels.

2.5. Characterization of Gels Containing UAE-Cp Extract

Both control and test formulations were subjected to stability studies (color, odor, homogeneity, microbial growth, pH, and spreadability) for 10, 20 and 30 days at 8 °C and 25 °C. The color and microbial growth of the gels were assessed visually, while homogeneity was evaluated through tactile inspection. The pH values were determined (in triplicate) by dispersing an aliquot of each formulation in distilled water (10%, w/v), and using a calibrated pH meter (CRISON micropH 2002, Barcelona, Spain). Spreadability was measured (in triplicate) following Slip and drag” method, as described by [8]. Tests were performed by weighing 0.5 grams of gel, which was then spread between two glass plates to form a circle with a 2 cm diameter. Then, a weighted object was placed on the slides to eliminate trapped air and create a consistent film between them. Excess gel was carefully removed from the edges. The top slide was then dragged with a force of 50 g, and the time required for the slide to move 6 cm was recorded. The spreadability (S) was determined by Equation (1).

$$S=m\times {\displaystyle \frac{l}{t}}$$

(1)

where m = weight applied to the top slide (20 g); l = distance moved by the slide (6 cm); t = time taken (seconds) to separate the slides.

2.6. Statistical Analysis

Results were expressed as means ± standard deviation (SD) of three parallel measurements and statistically analyzed by the SPSS statistic software, version 20.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was applied to investigate the differences between extracts and gels and post hoc comparisons of the means were performed with Tukey’s HSD test. Statistical significance was accepted at a level of p < 0.05.

3. Results and Discussion

3.1. Characterization of Chayote Peel Extracts

UAE, MAE and ME were employed for extraction of phenolic, carotenoid and related antioxidant compound from chayote peel. All Cp extracts were prepared using a solid to sample ratio of 1:30 g/mL (37% ethanol/water), temperature and extraction time of 55 °C and 30 min. UAE and MAE employed 224 W power, while ME did not consider this parameter. The comparative analysis of extraction yields and in vitro TPC, TC, and antioxidant activity of Cp extracts obtained from the three extraction techniques, UAE, MAE and ME is reported in

Table 1. Extraction yields, total phenolics Content (TFC), total carotenoids content (TCC), DPPH^• radical scavenging activity, ABTS^•+ radical scavenging activity and ferric-reducing antioxidant power (FRAP) of chayote peel extracts prepared by different technologies.

	UAE	MAE	ME
Extraction yield (%)	9.34 ± 1.22 a	6.56 ± 1.45 b	6.88 ± 0.64 b
TPC (mg GAE/ 100 g DW)	421.89 ± 10.64 a	307.11 ± 6.45 c	347.88 ± 8.54 b
TCC (mg/ 100 g DW)	10.14 ± 0.87 a	9.67 ± 0.84 b	6.67 ± 0.64 c
DPPH• (mg TE/ 100 g DW)	209.32 ± 12.07 a	203.56 ± 22.84 a	200.55 ± 9.64 a
ABTS•+ (mg AAE/ 100 g DW)	420.87 ± 23.64 a	306.59 ± 7.36 c	346.09 ± 31.65 b
FRAP (mg AAE/ 100 g DW)	85.78 ± 7.68 a	77.89 ± 10.64 a	71.89 ± 10.64 b

Values are expressed as mean ± standard deviation (n = 3). Different letters (a–c) in the same line indicate significant differences between extracts (two-way ANOVA, p < 0.05). Abbreviations: gallic acid equivalents (GAE); trolox equivalents (TE); ascorbic acid equivalents (AAE); ultrasound-assisted extraction (UAE); microwave-assisted extraction (MAE); maceration extraction (ME).

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The ethanolic ME resulted in an extraction yield of 6.88%, significantly higher (p < 0.05) than the 2.9% yield reported by [9] for Cp methanolic extracts. However, the temperature and extraction time were not specified in their study. Bellur et al. (2016) also used ME with ethanol, methanol, and water as solvents for 3 hours at a 1% solid to solvent ratio, reporting extraction yields of 0.5%, 1.2%, and 1.5%, respectively—much lower than the yield obtained in this study, which utilized 37% ethanol, 55 °C, and a 30-min extraction time. Nonetheless, their study found TPC values of 228, 358, and 387 mg TAE/100 g DW for ethanol, methanol, and aqueous Cp extracts, respectively [10], which are in the same range as obtained in this study. MAE, applied to chayote peel for the first time in this study, produced Cp extracts with antioxidant capacities (measured by DPPH and FRAP assays) comparable to those obtained by UAE. However, MAE requires more expensive equipment than the relatively simple ultrasound bath used in UAE, which may limit its feasibility. Overall, the results suggest that UAE is a promising green extraction technique for valorizing Cp, as it facilitates solvent penetration into plant cells and enhances the release of bioactive compounds. Despite the initial investment in equipment, the UAE technique could prove more profitable in terms of yield and stability of bioactive compounds from Cp. Based on these results, the UAE-Cp extract was selected to evaluate the growth of keratinocyte (HaCaT) cells using a MTT assay. This cell line is considered an appropriate in vitro model for evaluating the toxicity of substances or products designed for dermatological applications [6]. As shown in Figure 1, a significant increase in keratinocyte cell viability was observed after 24 hours of exposure to the UAE-Cp extract, compared to the negative control (NEGc). The UAE-Cp extract did not reduce cell viability at concentrations ranging from 0.1 to 100 μg/mL, but at 1000 μg/mL, cell viability decreased by approximately 54%. Based on these findings, a non-cytotoxic concentration of UAE-Cp extract was determined to be 100 μg/mL, which should be selected for the gel formulations. However, the UAE-Cp extract was incorporated into the gel at a concentration of 1% (w/w), ten times higher than the non-cytotoxic concentration, to account for any potential chemical interactions (matrix effects) with the gel's composition.

3.2. Characterization of Gel Containing UAE-Cp Extract

For consumer acceptance, stability studies of cosmetic formulations are essential to establish criteria for quality control, safety, and efficacy [11]. Control and test formulations were subjected to stability studies (color, odor, homogeneity, microbial growth, pH, and spreadability) for 10, 20 and 30 days at 8 °C and 25 °C. As observed in

Table 2. Characterization of test/control gels at variable storage conditions over a period of 30 days.

	Time (days)	8 °C ± 1		25 °C ± 1
		Test	Control	Test	Control
Color	DAY 0	Gr	T
	DAY 10	Gr	T	T	T
	DAY 20	Gr	T	T	T
	DAY 30	Gr	T	t	T
Odor, Look, Microbial growth	DAY 0	(-), T, NA	(-), T, NA
	DAY 10	(-), T, NA	(-), T, NA	(-), T, NA	(-), T, NA
	DAY 20	(-), T, NA	(-), T, NA	(-), T, NA	(-), T, NA
	DAY 30	(-), T, NA	(-), T, NA	(-), Tr, NA	(-), T, NA
pH	DAY 0	5.61 ± 0.32 a	5.21 ± 0.21 a
	DAY 10	5.61 ± 0.24 a	5.22 ± 0.31 a	5.59 ± 0.44 a	5.02 ± 0.44 a
	DAY 20	5.56 ± 0.45 a	5.14 ± 0.35 a	5.58 ± 0.43 a	5.23 ± 0.22 a
	DAY 30	5.52 ± 0.23 a	5.24 ± 0.33 a	5.45 ± 0.11 a	5.21 ± 0.31 a
Spreadability (g.cm/s)	DAY 0	4.01 ± 0.51 d	4.12 ± 0.31 a
	DAY 10	4.61 ± 0.24 c	4.22 ± 0.22 a	5.09 ± 0.44 bc	4.31 ± 0.14 a*
	DAY 20	5.56 ± 0.80 b	4.16 ± 0.31 a*	5.85 ± 0.33 b	4.56 ± 0.20 ab*
	DAY 30	6.03 ± 0.53 b	4.26 ± 0.27 a*	7.12 ± 0.34 a	5.03 ± 0.23 b*

Values are expressed as mean ± standard deviation (n = 3). For each parameter and each test/control formulation, different letters (a–d) indicate significant differences between conditions (Two-way ANOVA, p < 0.05). * indicates significant differences between test/control formulations (t-test, p < 0.05). Abbreviations: Gr (green); DGr (dark green); T (transparent); Tr (translucent); (-) absent; NA (not applicable).

, at the beginning of the experiment, gel containing UAE-Cp extract (test formulation) presented homogeneous aspect, greenish color, and a characteristic plant odor. After placing the gel containing UAE-Cp extract (test formulation) at 8 °C ± 1 and 25 °C ± 1, for 30 days, few changes in color and look were observed. Additionally, no odor or visual microbial growth was observed throughout the entire study period. While the antimicrobial potential of the UAE-Cp extract (and the test formulation) was not evaluated in this study, previous research has indicated that chayote exhibits antimicrobial properties [1]. Therefore, the UAE-Cp extract developed in this work could potentially act as a natural preservative, inhibiting microbial growth in the gel. Since phenoxyethanol was added in both the test and control gel formulations, further studies are needed to assess the specific antimicrobial contribution of the UAE-Cp extract in the gel formulation. Since the normal pH of skin is 5.5, ideally the topical formulation should be nearer to this pH range [4]. The pH of both control (5.21 ± 0.21) and test formulation (5.61 ± 0.32) shows minor changes when stored at 8 °C ± 1 and 25 °C ± 1 for 30 days. This result suggests that formulation containing the UAE-Cp extract is stable at both temperatures, supporting its application for topical use. Spreadability is a key characteristic of gels. A formulation with high spreadability ensures that the active substances are evenly distributed over a larger area, enhancing their effectiveness in delivering a therapeutic effect [4]. At the start of the experiment, the incorporation of UAE-Cp extract did not affect the gel's spreadability, as the values (4.01 ± 0.51 g.cm/s) were like those of the control test (4.12 ± 0.31 g.cm/s). This suggests that adding UAE-Cp extract (at a concentration of 1% w/w) to the gel formulation does not hinder its ability to spread with minimal shear force. In contrast, a slight increase in spreadability was observed for both storage conditions (8 °C ± 1 and 25 °C ± 1) over a 30-day period. The highest spreadability value (p<0.05) of 7.12 ± 0.34 g.cm/s was recorded for the gel stored at 25 °C ± 1 after 30 days, significantly higher (p<0.05) than the spreadability of the control gel (5.03 ± 0.23 g.cm/s).

4. Conclusions

Compared to microwave-assisted and maceration techniques, the ultrasound-assisted extraction was more effective in extracting phenolics, carotenoids and the ABTS^•+ value from chayote peels. Until a concentration of 100 μg/mL, the ultrasound chayote peel extracts did not experience a decrease in their keratinocytes cell viability, supporting their potential safety for cosmeceutical development. A stable gel containing the ultrasound chayote peel extract dispersed into Carbopol Ultrez-21^® gel was successfully developed and subjected to stability studies for 30 days. The physical stability of the gel formulation containing the ultrasound chayote peel extract was confirmed by the maintained appearance, odor, color, and pH after 30 days of storage at both 4 °C and 25 °C.

Further research is required to identify the optimal concentration of UAE-Cp extract for gel formulation, ensuring it does not alter the physicochemical and rheological properties. Once the optimal concentration is determined, the formulation should undergo additional characterization, including an evaluation of its rheological behavior over extended storage periods, in vitro permeation studies to assess the release profile of bioactive compounds from the Cp extract, and sensory acceptability tests (including human skin irritation studies) to evaluate its potential for cosmeceutical applications.