Madeira Island is a Portuguese territory that is located in the Atlantic Ocean, which has a temperate tropical climate, which allows for the cultivation of various species of passion fruit. The purple passion fruit (Passiflora edulis
) is one of the species used in the production of juices by the food industry. Only the passion fruit pulp is used in the production of the juice, and the discarded seeds generate thousands of tons of waste every year [1
]. The generation of waste has high costs in its treatment and, based on this, the use of this waste in other processes that can produce value-added products results in great interest for the society and scientific community [4
It is mentioned in the literature that the purple passion fruit seeds oil has antioxidant, anti-inflammatory, and skin lightening effect, among others [7
]. The oil is rich in stilbenes, vitamins, and catechin. It is described in the literature the presence of piceatannol and resveratrol in passion fruit from Japan and Brazil [10
Several studies highlight the effectiveness of resveratrol due to its antioxidant activity, anti-aging potential, neuroprotective effect, and anti-cancer properties, particularly in cases of leukemia, and in cancers of the breast and colon [11
The benefits of piceatannol have not been studied as extensively as in the case of resveratrol [20
]. Piceatannol (3,3′,4′,5-trans-tetrahydroxystilbene) is a polyphenolic compound that has been found in some plants, including grapes, passion fruit, white tea, rhubarb, peanuts, berries, and some mushroom species [10
Stilbenes are compounds that are considered to be phytoalexins, because they protect plants against fungi and toxins. The presence of an additional hydroxyl group in the piceatannol structure gives it greater antioxidant activity when compared to its prodrug, resveratrol [21
]. Piceatannol also promotes collagen production, preventing skin damage and inhibiting melanin synthesis [24
Yokozawa and Kim studies [25
] have shown that piceatannol has a better inhibitory activity of the tyrosinase enzyme, as well as decreases melanin production, better than resveratrol and kojic acid, a potent skin whitening agent.
In previous studies, we evaluated the antioxidant activity of passion fruit seeds extracts, which were obtained by two methods, Soxhlet and Ultrasound, using various solvents. Extracts that were obtained by both methods using ethanol and acetone were chosen, since they showed the highest antioxidant capacity [8
In view of the abundance of passion fruit waste as by-products in Portugal, sustainable management of these by-products is necessary. In this context, the objective of this work was to identify and quantify stilbenes, piceatannol, and resveratrol, by High Performance Liquid Chromatography (HPLC), in Passiflora edulis seeds oil from Madeira Island and compare with commercial passion fruit seeds oil, in order to evaluate the potential of these antioxidant compounds for further applications by the pharmaceutical and cosmetic industries.
3. Materials and Methods
3.1. Samples Preparation
Passion fruit seeds were obtained from the food industry of Madeira Island. These seeds were then dried in a stove and, after that, the extracts were prepared. These extracts were prepared according to Krambeck and collaborators [8
]. The extracts were prepared using ethanol and acetone, with two preparation methods: Soxhlet and ultrasound.
3.2. Chemicals and Standards
Piceatannol and resveratrol standards, as well as ethanol and formic acid, were obtained from Sigma Aldrich (London, UK). Acetone was purchased from Fisher Chemical (Loughborough, UK). Methanol was purchased from VWR Chemicals (Vila Nova de Gaia, PT).
The stock solutions containing 1mg/mL of piceatannol and the same concentration for resveratrol in ethanol were prepared. All of the solutions were stored at −4 °C. Subsequently, for the calibration curve, standard solutions with concentrations ranging from 1.25–20 µg/mL for piceatannol and 0.625–35 µg/mL for resveratrol were prepared.
The flow diagram for the extraction of the two stilbenes, piceatannol and resveratrol, can be seen in Figure 5
. Briefly, after preparing the extracts, these were compared with a commercial oil from Akoma (London, UK), regarding the content of the stilbenes studied through HPLC analysis. In addition to determining the presence of stilbenes, the content of these elements in the extracts were also quantified.
For the extracts that were obtained by Soxhlet, each selected solvent was heated to its boiling point, and the reflux was maintained for eight hours. For the extracts that were obtained by ultrasound, an ultrasound bath (35 kHz/80 W) (Sonorex RK100h, Bandelin, Germany) was used. The extraction time was 60 min at room temperature. At the end of all tests, the solvents were removed while using a rotary vacuum evaporator R-300 (Buchi, Flawil, Switzerland).
Reverse Phase High-Performance Liquid Chromatography (RP-HPLC)
RP-HPLC was carried out with some modifications, according to Lai and collaborators [38
], for the simultaneous determination of two polyphenols: piceatannol and resveratrol. Analyses were carried out using a high-performance liquid chromatography (HPLC) Waters 2690 Separations Module, with photodiode array detector (PDA-Waters 996), and a 20 µL aliquot of the extract was injected onto a Waters ACE Equivalence C18 column (250 × 4.6 mm i.d.; 5 µm particle size). The mobile phases consisted of (A) water with 0.1% formic acid and (B) methanol with 0.1% formic acid. The total run time was 46 min., being 0–10 min., 0–15% B; 10–20 min., 15% B; 20–30 min., 15–35% B; 30–35 min., 35–100% B; 35–40 min., 100% B; 40–41 min., 100–0% B; 41–46 min., 0% B. All of the measurements were carried out at a flow rate of 0.8 mL/min., using a wavelength of 320nm. Peaks corresponding to piceatannol and resveratrol were analyzed by comparison with the retention times and UV spectras of their respective standard solutions and then quantified through calibration curves. The results were expressed in µg/mL oil. All of the analyses were carried out in triplicate.
3.4. Statistical Analysis
The results were statistically evaluated by one-way analysis of variance (ANOVA), in which significant differences at the 5% level were analyzed by the Tukey’s test. SPSS Software (Version statistic 26, IBM SPSS, Chicago, IL, USA) was used for the statistical analysis in this study.
In this study, resveratrol and piceatannol were not detected either in the extracts of by-products of Passiflora edulis that were obtained by the Soxhlet method or in the commercial oil.
Extracts obtained by the ultrasound method using ethanol or acetone showed significant amounts of stilbenes such as piceatannol and resveratrol. Passion fruit by-products can be used in cosmetic and pharmaceutical industries having an added value, in addition to reducing the environmental pollution, avoiding the burning or landfill of waste.
The obtained results also suggest the possibility of production of Passiflora edulis seeds oil with green solvents and the potential interest of this product to industries, as it represents a low-cost ingredient.