Lychee peel extract obtained by ultrasound-assisted extraction: bioactive compounds and functional properties

. The purpose of this study was to evaluate the impact of extracting solvent composition on the concentration of bioactive compounds and functional properties of lychee peel extracts obtained by ultrasound-assisted extraction. Five conditions were evaluated with different solvent proportions: 100% water, 25% ethanol and 75% water, 50% ethanol and 50% water, 75% ethanol and 25% water, and 100% ethanol. The use of an equitable mixture of water and ethanol (50% ethanol and 50% water) resulted in extracts with a higher content of total phenolic compounds (10964.3 mg EAG 100 g -1 ), antioxidant activity (27.40%), and inhibition of α -glucosidase (94.31%), α -amylase (33.49%) and angiotensin-converting enzyme (ACE, 51.82%). This extract showed a diversity of phenolic compounds (quercetin 3-glucoside, procyanidin A2, B1, and B2, epicatechin, catechin, kaempferol 3-glucoside, trans-resveratrol, myricetin, and gallic acid). Therefore, extracts of lychee peel with potential anti-diabetic and anti-hypertensive activity can be obtained by ultrasound-assisted extraction. 50% water and 50% ethanol may be suggested as the extracting solvent.


Introduction
Lychee (Litchi chinensis) is a fruit predominantly from subtropical and tropical climates, with an estimated annual world production of 3.5 million tons (Morales-Trejo et al., 2022).It consists of seeds, a white and sweet edible pulp with a gelatinous characteristic, and a rough, red, and slightly rigid shell.Lychee is commonly consumed fresh, but it can be processed to obtain dried fruits, wines, and juices to meet market demand for new products and extend their shelf life (Zhao, Wang, Wang, Zhu, & Hu, 2020).
The profile and concentration of bioactive compounds in fruit peel extracts depend on cultivars and fruit maturation stage, environmental factors, and extraction methods (Yao et al., 2021).Extracts are commonly obtained using conventional solid-liquid extraction methods with organic solvents.However, these methods may change the chemical composition of the extracts and use high volumes of solvents (Silva, Garcia, & Franciscato, 2016).
The ultrasound-assisted extraction technique may have several advantages: shorter process time, higher yields, low-temperature extractions, and maintenance of the composition and bioactivity of the extracts (Kumar, Srivastav, & Sharanagat, 2021).Furthermore, it can be considered "environmentally friendly" regarding reagent and energy consumption compared to conventional extraction techniques (Živković, Šavikin, Janković, Ćujić, & Menković, 2018).The parameters of the extraction process are important in the quality of the obtained extract, such as sonication power, extraction time, frequency, ultrasonic wave distribution, type of solvent, solid-liquid ratio, particle size, and temperature, among others (Chemat et al., 2017;Ez zoubi et al., 2021).
Among the process parameters, the type of solvent was reported as the most critical variable, compared to temperature and process time (Silva et al., 2016).Extractions using only water as a solvent may have low yields, and mixing water with organic solvents, such as ethanol, is recommended.Ethanol has a high affinity with phenolic compounds and is the most recommended solvent for extracting these compounds from agroindustrial by-products.In addition, it has other advantages, such as being obtained from a plant source (sugar cane), low cost, and having GRAS (Generally Recognized as Safe) certification (Kumar et al., 2021).The rise in the proportion of ethanol in solvent mixtures is related to an increase in the concentrations of phenolic compounds in the extracts, up to a maximum limit, followed by a reduction in yield (Kumar et al., 2021).Thus, studying solvent composition (water and ethanol mixture) is important for evaluating ultrasound-assisted extraction processes.
Thus, the aim of the present article was to evaluate the effect of different concentrations of ethanol and water as a solvent on the functional properties and concentration of bioactive compounds of lychee peel extracts obtained by ultrasound-assisted extraction.

Raw material and processing of the lychee peels
The lychee fruits at commercial maturation were purchased in the city of Maringá -PR, Brazil (harvest of 2020/2021), sanitized and selected based on the absence of injuries.Subsequently, they were manually peeled, and the peels were dried at 45°C for 24h in a drying oven with air circulation.Afterward, they were ground in a mill (SL-30, Solab, SP, Brazil) (Silva et al., 2016), and the peel flour was standardized using Tyler series sieves, with an average diameter of 0.85 cm (20 mesh sieve).

Chemical composition of lychee peel flour (LPF)
The chemical composition of LPF was determined according to AOAC (2012).Moisture was determined in an oven at 105°C until constant weight (AOAC 934.06), lipids by Soxhlet using ethyl ether as the solvent (AOAC 920.39), ash by incineration at 550°C (AOAC 940.26), protein content by the micro-Kjeldahl method with a factor of 6.25 (AOAC 960.52) and carbohydrates by difference.

Ultrasound-assisted extraction
The extraction was performed using an ultrasonic bath of 2.8 L at a frequency of 40 kHz (USC 1400 A, Unique, SP.Brazil).The process parameters were a solid-liquid ratio of 1:20 (m/v) and an extraction time of 10 min.These parameters were selected based on previous studies with extracts of citrus (Ma, Chen, Liu, & Ye, 2009), pomegranate (Sharayei, Azarpazhooh, Zomorodi, & Ramaswamy, 2019), and jaboticaba (Rodrigues et al., 2015) peel.These studies reported maximized extraction of target compounds at these conditions, including phenolic compounds.Five conditions were tested, using water and ethanol as solvents in different proportions, being 100% water, 25% ethanol and 75% water, 50% ethanol and 50% water, 75% ethanol and 25% water and 100% ethanol.

Statistical analysis and experimental design
The experiment followed a completely randomized design with two replications.Analyzes were realized in triplicate.The TPC data, antioxidant, and enzyme inhibitory activities were submitted to Analysis of Variance (ANOVA) and Tukey or Fisher's multiple comparison test (p=0.05)using the XLXTAT 2022 software (Adinsoft, Paris, France).

Chemical composition of LPF
The chemical composition of LPF is shown in Table 1.The LPF had a moisture content of 10.64 g 100 g -1 and a carbohydrate concentration of 74.17 g 100 g -1 , which was the primary nutrient in LPF.The carbohydrate content is intrinsically linked to the production of compounds by the tissues that make up the pericarp or epicarp of fruits for protection, such as polysaccharides (pectin, cellulose, and hemicellulose) and dietary fibers (Pareek, 2016;Queiroz, Abreu, Santos, & Simão, 2015).Studies involving the characterization of litchi peel are still scarce (Chukwuma et al., 2021), and significant concentrations of phenolic compounds have been reported, such as procyanidins, anthocyanins, and flavones (Liu et al., 2021).
However, the composition may change the extracts based on the extraction method and process parameters (Devequi-Nunes, 2018; Lazarjani, Young, Kebede, & Seyfoddin, 2021).In this way, LPF may have important nutrients associated with health benefits to consumers, suggesting its utilization as a raw material for extracts.

TPC, phenolic compounds profile, antioxidant activity (DPPH and ABTS) and organic acids of lychee peel extracts
The total phenolic compounds and antioxidant activity (DPPH and ABTS) of lychee peel extracts are shown in Table 2, while the inhibitory effect on α-glucosidase, α-amylase and ACE enzymes is shown in Table 3.The extracts showed TPC of 6426.1 to 10,964.4 mg EAG 100 g -1 , values close to those reported in tangerine extracts, 14,000 mg EAG 100 g -1 (Nipornram, Tochampa, Rattanatraiwong, & Singanusong, 2018) and higher than those reported for mango, 400 mg EAG 100 g -1 (Sánchez-Camargo et al., 2021), araticum, 489 to706 mg EAG 100 g -1 (Arruda et al., 2019) and orange, 46 to 149 mg EAG 100 g -1 (Barrales et al., 2018).Furthermore, the extracts showed antioxidant activity of 86.22 to 90.89% of radical inhibition in the DPPH analysis and 16.61 to 27.40% of radical inhibition in the ABTS analysis.Finally, the extracts showed 28.63 to 33.49% inhibition of α-amylase, 73.92 to 94.31% inhibition of αglucosidase, and 33.93 to 51.82% inhibition of ACE.Inhibition of α-glucosidase and α-amylase promotes a reduction in the rate of glucose absorption in the small intestine, decreasing postprandial hyperglycemia (Aleixandre, Gil, Sineiro, & Rosell, 2022).At the same time, ACE is an enzyme that plays a vital physiological role in the degradation of the vasodilator bradykinin leading to hypertension and in the regulation of blood pressure by converting angiotensin-I to angiotensin-II, a potent vasoconstrictor.For this reason, there is a demand for foods with ACE-inhibitory activity (Kessy, Wang, Zhao, Zhou, & Hu, 2018;Rong et al., 2018).The higher inhibition of α-glucosidase compared to α-amylase is desired, as strong or complete inhibition of the latter may cause undesirable side effects after consumption of the extract (Oboh, Ademiluyi, Agunloye, Ademosun, & Ogunsakin, 2018).Our results demonstrate that the extraction process used in the present study could extract the bioactive compounds commonly reported for lychee peel, resulting in an extract with important antioxidant activity and potential anti-diabetic and anti-hypertensive properties.
The increase in the concentration of ethanol in the solvent mixture caused an increase in TPC and antioxidant activity (ABTS) up to a concentration of 50%, with a subsequent decrease (p < 0.05).It was observed the same behavior for the inhibition of α-amylase, α-glucosidase, and ACE enzymes.Therefore, to obtain an extract with higher antioxidant activity and anti-diabetic and anti-hypertensive properties, it would be interesting to use a mixture of 50% water and 50% ethanol as solvent.The increase in ethanol concentration promotes an increase in the diffusivity and solubility of phenolic compounds in the plant matrix due to the reduction in the dielectric constant of the solvent.However, high ethanol concentrations can cause protein denaturation and dehydration of plant tissue, resulting in reduced yields (Kumar et al., 2021).The presence of ethanol in the solvent promotes the recovery of bioactive compounds.When used together (binary systems), it turns out that fewer polar compounds are extracted in ethanol, while polar compounds are better recovered with water.When water and ethanol are used together, there is an advantage over their pure constituents since a synergistic effect is obtained for both solvents in the phytochemical extraction (Lim, Cabajar, Lobarbio, Taboada, & Lacks, 2019).
Our results demonstrate the importance of evaluating the type and composition of solvents, as increases of 17.08 to 66.42% radical inhibition were observed in the antioxidant activities of 50% water and 50% ethanol condition concerning the other conditions.Furthermore, increases of 1.96 to 4.86% in α-amylase inhibition, 4.00 to 20.39% in α-glucosidase inhibition, and 7.27 to 17.89% in ACE inhibition were observed for this condition.
Thus, the 50% water and 50% ethanol condition was used to evaluate phenolic compounds and organic acid profiles to correlate its composition with the observed potential health effects.The phenolic compound profile is presented in Table 4.The lychee peel extract showed the highest concentration of procyanidin A2, followed by procyanidin B2, epicatechin, quercetin 3-glucoside, kaempferol 3-glucoside, catechin, gallic acid, trans-resveratrol, procyanidin B1 and myricetin.Procyanidin oligomers are compounds commonly reported in lychee peels, including epicatechin and procyanidins A (mainly A2) (Yao et al., 2021).Quercetin and its derivatives, epicatechin, catechin, procyanidins A, and epigallocatechin gallate, have been reported to be the phenolic compounds with the most significant α-amylase inhibitory activity.At the same time, procyanidins A2 would have a high capacity to inhibit α-glucosidase (Chukwuma et al., 2021).Finally, some flavonoids (rutin, quercetin, procyanidins, catechin, and epicatechin) and gallic acid have exhibited inhibitory action on the ACE enzyme (Kessy et al., 2018;Yao et al., 2021).
The suggested mechanisms of action of phenolic compounds on antioxidant, antidiabetic, and antihypertensive properties have been studied.Procyanidin oligomers may reduce oxidative processes through the chelation of metal ions and the sequestration of reactive oxygen species, resulting in high antioxidant activities (Chukwuma et al., 2021).It has been described that A-type dimeric procyanidins (procyanidins A2) could have a greater antioxidant activity due to the number of hydroxyls in their structures (Zhang et al., 2020).Kaempferol 3-glucoside may show antioxidant activity due to a hydroxyl group in the C ring, contributing to its radical scavenging activity (Mukemre, Konczak, Uzun, & Dalar, 2020).
Procyanidin oligomers may also inhibit α-glucosidase and α-amylase by binding to their active sites (Chumsri, Chaijan, & Panpipat, 2021), resulting in improved glucose homeostasis, suppressing glucose production in the liver, modulating protein expression, and increasing glycolysis.Procyanidins A showed more significant activity than procyanidins B (Yao et al., 2021).The inhibition of α-glucosidase may be related to the molecular structure of the phenolic compound and glycosylation, and lower glycosylation and smaller molecular structures show higher inhibitory activity (Zhao et al., 2020).The anti-hypertensive activity of phenolic compounds is related to the number of hydroxyl groups, their position, and the presence of double bonds in the rings, resulting in stable complexes that chelate zinc in the ACE-I active site (Escher et al., 2020).Furthermore, phenolic compounds may interact with the disulfide bridge residue at the protein's active site (ACE), resulting in enzymatic inhibition (Oboh et al., 2018).Thus, the higher concentration of procyanidins, mainly procyanidin A2, in the 50% water and 50% ethanol extract (6.786 mg 100 g-1) may be related to the observed antioxidant, α-glucosidase and α-amylase inhibitory activities.Therefore, the present study demonstrated that lychee peel extract has phenolic compounds that could exert antidiabetic and anti-hypertensive activity.
The incidence of these compounds implies that the extracts obtained are potential sources for isolating bioactive compounds that may have important therapeutic properties for numerous diseases.Pharmacological perspectives include antioxidant, hypolipidemic, antidiabetic, anticancer, antiinflammatory, neuroprotective, and antimicrobial properties possibly mediated by its constituent polyphenols (Chukwuma et al., 2021).
Malic acid, a dicarboxylic organic acid, is naturally present in vegetables and fruits such as pears and apples and was the predominant acid in the 50% water and 50% ethanol extract (17.08 mg mL-1).During the Krebs cycle, it is produced in the body, helping the body to produce energy (Muhamed, Moussa, Aboasy, & Gaweesh, 2022) enhancing the activities of digestive enzymes and could aid digestion and absorption by various chelating cations (Ren et al., 2021).Other effects of malic acid have been reported by different authors, such as decreasing the quantity of glucose absorbed by the gastrointestinal system (Guevarra & Panlasigui, 2000), stimulating insulin secretion (Édouard & Kouassi, 2009), and decreasing the absorption of glucose through inhibition of the α-glucosidase enzyme (Gou et al., 2015).

Conclusion
This is the first study to verify the effect of solvent type (water and ethanol mixtures) on the concentration of bioactive compounds and functional properties of ultrasonic lychee peel extract.A combination of 50% water and 50% ethanol is recommended, resulting in extracts with a greater content of total phenolic compounds, inhibition activity of α-amylase, α-glucosidase, ACE and antioxidant activity.In addition, this extract was presented as a source of phenolic compounds (kaempferol 3-glucoside, procyanidin A2, B1, and B2, epicatechin, catechin, quercetin 3-glucoside, trans-resveratrol, myricetin, and gallic acid).Therefore, the extract obtained could be recommended for glycemic control and arterial hypertension.However, in vivo studies are needed to prove these effects.

Table 1 .
Chemical composition of lychee peel flour.

Table 2 .
Content of total phenolic compounds and antioxidant activity of lychee peel extracts.
cData are presented as mean ± standard deviation.Values followed by different letters in the column indicate a significant difference (p < 0.05, n = 6).

Table 3 .
Inhibitory activity of lychee peel extracts on alpha-amylase and angiotensin-converting enzyme (ACE).

Table 4 .
Profile of the phenolic compounds (mg 100 g -1 ) and organic acids (mg ml -1 ) of the 50% water and 50% ethanol condition.