Araçá (Psidium cattleianum Sabine): bioactive compounds, antioxidant activity and pancreatic lipase inhibition

“Araçá” has been reported with different biological activities such as antioxidant, antiproliferative and antimicrobial as well as inhibitors of digestive enzymes. The digestive pancreatic lipase enzyme plays a fundamental role in lipid metabolism, and its inhibition has been studied as a target for obesity treatment. This study quantified the bioactive compounds present in different parts of “araçá” fruit and evaluated their antioxidant activity and lipase inhibition properties. Three samples were analyzed for total anthocyanins, total phenolic content, antioxidant activity and pancreatic lipase inhibition. Anthocyanins were reported only in pulp-peel of red “araçá” sample. Phenolic compounds concentration was higher in pulp-peel than in seeds for all samples. The antioxidant activity followed the same trend. A positive correlation was observed between total phenolic content and both antioxidant activity and lipase inhibition. Lipase inhibition activity was higher for pulp-peel compared to the seeds. Overall, the results showed that “araçá” fruit extracts could be beneficial for the treatment of obesity.


INTRODUCTION
"Araçá" (Psidium cattleianum Sabine) is a native Brazilian fruit from the Myrtaceae family originating in the south of Brazil, that is harvested between January and May (PEREIRA et al., 2018). "Araçá" peel colour can be yellow or red, and its pulp is mucilaginous, aromatic, and contains many seeds (MEREGALLI et al., 2020). Psidium cattleianum is known by different names such as "araçá", "araçá-rosa", "araçá-de-comer", "araçá-dapraia" and "araçá-coroa" (PEREIRA et al., 2018) and is consumed in natura, and preserved as jams, jellies, and juices (REISIG et al., 2016). Due to its high content of vitamin C and bioactive compounds with an array of properties (e.g. antioxidant, Pereira et al. anticarcinogenic, analgesic, and antimicrobial), it also merits exploration by the pharmaceutical industry (ALMEIDA LOPES, DE;SILVA, E. De O., 2018;MEDINA et al., 2011;PEREIRA et al., 2018). In vivo research has shown that "araca" can improve blood glucose levels, reduce low-density lipoprotein (LDL), and total blood cholesterol levels, and decrease fat deposition in the liver when administered in the diet of rats (DALLA-NORA et al., 2014).
The biological activities reported for "araçá" are related to phenolic compounds and carotenoids reported to be present in "araçá" fruits (ALMEIDA LOPES et al., 2018;CORRÊA et al., 2011). Consumption of foods rich in phenolic compounds is associated with reduced risk of health disorders due to their neutralization of excess free radicals and reactive oxygen species (BELISÁRIO et al., 2020;SHAHIDI & AMBIGAIPALAN, 2015). Phenolic compounds can also inhibit specific digestive enzymes (WU et al., 2020). Pancreatic lipase acts in the breakdown of fats, including triglycerides and phospholipids, playing an important role in lipid metabolism. Thus, inhibitors of this enzyme represent a potential therapeutic route for obesity due to their reduction of lipid digestion and absorption at the peripheral level. Considering that 50-70% of total dietary fat hydrolysis is performed by pancreatic lipase, inhibition of this enzyme is a potential stand alone treatment for obesity (ALIAS et al., 2017). Orlistat, obtained from Streptomyces toxytricini, is currently the only pancreatic lipase inhibitor approved for clinical use (FINER et al., 2000;ZHANG et al., 2018). It can produce side effects including flatulence, oily spots, abdominal cramps, urgency, faecal incontinence, and steatorrhea (CHAPUT & TREMBLAY, 2007) that reduce patient compliance with treatment regimens. Alternatives are needed, potentially including the use of natural products.
Antioxidant compound content is dependent on the part of the fruit analyzed. Some phenolic compounds are present in greater quantities in the pulp and bark than in the seeds of Momordica cochinchinensis and other exotic fruits from Colombia (CONTRERAS-CALDERÓN et al., 2011;KUBOLA & SIRIAMORNPUN, 2011). Genetic variability of this fruit should also be considered, because there are variations in alleles within the species, both between different populations of the same species, and within single populations (GRIFFITHS et al., 2000). These factors may influence fruit composition and biological activity. Thus, this study verified the potential of different genotypes, and different parts of "araçá" fruit to inhibit pancreatic lipase activity, and to broadly assess "araca" bioactive compound and antioxidant activity content.

"Araçá" samples
Three samples of "araçá" were obtained from the Active Germplasm Bank of native fruits at Embrapa Clima Temperado (31°40′47"S, 52°26′24"W, RS, Brazil, accession numbers AC 44 and AC 87 (red genotype), and Bicudo cultivar (yellow genotype)). Several fruits from three plants of each cultivar or accession (a new plant variant obtained via tissue culture, chemical treatment, or any classical breeding practice, not yet characterized, but assigned an accession number) were harvested when ripe between March and April of 2016. Samples were transported to the laboratory in boxes at 25°C, within 30 min. Pulp-peel were manually separated from the seeds, and both were frozen at -20ºC. Samples were lyophilized and ground (particles diameter < 5 μm) under liquid nitrogen using a ball mill, and stored at -80°C until analyzed.

Nitric oxide radical inhibition
Nitric oxide radical scavenging activity was measured using a previously described method (VINHOLES et al., 2017). Lyophilized "araçá" samples (62.5 mg) were stirred for 5 min in 10 mL of 50% ethanol (1:40, w/v). Samples were filtered (paper filter, Whatman n o 4) and stored at -20°C until analyzed. A 50 µL aliquot of each extract (6.25 mg/ mL) or the 50% ethanol control were mixed with 50 µL of 20 mM sodium nitroprusside, and incubated for 60 min at room temperature under light. Fifty microliters of Griess reagent (0.1 % naphthylethylenediamine dihydrochloride and 1 % sulphanilamide in 2% phosphoric acid) was then added, and mixtures were incubated at room temperature in the dark for 10 min. Absorbance at 562 nm was measured, and results were expressed as percent inhibition (I%) using equation 1: (1) where A control is absorbance of the control reaction (all reagents, except the extract), and A sample is absorbance of the dissolved "araca" extract.

Pancreatic lipase inhibition
Lipase inhibition was measured using the procedure described in the Lipase Activity Assay Kit (MAK046; Sigma-Aldrich). Lyophilized "araçá" samples (250 mg) were stirred for 5 min in 10 mL of ethanol (50 %) (1:40, w/v). Samples were filtered (paper filter, Whatman n o 4) and stored at -20°C until analyzed. Extract samples (2.5 mg/mL) were added separately to the lipase mixture. Absorbance was recorded in a microplate reader and compared with that of the lipase mixture without extract (control). Absorbance was measured at 570 nm at T1 to read A1 and at T2 after incubating the reaction at 37°C for 40 min. Change in A 570 Between T1 and T2 (A2−A1) represents glycerol oxidation. Lipase activity was calculated using equation 2, (2) in which B is the glycerol concentration in the standard curve (nmol), V is the pre-treated sample volume (mL) added to each reaction well, T1 is the time of the initial reading (A1) (min), and T2 is the time of the second reading (A2) (min). One unit is defined as the amount of lipase needed to hydrolyse triglycerides at a rate yielding 1.0 μmol of glycerol per min at 37°C. Orlistat (Xenical) at a final concentration of 0.24 mg/mL was used as a positive control for lipase inhibition, based on a value reported in the literature (CHATER et al., 2016). Percent inhibition of lipase extracts was calculated using equation 3: (3)

Statistical analysis
Analyses were performed in triplicate (n=3) and results (means ± standard deviation) were calculated using Microsoft Excel. Results for total anthocyanin content, total phenolic content, antioxidant activity, nitric oxide radical inhibition, and lipase inhibition were submitted to analysis of variance. Means were compared between "araçá" genotypes and parts (pulp-peel vs. seeds) for each assay by Tukey test at a 0.05 confidence level using WinStat 2.11.

RESULTS AND DISCUSSION
The increased prevalence of overweight people in the world's population reinforces interest in identification of foods with high levels of bioactive compounds (INOUE et al., 2018). Consumption of foods high in phenolic compounds has been associated with reduced risk for development of chronic diseases such as cancer, cardiovascular Pereira et al. diseases, obesity, and atherosclerosis (SHAHIDI & AMBIGAIPALAN, 2015).
All "araçá" samples afforded considerable amounts of phenolic compounds. Anthocyanins were detected only in the pulp-peel of red genotype "araçá", with concentrations of 42.2 and 43.7 mg/100 g dried sample (Table 1). This was expected, because these compounds are responsible for the red color of AC 44 and AC 87 samples (VEBERIC et al., 2015). Total phenolic content was similar among all genotypes. However, higher content was observed in pulp-peel fractions than in seeds among all genotypes. Values for this parameter varied from 1418.5 to 1533.4 mg/100g in seeds, and 1933.3 to 2088.5 mg/100g in pulp-peel (Table 1). These values are higher than those reported by Denardin et al. (2015) (660,19 mg/100g). By contrast, CHAVES et al. (2018) reported higher concentrations of phenolic compounds in the red genotype (719,00 mg/100g) compared to the yellow genotype (382 mg/100g). Phenolic compounds including catechin and ellagic acid were found in high quantities in pulp-peel extracts of AC44, AC87, and Bicudo, while catechin, ellagic acid, and quercetin were the major compounds in seed extracts (PEREIRA et al., 2020).
All "araçá" samples showed scavenging capacity towards DPPH radical. However, for all samples, the pulp-peel extracts were more active than the seed extracts. The yellow genotype (Bicudo) and AC 87 showed the highest activity (Table 1). Values for antioxidant activity varied from 154.9 to 330.3 μg/g in seeds, and 1097.7 to 1277.0 μg/g in pulppeel. The antioxidant activity assay using DPPH• stable radical is widely used to assess native fruits (MEDINA et al., 2011;VINHOLES et al., 2017). "Araçá" showed higher antioxidant activity than "uvaia" or "guabiroba", with a value of 389.7 µg/g, similar to results observed for seeds in the present study (PEREIRA et al., 2012).
The antioxidant activity in the pulp-peel of "camu-camu" and "araçá-boi" fruits, both from the Myrtaceae family, yielded values of 34.79 µg trolox/g and 6048.26 µg trolox/g dried fruit (NEVES et al., 2015). The peels of both samples showed higher antioxidant activities than their pulps. In one other study of fruits from the Myrtaceae family, antioxidant activity values were between 3455.85 µg trolox/g and 16997.37 µg trolox/g of fruit (BARROS et al., 2017).
Nitric oxide radical (NO•) inhibition was also higher in pulp-peel extracts, but did not differ significantly among genotypes, with inhibition ranging from 72.1% to 73.0%. Inhibition by seed extracts varied from 18.3% to 50.0%, with the yellow genotype (Bicudo) being the most active Table 1 -Total anthocyanins content and total phenolic content, antioxidant activity and inhibition of pancreatic lipase of differents "araçá" genotypes [a] .

Sample
Total anthocyanins content [b] Total phenolic content [c] Antioxidant activity DPPH • [d] NO [a] Mean values (n= 3) in a column followed by the same letter are not significantly different from each other (< 0.05 ANOVA followed by Tukey test). [b] Total anthocyanins expressed as mg of cyanidin-3-O-glucoside equivalents/100 g dry basis. [c] Total phenolic content expressed as mg of chlorogenic acid equivalent/100 g dry basis. [d] Antioxidant activity expressed as μg of Trolox equivalents /g dry basis.
( Table 1). The NO• radical is a signaling molecule produced in the body that is responsible for different physiological and pathological processes. In pathological conditions, this radical is produced in excess, causing damage such as DNA fragmentation, cell damage, and neuronal cell death. In addition, NO• is neurotoxic, mediating pathological processes such as cerebral ischemia, epilepsy, Alzheimer's disease, and Parkinson's disease. Thus, its inhibition is of great importance (RADÜNZ et al., 2020).
Nitric oxide radical inhibition apparently correlates with levels of bioactive compounds present in "araçá" fruit (VINHOLES et al., 2017;VINHOLES et al., (2018). It has been reported that even after gastrointestinal digestion, one-third of the phenolic compounds present in "araçá" extracts remains, suggesting strong prospects for maintaining its antioxidant activity.
All "araçá" samples were able to inhibit pancreatic lipase, with greater potency in pulp-peel extracts than in seed extracts (except in AC 87), with percent inhibition above 54% (Table 1). Moreover, all samples, except for Bicudo seed extract, showed inhibitory values significantly higher than that of the positive control orlistat (31.2 ± 3.3 %). AC 44, with high enzymatic inhibition, has cyanidin-3-O-glucoside and catechin as major constituents (PEREIRA et al., 2020). Bicudo and AC 87 contain high catechin concentrations (PEREIRA et al., 2020).
To our knowledge, this is the first report of "araçá" (Psidium cattleianum) ability to inhibit this enzyme. Polyphenols are the main class of pancreatic lipase inhibitors (BUCHHOLZ & MELZIG, 2015) due to chemical characteristics that facilitate bond formation between these compounds and the enzyme (BUCHHOLZ & MELZIG, 2015). Compounds such as myricetin, kaempferol glucosides, catechin/ epicatechin, and procyanidins are associated with pancreatic lipase inhibition (BUCHHOLZ & MELZIG, 2015;CAMARGO et al., 2017;GENDARAM et al., 2017;YOSHIKAWA et al., 2009;ZHANG, B. et al., 2015). The antihyperlipidemic action of catechin is due to its inhibition of key enzymes involved in lipid biosynthesis, and its ability to reduce intestinal absorption of lipids (ANANDH BABU;LIU, 2008). Other studies have reported ellagic acid, quercetin (MARTINEZ-GONZALEZ et al., 2017), and anthocyanins to be lipase inhibitors (YOU et al., 2011). Different biological properties such as thermogenic activity, fat oxidation ability, appetite control, obesity-related hormone level regulation, and inhibition of digestive enzymes involved in the absorption of carbohydrates and lipids are ascribed to the phenolic compounds chlorogenic acid, the catechins, and quercetin, present in "araçá" (SIMÃO et al., 2017).
Correlation analysis showed that the antioxidant activity, nitric oxide radical inhibition, and lipase inhibition positively correlated with total phenolic content (Table 2). Different pharmacological properties have been ascribed to "araçá" that are associated with its chemical composition. In fact, the Psidium genus has been described as having antioxidant, antidiabetic, anticancer, antimicrobial, anti-inflammatory, and anti-aging activities, among others (PEREIRA et al., 2018). These properties showed this fruit's potential applications in the food and pharmaceutical industries. Since few drug options exist for treating obesity, and orlistat has side effects, research into, and development of natural products with pancreatic lipase inhibition are an inspiring avenue of research in pursuit of new therapies. Natural products containing phenolic compounds have already shown efficient inhibition of pancreatic lipase. More detailed studies are still needed. This research may provide some guidance in the development of other studies, given the promise of pancreatic lipase inhibitors for treatment of obesity and related disorders.

CONCLUSION
The present study described bioactive compounds (total anthocyanins and total phenolic content), antioxidant activity, nitric oxide radical neutralization, and pancreatic lipase inhibition of three "araçá" samples. Anthocyanins were present only in red "araçá" genotypes. Total phenolic content was similar among genotypes, but was tissue type dependent within the fruit. Pulp-peel extracts showed higher phenolic content and antioxidant and antiradical activities. This study provided, to our knowledge, the first assessment of pancreatic lipase inhibition by this species. All extracts inhibited lipase more efficiently than orlistat. Thus, "araçá" may provide considerable amounts of phenolic compounds with antioxidant activity and lipase inhibitory properties. Research is being conducted to develop a functional food containing "araçá" extracts, which preserves its biological properties, to aid in controlling and combating obesity.