Phytochemical profiling, antidiabetic, antitumoral and cytotoxic potential of Psidium cattleianum Afzel. ex Sabine leaves of red variety

Abstract In this study, phytochemical profiling, and antidiabetic, antitumoral and cytotoxic potential of aqueous extracts of leaves of red variety of Psidium cattleianum Afzel. ex Sabine were investigated. The extracts were obtained using a cellulase complex. The total phenolic compounds (TPC) were determined, and the individual phenolic compounds were quantified by HPLC-ESI-MS/MS. For the TPC, the amounts varied from 85.91 to 106.33 mg EAG g−1. Eighteen compounds have been identified. The compounds with the highest concentrations were gallic acid, quercetin and protocatechuic acid. Antidiabetic activity was obtained through α-amylase and α-glucosidase inhibition tests. The extract inhibited 17.94% of α-amylase activity and 73.34% of α-glucosidase activity. The antitumoral activity in cells of cutaneous melanoma (SK-MEL-28) and the cytotoxic activity was determined in peripheral blood mononuclear cells (PBMC). The cellular migration was determined for cells SK-MEL-28. Antitumoral effects on cells SK-MEL-28 were observed and the absence of cytotoxicity on the PBMCs. Graphical Abstract


Introduction
The genus Psidium, is composed of about 150 fruitful species, with 60 found in Brazilian territory such as Psidium cattleianum Afzel. ex Sabine (Arac¸ a or Cattley guava). P. cattleianum exists in two morphotypes: with red fruits (red variety) and with yellow fruits (yellow variety). In P. cattleianum, red variety, the main identified phenolic substances are tannins and flavonoids (Medina et al. 2011;Denardin et al. 2015).
The biological activities of P. cattleianum fruits, such as antioxidant and antimicrobial activities, are attributed to their high content of phenolic compounds (Pereira et al. 2018), however, for the leaves, the information in the literature is scarce for some activities and nonexistent for others. Dacoreggio et al. (2019), when studying the aqueous extracts of leaves of P. cattleianum (yellow variety), verified promising results for allelopathic activity, antimicrobial activity, and disinfection potential. The antidiabetic, antitumor (melanoma) and cytotoxic activities of extracts from the leaves of P. cattleianum (red variety) are not reported in the literature. In this work, we report the phytochemical profile, antidiabetic, antitumor and cytotoxic potential of the aqueous extract from the leaves of P. cattleianum, red variety.

Extraction and TPC
In the leaves, after the enzyme-assisted extraction, smaller granules with some breaking points are observed, resulting from the hydrolysis ( Figure S1). This shows that there was hydrolysis of the leaves by cellulases. The hydrolysis provided the migration of phenolic compounds from the leaves to the aqueous medium. The extract presented 106.33 ± 0.842 mg EAG g À1 for TPC. Dacoreggio et al. (2019), for aqueous leaf extracts of P. cattleianum, yellow variety, using the same complex of enzymes, obtained 101 mg EAG g À1 . As the same extraction condition was used, the total phenolic content is similar, for the red and yellow morphotypes of P. cattleianum. Table S1 shows the results of the identification and quantification (mg L À1 ) of the phenolic compounds of the aqueous extract of leaves of P. cattleianum, red variety. 18 phenolic compounds were identified, with the largest concentration of gallic acid, followed by quercetin and protocatechuic acid. The first and third classified as phenolic acids and the second as flavonoid. Important compounds, already reported in the literature as promising molecules for drug development, are present in leaves of P. cattleianum.

Antidiabetic activity
Phenolic compounds have a direct relation with antidiabetic activity since they can inhibit specific enzymes (Valko et al. 2007). The results of the inhibition of a-amylase and a-glucosidase for the extract of P. cattleianum leaves are shown in Table S2. The largest values were obtained for extracts in the concentrations of 50 and 100 mg mL À1 and were statistically equal (p < 0.05). For a-glucosidase, the behavior was like the one of a-amylase, with a larger percentile of inhibition for the concentration of 50 and 100 mg mL À1 , statistically equals (p > 0.05), however with superior value to the a-glucosidase. Bailão et al. (2015) identified quercetin and gallic acid in extracts of Myrciaria cauliflora (DC) Berg, also from the Myrtaceae family. The extract showed significant inhibition values for the main enzymes involved in carbohydrate digestion, a-glucosidase, and a-amylase. In the literature, there is evidence that quercetin is a good inhibitor of a-glycosidase (Tadera et al. 2006) and is considered a good inhibitor of digestive enzymes (Vinholes et al. 2017) as well as the protocatechuic acid (Montagut et al. 2010).

Antitumoral activity and cytotoxicity
The PBMC and the SK-MEL-28 were treated with the extract of P. cattleianum leaves in different concentrations and times of exposition. For the PBMC we observe an increase of the cellular viability in the different times and tested concentrations ( Figure S2.). For the 24 h, there was an increase of the number of viable cells, compared with the control, for all treatments, being 12.10%, 4.03%, 37.17%, 68.59%, and 91.64% for the extract in the concentrations of 5 mg mL À1 , 50 mg mL À1 , 100 mg mL À1 , 250 mg mL À1 , and 500 mg mL À1 , respectively. However, for 5 mg mL À1 and 50 mg mL À1 there was no significant statistical difference (p > 0.05) concerning the control. For the 48 h and 72 h of exposure, there was similar behavior, with the increase of the cellular viability, although lower than the occurrence in 24 h of exposure to the extract in different concentrations. There was no significant statistical difference (p > 0.05) between the results for 48 h and 72 h, for one concentration, with a maximum increase of around 40% for the viable cells (500 mg mL À1 ). For SK-MEL28 cells, a reduction in the cellular viability occurred ( Figure S3) of 14.39% and 5.19% for the viable cells exposed for 48 h to the extract in concentrations of 5 mg mL À1 and 500 mg mL À1 . For the remaining concentrations in the studied times of exposure, the number of viable cells remained statistically equal to the control (p < 0.05).
In Figure S4 we show the images of the wounds in the cellular monolayer of SK-MEL-28 before and after the treatment with the P. cattleianum leaves extract in the concentrations of 5, 50, 100, 250, and 500 mg mL À1 , in 24 h and 72 h. When we analyse the images of cellular migration, it is possible to observe that the extracts inhibited the cellular migration mainly in the highest concentrations, with a highlight to the images of 500 mg mL À1 of the 24 h and 72 h, confirming the results obtained to the cellular viability. According to Link et al. (2010), polyphenols can play an important role in the prevention of cancer by epigenetic mechanisms, mainly by methylation of DNA, hindering the modification of histones and the regulation of the expression of RNA. Medina et al. (2011) tested the antiproliferative effect of extracts of fruits of P. cattleianum in human cancerous cells MCF-7 (breast) and Caco-2 (colon), using cells of embryonic fibroblasts of 3T3 rat as control. The aqueous extracts were tested in concentrations of 80, 60, and 40 mg mL À1 . All the P. cattleianum extracts reduced the survival rates of breast cancer cells (MCF-7) and colon cancer cells (Caco-2), through a different mechanism of toxicity, since these extracts do not affect the fibroblastic cells (3T3), like what was seen in our study.

Experimental
The experimental section is available in supplementary material.

Conclusion
The leaves of P. cattleianum Afzel. ex Sabine, red variety, contain an expressive amount of total phenolic compounds. Among the phenolic compounds are gallic acid, quercetin and protocatechuic acid. Using enzyme-assisted extraction, it is possible to obtain aqueous extracts, which can be directly applied in determining biological activities and in characterisation. The extract presented promising antidiabetic activity, mainly regarding the inactivation of a-glucosidase. Moreover, the antitumoral effect on cells SK-MEL-28 and absence of cytotoxicity on the PBMCs were observed, indicating therapeutic potential.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was financed in part by the Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC -Termos de outorga 2019TR648 e 2021TR797). Registration Number in SisGen (Sistema Nacional de Gestão do Patrimônio Gen etico e do Conhecimento Tradicional Associado) AC525B6.