Evaluation of antitumoral and antioxidant activities of the hydroalcoholic extract and fractions obtained from the fruit pericarp of Sapindus saponaria L.

Abstract The fruits of Sapindus saponaria L., popularly known as ‘saboeiro’, have been used in medicine. This study evaluated the antioxidant and antitumor activities of the hydroethanolic extract (HAE) and fractions obtained from the fruit pericarp of S. saponaria. The HAE was obtained from the S. saponaria fruit pericarp by maceration; this was followed by fractionation using reversed-phase solid-phase extraction, resulting in fractions enriched with acyclic sesquiterpenic oligoglycosides (ASOG) and saponins (SAP1, and SAP2), confirmed by mass spectrometry with electrospray ionization (ESI-QTOF-MS). The greatest citotoxic activity was observed with the SAP1 fraction against the CaCo2 cell line with a GI50 of 8.1 µg mL−1, while the SAP2 fraction had a GI50 of 13.6 µg mL−1 against CaCo2. The HAE demonstrated the greatest antioxidant activity. S. saponaria has potential therapeutic use in the pharmaceutical industry as a natural anti-oxidant or antitumor product. Graphical Abstract


Introduction
Sapindus saponaria L. belongs to the Sapindaceae family that occur in tropical and subtropical regions (Acevedo-Rodríguez et al. 2010;Somner et al. 2015).S. saponaria is popularly known as 'sabão-de-macaco, saboeiro, saboneteiro, fruta de sabão, or sabão-de-soldado' , is a small tree occurring in the states of the North, Northeast and Center-West of Brazil, used in landscaping (Albiero et al. 2001).
Sapindus saponaria is known for the great presence of saponins, a natural surfactant found in its seeds and fruits (Albiero et al. 2001;Pelegrini et al. 2008), and acyclic sesquiterpenic oligoglycosides (Murgu and Rodrigues-Filho 2006).The plant has been widely used by the inhabitants of the north and northeast regions of Brazil for the treatment of inflammations, injuries and even cancer.The fruits, in particular, are used by the population to manufacture soap and as remedies against ulcers, skin lesions and inflammations (Albiero et al. 2001;Pelegrini et al. 2008).The bark, root, and fruits of S. saponaria specifically are used in folk medicine as a tranquilizer, astringent, diuretic, expectorant, tonic, blood purifier, and to fight coughs (Abreu Guirado 2005), in addition to cytotoxicity and genotoxicity (Albiero et al. 2001;Rashed et al. 2013a;Santos et al. 2020).
Considering the biological potential reported in the literature, the aim of the study was evaluate the antioxidant and antitumor activities of the hydroalcoholic extract and fractions from the fruit pericarp of S. saponaria.

Characterization by mass spectrometry with electrospray ionization (ESI-QToF-MS)
As demonstrated by Murgu and Rodrigues-Filho (2006), the mass spectrum of the HAE revealed two main chemical families.This first region was called the acyclic sesquiterpene oligoglycoside (ASOG) region, which was formed by intense peaks of m/z from 1100 to 1550, with each peak in the cluster separated by 42 Da.The other region, named the saponin (SAP) region, contained less intense peaks from m/z 700 to 1100, with some of the peaks also showing the pattern of 42 Da mass differences.
In the ASOG fraction, a prevalent presence of peaks above m/z values of 1100 was observed, with each peak separated by 42 Da (equivalent to repeated losses of COCH 2 ), confirming the enrichment of the ASOG fraction with acyclic sesquiterpene oligoglycosides (Figure S1).The putative identification by ESI-QTOF-MS showed four ASOG in negative mode (Table S1).In the SAP fractions, a prevalent presence of a extensive presence of peaks below the m/z of 1100 was observed, confirming the enrichment of the SAP fractions with saponins (Figure S2).The phytochemical profiling enable the putative identification of eleven saponins in negative mode (Table S2).

Antioxidant activity
The HAE, SAP1, SAP2 and ASOG fractions from the fruit pericarp of S. saponaria showed antioxidant activity (Table S3), preferentially by the TBARS assay, with the best result with the HAE (IC 50 of 2.30 mg mL −1 ), followed by the SAP1 and SAP2 fractions (2.43 and 2.49 mg mL −1 , respectively) and ASOG fraction (2.64 mg mL −1 ) all of which had better activity than the Trolox control (9.10 mg mL −1 ).This TBARS assay is widely used to measure lipophilic compound oxidation and antioxidant activity of natural products (Ghani et al. 2017).The OxHLIA assay is based on the inhibition of free radical-induced membrane damage in erythrocytes by antioxidants (Takebayashi et al. 2010).In the OxHLIA assay only the ASOG fraction presented activity with an IC 50 of 89.04 µg mL −1 .The HAE and SAP fractions did not reveal any potential in protecting the erythrocyte population from haemolytic action A number of species of the genus Sapindus have also shown pharmacological antioxidant activity, including S. mukorossi (Shah et al. 2017;Kora 2020), S. trifoliatus (Sahoo et al. 2010;Pradhan 2014), and S. emarginatus (Srikanth and Muralidharan 2010) and S. Saponaria (Niloufer and Lakshmi 2021).

Cytotoxic activity
The HAE, SAP1 and SAP2 fractions from S. saponaria showed activity against tumor cell lines (Table S4), which may be related to the saponins.up to 400 µg mL −1 of the ASOG fraction, on the other hand, did not show capacity to inhibit the proliferation of any of the tumor cell lines tested.Moreover, up to 400 µg mL −1 of the ASOG fraction was not toxic for non-tumor cells (Vero, GI 50 > 400 µg mL −1 ).
The HAE and fractions from S. saponaria also demonstrated activity against non-tumor cells (Vero, GI 50 between 14.3 to 127.0 µg mL −1 ; Table S4).The SI is an excellent tool to assess the selective power of samples against tumor and non-tumor cells, and this number is expressive when near or ≥ 2.0 (Suffness and Pezzuto 1990).Given this aspect, the most promising results found for the SI were the HAE against AGS at 1.9 and against CaCo2 at 1.8; and and with the SAP2 fraction against CaCo2 at 2.1 and AGS at 1.7; and with the SAP1 fraction against CaCo2 at 1.7 according to Table S4.
According to Albiero et al. (2001), the extracts and fractions of fruits from S. saponaria that demonstrated significant activity against an Ehrlich ascitic tumor.Saponins belong to a diverse group of compounds with high chemoprevention potential, and are characterized by a wide spectrum of anti-tumor activities (Koczurkiewicz et al. 2015).The action of saponins is related to their ability to interact with cell membranes, resulting in short-term membrane rearrangements or complete destruction.This ability is responsible for both the cytotoxic and hemolytic activity of saponins (Lorent et al. 2014).

Conclusion
The results of this study showed that the SAP1 and SAP2 fractions (enriched with saponins) showed higher cytotoxic activity in relation to the HAE and the ASOG fraction (enriched with acyclic sesquiterpene oligoglycosides).The latter of which showed significant antioxidant activity.Therefore, S. saponaria has pharmacological potential for use in the pharmaceutical industry, such as in obtaining a natural antitumor or antioxidant product.