Pharmacological, toxicological and phytochemical analysis of Spondias dulcis parkinson

Abstract Spondias dulcis Parkinson have been used in traditional medicine in Asia, Oceania, and South America, for different diseases conditions and as a functional food. The scientific literature described as different potential pharmacology such as antioxidant, anti-inflammatory, antimicrobial, thrombolytic and enzymatic inhibitor. This study aimed to: (1) establish the pharmacological activity in intestinal motility in vivo and antioxidant activity in vitro; (2) perform the acute toxicology test in mouse; (3) characterize the phytochemical profile based on counter-current chromatography (CCC) and NMR analysis. The results revealed a laxative effect of S. dulcis extract and a high antioxidant activity (IC50 = 5.10 for DPPH assay and 14.14 for hydrogen peroxide scavenging test). No side effects were observed in the oral acute toxicity test for a dose up to 2000 mg/kg. The chemical profile was identified by CCC and NMR, and the comparison of the data obtained with previous literature revealed the presence of the flavonoid rutin (Quercetin-3-O-rutinoside) in the extract. Graphical Abstract


Introduction
Spondias dulcis parkinson (English: golden apple; portuguese: cajarana, cajá-manga) is part of the Anacardiaceae family, originally from oceania and asia and distributed in the Caribbean and South america (Fernandes et al. 2018).the species and the genus Spondias were used in traditional medicine for the treatment of numerous diseases and in food industry (Sameh et al. 2018).the scientific literature described biology potential of S. dulcis such as antioxidant, antitumoral, antimicrobial, enzymatic inhibitor, immunomodulation and antithrombogenic activity (islam et al. 2013;Yolande et al. 2018;Santos et al. 2023).However, there is limited information about the pharmacological activity, the toxic effects, and the chemical composition of S. dulcis leaves. in this context, the present study investigates possible new biological properties of the extract from the leaves of S. dulcis through intestinal motility activity, antioxidant effects and acute toxicology.this study also established the performed phytochemical profile of S. dulcis using countercurrent chromatography (CCC) and nmR analysis.

Antioxidant activity
the antioxidant activity (table S2) was assessed by the dppH test (Chaves et al. 2016) and by hydrogen peroxide scavenging (mukhopadhyay et al. 2016).the dppH test of the S. dulcis extract provided an iC 50 value of 5.10.the iC50 value obtained from the extract was like those of the controls used in the test (quercetin and gallic acid).Quercetin and gallic acid are compounds with a good antioxidant activity as described in the literature (Chaves et al. 2016;Fernandes and Salgado 2016).the hydrogen peroxide scavenging test resulted in an iC50 of 14.14, similarly to the value obtained in the control group quercetin.

Pharmacology activity
the pharmacological activities evaluated in the present study were intestinal motility and antioxidant effect as mentioned previously in the text (table S1).Castor-oil induced intestinal motility (umer et al. 2013) and non-induced intestinal motility were available (marona and Lucchesi 2004). in the induced intestinal motility with castor-oil, a synergic effect was observed when administered with S. dulcis extract.animals presented a decrease in the time to start defecating was, a slight increase in defecation frequency together with decrease in the weight of the faeces.in the non-induced intestinal motility group, there was a statistically significant difference between the control group and the animals treated with the extract of S. dulcis, presenting the latest the beginning of intestinal movements in a shorter time.

Acute toxicology
no death was observed in both groups of animals treated with a single dose of 2000 mg kg −1 the S. dulcis extract neither in the control group (saline) during the 14 days of trials (El-ishaq et al. 2020).there were also no changes in the weight of the heart, the lungs, the kidneys, the liver, and the stomach in both groups.Liver biochemistry test of the enzymes aSt and aLt was not affected by the treatment with S. dulcis extract (table S3).However, it was found a reduction in body weight the group treated with the extract (Figure S1), which can be an indicative of the laxative activity of S. dulcis parkinson.

Phytochemical characterization
the phytochemical characterization was performed using a modified method of high counter-current chromatography as described in Soares et al. 2019.the mobile phase was composed by ethyl acetate:butanol:water in a 4: 1: 5 ratio.Eighty fractions were obtained which were grouped by affinity and visualized with thin layer chromatography using ethyl acetate/acetic acid/water as a mobile phase.the Fractions 28 to 38 were subjected to a column chromatography, resulting in 50 subfractions grouped by affinity and again eluted in thin layer chromatography (Figure S2). a fraction with a yellow-golden colour and a Rf of 0.76 was submitted to carbon ( 13 C nmR) and hydrogen ( 1 H nmR) nmR spectra for compound elucidation.Based on literature (oliveira et al. 2013;Sameh et al. 2018) the compound corresponded to the rutin (supplement material; Figures S3-S5).the rutin is common compound of Spondias genus and have important bioactive properties, such as antioxidant potential (Santos et al. 2023).Rutin can undergo hydrolysis caused by the intestinal flora or action of digestive enzymes, producing quercetin and sugars (amaretti et al. 2015).Kim et al. (2018) suggest the potential of quercetin in gastrointestinal motility based in possible relation of muscarinic acetylcholine receptors m2 and m3.

Conclusion
the present study described the biological properties and the phytochemical profile from the extract obtained from the leaves of S. dulcis parkinson.the pharmacology revealed a good laxative effect (using an induced and a non-induced models), an antioxidant activity and no mortality in the acute toxicology test.the chemical characterization of the extract of S. dulcis parkinson resulted in the isolation and structural elucidation of the rutin (Quercetin-3-o-rutinoside). the results obtained in this study demonstrate the potential of the plant for medical and pharmaceutical applications and highlight the need to perform further investigations with the S. dulcis parkinson.