Metabolomic Profiling of Mango (Mangifera indica Linn) Leaf Extract and Its Intestinal Protective Effect and Antioxidant Activity in Different Biological Models

Mangifera indica Linn popularly known as mango is used in folk medicine to treat gastrointestinal disorders. The aim of this study was to identify the metabolomic composition of lyophilized extract of mango leaf (MIE), to evaluate the antioxidant activity on several oxidative stress systems (DPPH, FRAP, TBARS, and ABTS), the spasmolytic and antispasmodic activity, and intestinal protective effect on oxidative stress induced by H2O2 in rat ileum. Twenty-nine metabolites were identified and characterized based on their ultra-high-performance liquid chromatography (UHPLC) high-resolution orbitrap mass spectrometry, these include: benzophenone derivatives, xanthones, phenolic acids, fatty acids, flavonoids and procyanidins. Extract demonstrated a high antioxidant activity in in-vitro assays. MIE relaxed (p < 0.001) intestinal segments of rat pre-contracted with acetylcholine (ACh) (10−5 M). Pre-incubation of intestinal segments with 100 µg/mL MIE significantly reduced (p < 0.001) the contraction to H2O2. Similar effects were observed with mangiferin and quercetin (10−5 M; p < 0.05) but not for gallic acid. Chronic treatment of rats with MIE (50 mg/kg) for 28 days significantly reduced (p < 0.001) the H2O2-induced contractions. MIE exhibited a strong antioxidant activity, spasmolytic and antispasmodic activity, which could contribute to its use as an alternative for the management of several intestinal diseases related to oxidative stress.

3 Bellidin, can react by carbon-carbon coupling reactions of the activated ortho position of its 1,3 biphenol moiety 4 with a glucose, to form mangiferin, peak 11. One galllic acid can be added to glucosa moiety of the later, to form 5 peak 13, which loses an OH moiety to give peak 12. Another phenolic derivative, iriflophenone, can be also C-6 glycosilated to form peak 6, which in turn reacts with a gallic acid molecule to form peak 9, and this, in turn, forms 7 peak 14 by esterification of an alcohol of the glucose moiety with a gallic acid molecule ( Figure S2).

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The TPC of mango leaf extract (MLE) was determined according to the procedure reported previously [8] with 13 some modifications. The method uses gallic acid as a reference standard. Briefly, 30 µL of sample was mixed with 150 14 µL of 10% Folin-Ciocalteu reagent (v/v). After 4 minutes, 120 µL of 7% sodium carbonate solution (w/v) was added, 15 followed by 2 hours of reaction at room temperature. The absorbance of the mixture was measured at 760 nm in a 16 microplate reader spectrophotometer (accuSkan GO UV/Vis; Fisher Scientific; PA USA). The TPC value was expressed 17 in micrograms of Gallic Acid equivalents (µg GAE). All measurements were performed in triplicate. The free radical scavenging capacity of the extract was determined by DPPH assay as previously described

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Egg yolk homogenates were used as a lipid-rich medium, according to the method previously reported [10]. Initially, 29 500 µL of this 10% (w/v) tissue homogenate and 100 µL of sample solutions to be tested were added. It was made up to 30 1 mL with distilled water, and 50 µL of AAPH solution (0.07 M) was added to induce lipid peroxidation. Then 1.5 mL 31 of 20% TCA (pH 3.5) and 1.5 mL of 0.8% TBA (w/v) in 1.1% DDS solution (w/v) were added and the resulting mixture 32 was vortexed and then heated at 95 °C for 60 minutes. It was then cooled to 2 °C, then 5 mL of n-butanol was added to 33 each tube, vortexed and centrifuged at 3000 rpm for 10 minutes. The absorbance of the upper organic layer was measured 34 at 532 nm. The calibration curve was performed with 1,1,3,3-tetraethoxypropane or malondialdehyde (MDA). In the 35 case of the control, the same procedure was followed with the replacement of distilled water (100 µL

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The effect on the basal tone was evaluated by cumulative concentration-response curves for the lyophilized MLE at 61 various concentrations (0.1, 1, 10, 100 and 1000 µg/mL) with an administration interval of 5 minutes.   Tyrode solution to stabilize the tissue again, waiting for at least 10 minutes.

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The tension was adjusted again to 1 g if necessary. Subsequently, 100 µg/mL MLE was added for 20 minutes 82 in the same normal Tyrode solution. Then, it was replaced again with Ca 2+ free Tyrode solution for 10 minutes 83 and 100 µg/mL MLE was added. It contracted |with ACh 10 -5 M previously and waited 5 minutes to see the 84 response. Finally, CaCl2 (0.1 mM; 0.3 mM; 0.6 mM, 1 mM) was added.

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After 1 hour of stabilization, the protocol was initiated by generating a contractile plateau with ACh 10 -5 M 87 (as 100% of maximal response), then tissue was washed with fresh tyrode solution (4-5 times) and returned to its 88 initial tension of 1 g. Subsequently, MLE (100 µg/mL) was incubated in the organ bath for 20 minutes, then H2O2 89 (10 -10 to 10 -4 M) were added in cumulative concentrations. To study the role of the main metabolites of MLE on 90 oxidative damage induced by H2O2 in rat ileum, mangiferin, quercetin and gallic acid were used. The ileal strips 91 were pre-incubated with mangiferin 10 -5 M, quercetin 10 -5 M, or gallic acid 10 -5 M for 20 min before the experiment.

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Then, tissues were exposed to cumulative concentrations of H2O2 (10 -10 to 10 -4 M). In parallel, the effect of H2O2 93 was studied in an intact portion of the ileum in the absence of MLE or its main metabolites, which served as a 94 control.