Rutin derivatives obtained by transesterification reactions catalyzed by Novozym 435: Antioxidant properties and absence of toxicity in mammalian cells

Abstract

Flavonoids are one of the most important and diversified phenolic groups among products of natural origin. An important property of this metabolite class is the antioxidant action. This study evaluated the antioxidant and cytotoxic activities and oxidative stress of transesterification products of the flavonoid rutin, catalyzed by Novozym® 435. The presence of monoacetate and diacetate was confirmed by quantitative evaluation of the retention times (rutin, 15.68 min; rutin monoacetate, 18.14 min; and rutin diacetate, 18.57 min) and by the data from LC-MS and NMR 1H and 13C. The experiment showed excellent conversion values of 96% in total acetates (rutin monoacetate and diacetate). These results confirmed that rutin derivatives have antioxidant potential, as evaluated by the ORAC method (rutin standard: 0.53 ± 0.08 μM Trolox/g and rutin derivatives: 2.33 ± 1.08 μM Trolox/g) and also show low cytotoxicity in human and animal cells. Rutin derivatives reduced the production of reactive oxygen species in RAW macrophages as well. Many qualities attributed to rutin derivatives make them promising potential candidates for use as nutraceuticals, including their high amounts of antioxidants, biological potential and low toxicity, which contribute to the reduction of oxidative stress.

Fig 1. TLC (Butanol (4): Water (0.25); ethanol (0.25) and acetic acid (0.5)) of the transesterification reaction of the flavonoid rutin with the acyl donor vinyl acetate catalyzed with the enzyme Novozym 435, and the respective products.

Fig 2. Analysis by High-Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-DADMS/MS) of the reaction products obtained from rutin acetylation reaction (120 h) at 190 nm: Rutin (1), RT: 10.80 min; rutin monoacetate (2), RT: 12.08 min; and rutin diacetate (3), RT: 13.79 min.

Fig 3. Mass spectra of the reaction products (72 h) obtained by HPLC-MS analysis.

a) Rutin monoacetate (m/z: 651.34) and b) Rutin diacetate (m/z: 693.53).

Fig 4. Analysis of the fragments obtained by mass spectrometry of: A-rutin: m/z = 609.2 [M-H]-; m/z = 301.0 [M-H-(Rha + Glu)]-; B-rutin monoacetate: m/z = 651.2 [M-H]-; m/z = 609.1 [M-H-(COCH2)]-; m/z = 463.1 [M-H-(Rha-OCOCH2—H2O)]-; m/z = 301.0 [M-H-(Rha-OCOCH3 + Glu)]-; C-rutin diacetate: m/z = 693.2 [M-H]-; m/z = 651.2 [M-H-(COCH2)]-; m/z = 505.0 [M-H-(Rha-OCOCH2)]-; m/z = 301.0 [M-H-(Rha-OCOCH3 + Glu-OCOCH2)]-.

Fig 5. Conversions (%) of the acetates in different reaction times based on calculation of the relative area percentage detected at 190 nm by HPLC-DAD-MS.

The analyses were performed in triplicate. *No significant difference. Bonferroni Test p< 0.05.

Fig 6. Chromatograms obtained by HPLC-UV (C18 Column) from quantitative analysis of the fractions (4–13) obtained by the chromatographic purification step of the scale-up reaction.

Peaks of fractions 4 (FR4) and 5 (FR5): rutin diacetate (87% purity) eluted at 13.70 and of fraction FR6-13: rutin monoacetate eluted at 12.36 min.

Fig 7. HMBC H-C (J2-J3) correlation signals of rutin diacetate.

Fig 8. Expansion of HSQC H-C (J1) spectra showing the correlations for diagnostic signals of rutin diacetate.

Fig 9. Expansion of HMBC H-C (J2-J3) spectra showing the correlations for diagnostic signals of rutin diacetate.

Fig 10. Scheme with optimized reaction conditions and enzyme Novozyme 435 as biocatalyst by 96 h.

1: quercetin-3-O-rutinoside (rutin); 2: quercetin-3-O-rutinoside monoacetate (rutin monoacetate); and 3: quercetin-3-O-rutinoside diacetate (rutin diacetate).

Fig 11. Cytotoxicity of reaction mixture and rutin on RAW, Vero and Hep G2 cells.

Initially, mammalian cells (10⁵ cells) were incubated in a 96-well plate for 24 h in the absence (white bars) or in the presence of single doses of the test compounds at different concentrations, as indicated (black bars). After the incubation, the viability of each type of cell was determined spectrophotometrically at 570 nm (ABS, absorbance) by MTT assay. Data shown are the mean ± standard deviation (SD) of three independent experiments performed in triplicate. Asterisks represent significant differences in relation to control (P < 0.05).

Fig 12. Effect of rutin and reaction mixture (monoacetate and diacetate) on the production of reactive oxygen species (ROS).

(A) The RAW macrophages were pre-treated for 24 h with rutin esters and rutin before stress induction with hydrogen peroxide (H₂O₂) (10 mM) for 1 h. (B) Post-treatment (24 h) of macrophages with both compounds after stress induction with 10 mM H₂O₂ for 1 h. The production of ROS in each system was measured fluorometrically in the control and treated cells incubated with the green fluorescent probe H2DCFDA. Non-treated cells and cells stressed with 10 mM H₂O₂ were used as positive controls for the intracellular generation of ROS. The results are expressed as fluorescence arbitrary units (FAU). Values represent mean ± standard deviation of three independent experiments. Asterisks represent significant statistical differences from the negative controls (white bars), and stars represent statistical differences from the positive controls (hatched bars) (P< 0.05).

Fig 13. Reuse of Novozym 435 of reaction transesterification.

Description of the conversion of the monoacetate (m/z: 651) and diacetate (m/z: 693) product obtained from the transesterification reaction after five reuse reactions (120 h, 60°C, 200 rpm).